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and Genetics Development Meeting 6th European Zebrafish Rome 15th - 19th July 2009 Program & Abstracts Genetics and Development Meeting 6th European Zebrafish Rome 15th - 19th July 2009 Program & Abstracts Contents Intro Organizing Committee ................................................................................ Pag. Registration ................................................................................................. Pag. Meeting information .................................................................................... Pag. Congress map .............................................................................................. Pag. Sponsors ...................................................................................................... Pag. Exhibitors and Exhibition area ..................................................................... Pag. Information for Authors................................................................................ Pag. Aknowledgements ....................................................................................... Pag. Program at a glance ..................................................................................... Pag. Scientific program ....................................................................................... Pag. ABSTRACTS .............................................................................................. Pag. TALKS Session I: Session II: Session III: Session IV: Session V: Session VI: Early development, gastrulation and segmentation .................. Pag. Signaling ................................................................................. Pag. Gene regulation ...................................................................... Pag. Organogenesis ........................................................................ Pag. Emerging technologies ............................................................ Pag. Genomic workshop ................................................................. Pag. 37 43 49 55 61 67 74 80 86 92 98 4 5 6 8 9 10 11 12 14 17 35 Session VII: Cancer .................................................................................... Pag. Session VIII: Disease models ....................................................................... Pag. Session IX: Cardiovascular system ............................................................. Pag. Session X: Hematopoiesis and immune system ........................................ Pag. Session XI: Neurobiology I: patterning and behaviour ............................... Pag. Session XII: Neurobiology II: sensory organs and regeneration ................... Pag. 104 2 POSTER Husbandry workshop .................................................................................. Pag. 143 Gern cells and maternal determinants ......................................................... Pag. 147 Early development and gastrulation ............................................................. Pag. 149 Signaling ..................................................................................................... Pag. 164 Organogenesis............................................................................................. Pag. 188 Neurobiology: patterning ............................................................................ Pag. 223 Neurobiology: sensory organs .................................................................... Pag. 268 Behaviour .................................................................................................... Pag. 293 Cardiovascular system ................................................................................. Pag. 297 Hematopoiesis and immunology ................................................................. Pag. 325 Disease models ........................................................................................... Pag. 351 Cancer......................................................................................................... Pag. 405 Regeneration ............... 26 Scientific Program Friday July 17th, 2009 SALONE DELLA CULTURA - EXHIBITION AREA 17:30 - 18:00 Coffee Break AUDITORIUM DELLE SCIENZE 18:00 - 19:30 Keynote lecture II Chair: Karuna Sampath, Singapore Enhancing mammalian regeneration Nadia Rosenthal, Rome, Italy 27 July 17th Scientific Program Saturday July 18th, 2009 AUDITORIUM DELLE SCIENZE 09:00 - 10:30 Session VIII Disease models Session Chairs: Ela Knapik, Nashville, USA Derek Stemple, Cambridge, UK CHEMICAL COLITIS MODELS IN ZEBRAFISH LARVAE S. Oehlers, Auckland, New Zealand DISRUPTION OF THE TROPONIN COMPLEX LEADS TO LOSS OF SARCOMERIC INTEGRITY IN SKELETAL MUSCLE M.I. Ferrante, Cambridge, UK GENETIC ANALYSIS REVEALS A ZEBRAFISH MODEL FOR FRASER SYNDROME AND IDENTIFIES POTENTIAL NOVEL DISEASE GENES T.J. Carney, Singapore CEYLON: A ZEBRAFISH MUTANT WITH SHWACHMANDIAMOND SYNDROME-LIKE BONE MARROW FAILURE N.S. Trede, Salt Lake City, USA CELLULAR SENESCENCE AND DNA DAMAGE IN A ZEBRAFISH MODEL OF COSTELLO SYNDROME IS LINKED TO UBIQUITINMEDIATED PROTEOSOMAL DEGRADATION OF ONCOGENIC HRAS M. Mione, Milan, Italy A MODIFIED ACETYLCHOLINE RECEPTOR DELTA-SUBUNIT ENABLES A NULL MUTANT TO SURVIVE BEYOND SEXUAL MATURATION J.M. Urban, Bethesda, USA 43 44 45 July 18th 46 47 48 SALONE DELLA CULTURA - EXHIBITION AREA 10:30 - 11:00 Coffee break 28 Scientific Program Saturday July 18th, 2009 AUDITORIUM DELLE SCIENZE 11:00 - 12:30 Session IX Cardiovascular system Session Chairs: Massimo M. Santoro, Turin, Italy Stefan Schulte-Merker, Utrecht, The Netherlands ROLE OF NOTCH SIGNALING IN EARLY ZEBRAFISH CARDIOGENESIS K.A. Nembhard, Durham, USA BUNGEE - A NOVEL REGULATOR OF CARDIAC VALVE FORMATION IN ZEBRAFISH I.M. Berger, Heidelberg, Germany CHEMOKINE SIGNALING CONTRIBUTES TO REGIONAL PATTERNING OF THE FIRST EMBRYONIC ARTERY A.F. Siekmann, Muenster, Germany July 18th 29 MiR-1 REGULATES ANGIOGENESIS BY MODULATING VEGF SIGNALING A.J. Giraldez, New Haven, USA THE ZEBRAFISH FULL-OF-FLUID MUTANT IDENTIFIES A SECRETED PROTEIN ESSENTIAL FOR LYMPHANGIOGENESIS IN ZEBRAFISH AND HUMANS B.M. Hogan, Utrecht, The Netherlands CHARACTERIZATION OF VASCULAR MURAL CELLS DURING ZEBRAFISH DEVELOPMENT M.M. Santoro, Turin, Italy 49 50 51 52 53 54 SALONE DELLA CULTURA - EXHIBITION AREA 12:30 -14:00 Lunch Scientific Program Saturday July 18th, 2009 POSTER AREA AMBULACRO DELLA PITTURA AND AMBULACRO DELLA LETTERATURA 14:30 - 16:00 Poster Presentation EVEN NUMBERS AUDITORIUM DELLE SCIENZE 16:00 - 17:30 Session X Hematopoiesis and immune system Session Chairs: Graham Lieschke, Parkville, Australia Maria Vega Flores, Auckland, New Zealand BOTH PRIMITIVE AND DEFINITIVE HEMATOPOIESIS ARISE FROM HEMOGENIC ENDOTHELIAL CELLS IN THE ZEBRAFISH EMBRYO J.Y. Bertrand, La Jolla, USA SDF-1 EXPRESSING CELLS ESTABLISH THE HEMATOPOIETIC STEM CELL (HSC) NICHE AND PLAY A ROLE IN HSC HOMING FOLLOWING ADOPTIVE CELL TRANSFER IN A ZEBRAFISH MODEL OF BONE MARROW TRANSPLANT T.C. Lund, Minneapolis, USA LIVE IMAGING REVEALS THAT DEFINITIVE HAEMATOPOIETIC STEM CELLS EMERGE DIRECTLY FROM HAEMOGENIC ENDOTHELIAL CELLS IN ZEBRAFISH EMBRYOS M.V. Flores, Auckland, New Zealand DEVELOPMENT OF AN IMMUNE-MATCHED TRANSPLANTATION MODEL TO DETECT HEMATOPOIETIC STEM CELL ACTIVITY IN ZEBRAFISH J.L.O. de Jong, Boston, USA THYMUS COLONIZATION IS UNDER THE CONTROL OF CHEMOKINES AND CHEMOKINE RECEPTORS B. Bajoghli, Freiburg, Germany A ZEBRAFISH TRANSGENIC MODEL TO STUDY ANTIGENPRESENTING CELLS: B LYMPHOCYTES, MACROPHAGES AND DENDRITIC CELLS V. Wittamer, San Diego, USA 55 56 July 18th 57 58 59 60 30 Scientific Program Saturday July 18th, 2009 AUDITORIUM DELLE SCIENZE 17:30 - 18:00 Community Meeting Chair: Marina Mione, Milan, Italy CENTRALE RISTOTHEATRE 20:00 - 02:00 Social Dinner 31 July 18th Scientific Program Sunday July 19th, 2009 AUDITORIUM DELLE SCIENZE 09:30 - 11:00 Session XI Neurobiology I: patterning and behaviour Session Chairs: Corinne Houart, London, UK Marnie Halpern, Baltimore, USA NEGATIVE REGULATION OF NEUROGENESIS BY AN FGF FROM NEURONS R. Gonzalez-Quevedo, London, UK SEGREGATED PROCESSING OF VISUAL INPUTIN Ilocalized to 2 cells by the 4-cell stage, and predicts the embryonic dorsal. This suggests that asymmetric localization of sqt RNA, and possibly other factors, is an early step in dorsal specification (Gore et al., Nature, 2005). The sqt 3' untranslated region (UTR) is necessary and sufficient for dorsal localization and the dorsal localization element lies within the first 50 nt of the sqt 3' UTR. Phylogenetic footprinting of the sqt 3'UTR from ~30 cyprinid species revealed multiple conserved blocks, and a potential conserved secondary structure. RNA gel-shift and UV cross-linking assays show multiple RNA-binding factors which bind specifically to distinct regions within the sqt 3'UTR. One of these, sqt-RNA binding factor 1 (Srbf1), binds to the sqt dorsal localization element. We have purified Srbf1 from oocyte extracts by chromatography, and determined its peptide sequence. Srbf1 is a nucleic acid-binding protein. Analysis of deletions and point mutations in Srbf1 shows that the N-terminus of Srbf1 is necessary and sufficient for binding to sqt RNA. We have also identified key residues in Srbf1 that are essential for binding to sqt RNA. An ENU-induced mutation in the srbf1 locus has been recently obtained by TILLING, and characterization is in progress. Interestingly, over-expression of mutant but not wild type Srbf1, in embryos leads to embryonic defects. Current efforts are aimed at purifying the other factors that bind to sqt RNA, and understanding the mechanisms by which Srbf1 binds and localizes sqt RNA to dorsal. 45 Talks 10 SESSION II Signaling Calcium Signaling in Zebrafish Left/Right Patterning D. Gunther1, M. Jurynec1, J. Amack2, H. J. Yost2, D. J. Grunwald1 1 Departments of Human Genetics and 2Department of Neurobiology & Anatomy, University of Utah, USA Directional fluid flow in Kupffer's Vesicle (KV) is required for the initiation of the left side-specific cascade of gene expression that leads to left-right asymmetries in zebrafish. We still have little understanding of how the information of fluid flow is transmitted from the KV to the lateral plate mesoderm, where left side-specific gene expression arises. Here we show that left-side-specific mobilization of intracellular calcium is dependent on normal fluid flow in KV and is required for asymmetric gene expression and subsequent left-right patterning. Ryanodine Receptors (RyRs) are one class of intracellular calcium release channels that are best known for their role in excitation contraction coupling in vertebrate cardiac and skeletal muscle. However RyRs are widely expressed in many non-muscle cell types suggesting they are involved in other processes as well. Here we show that the entire cascade of left-right asymmetric gene expression and organ morphogenesis requires left-side-specific elevation of intracellular calcium dependent on RyR function. At the 5-8 somite stage of zebrafish embryos, free intracellular calcium levels are elevated specifically on the left side of Kupffer's Vesicle. The mobilization of calcium depends on RyR function: When RyR expression or function is inhibited widely, or specifically in the KV, left-side-specific mobilization of calcium is blocked, along with left-side-restricted patterns of gene expression and subsequent organ morphogenesis. Calcium release appears to be part of the signaling pathway between fluid flow and asymmetric gene expression: First, pharmacologic inhibitor studies indicate that RyR function in left-right patterning is required during mid-somitogenesis, at the time of fluid flow. Second, left-sidespecific calcium mobilization depends on fluid flow in KV. Third, inhibition of RyR expression or function does not affect KV structure, ciliogenesis or fluid flow. Together these experiments indicate that intracellular calcium mobilization on the left side of the KV is a required step in normal left-right patterning. In additional experiments, we show that elevation of intracellular free calcium levels on the right side of KV is sufficient to drive left-side specific gene expression on the right sidel gland but also throughout the body and in cell lines, by an unknown mechanism. In order to understand the mechanism underlying light-induced per2 expression, functional analysis of the per2 promoter was performed by transient and transgenic expression of per2 promoterreporter constructs in zebrafish embryos and by means of stable transfection of PAC-2 zebrafish cells which are known to contain directly light-entrainable circadian clocks. This analysis has revealed a light-responsive module (LRM) in the per2 promoter which is both necessary and sufficient for light induction. Interestingly, the LRM sequence is highly conserved throughout evolution and the human LRM can substitute for its zebrafish counterpart to confer direct light regulation of gene expression in zebrafish cells. Functional analyses revealed that within the LRM, a D box enhancer is critical for its light-induced expression, while an E-box element is responsible for its clock-regulated expression. Using in vivo approaches complemented by in vitro techniques, transcription factors that bind to these elements were identified, characterized and functionally tested. The findings of these studies suggest a hierarchic mechanism in which light enables clock regulation of per2. This study extends the understanding of the mechanisms underlying light-entrainment of the circadian clock and contributes to a general understanding of how clock gene regulation has evolved in vertebrates as well as of general photic cellular responses. 49 Talks 14 SESSION III Gene regulation Conditional Gene Inactivation Reveals a Crucial Role For zgc:112094 in Zebrafish Development B. Zhang1, W. Liang1, J. Ren1, X. Ren1, M. Liu1, Z. Zhu1, and S. Lin1, 2 1 Key Laboratory of Cell Proliferation and Differentiation, Center of Developmental Biology and Genetics, College of Life Sciences, Peking University, Ministry of Education, Beijing, P. R. CHINA; 2 Department of Molecular, Cell & Developmental Biology, University of California Los Angeles, Los Angeles, USA. We have performed a pilot screen for gene trap in zebrafish using a Tol2 based construct that contains unidirectional Cre/loxP system coupled with a splice acceptor (SA) and poly-adenylation signal to stop transcription. When the insertion hits a gene of interest in one of its introns with the orientation of the SA sequence being opposite to the gene (R-SA), Cre recombinase can be applied to invert the gene trap cassette to disrupt the endogenous gene expression. Similarly, if the insertion is in the same direction of transcription (F-SA), causing abnormal splicing, Cre recombinase can be used to rescue the phenotype by reversing the insertion. From mapping 94 unique insertions, we obtained 7 in exons and 15 in introns. Among the R-SA intron insertions, one was identified as a conditional mutant with embryonic lethal phenotype only being apparent by reversing the insertion with Cre mRNA injection. RT-PCR and mRNA rescue experiment confirmed that the mutant phenotype was caused by the insertion of the conditional gene trap construct in the first intron of the gene zgc:112094, which encodes an ASB-5 homologous protein of E3 ligase. Expression of this gene was maternally detectable and became restricted to heart and somites after 22hpf. RNA in situ hybridization analysis with markers for different tissues, including flk1, etsrp and gata1, showed that blood vessel formation in the mutant embryos was severely affected. Angioblasts were reduced and failed to migrate to midline although they were present within the lateral mesoderm, while blood cell development remained normal. These data suggest that zgc:112094 is required for vasculogenesis. To our knowledge, this is the first Cre/ loxP mediated conditional mutation in zebrafish and the method could be scaled up for genome wide screen of mutations. The potential role of zgc:112094 in cell migration and/or proliferation may be related to cancer and human vascular diseases. Talks 50 SESSION III Gene regulation 15 Transcriptional control of bone formation through the regulatory gspecies. Nevertheless, the role of HES/her genes in somitogenesis remains unclear. Eleven her genes oscillate in zebrafish posterior PSM, in contrast to four known in mouse. This large number raises the question whether they function redundantly or play independent roles in different aspects of somitogenesis. To answer this we characterized her2, one of nine cyclic Hes5 sub-family genes, and compared it to known core components of the zebrafish segmentation clock, her1 and her7. In contrast to her1 and her7, cyclic expression of her2 is restricted to the posterior PSM, similar to the other Hes5 sub-family genes. Whereas over-expression of her1 or her7 causes weak disturbances, her2 over-expression results in strong disruption of somitogenesis. Loss of function of her1 or her7 yield distinct somitogenesis phenotypes, but somitogenesis is not affected in a her2 mutant, suggesting that other Hes5 family members compensate. This hypothesis is tested by knockdown of all cyclic Hes5 genes. In conclusion, these data indicate a role for her2 in somitogenesis that is distinct from known her genes. We propose a model in which the duplicated Hes5 sub-family members form an oscillatory loop conferring robustness to the fast segmentation clock of the zebrafish, a function not required in slower clocks such as the mouse. Talks 52 SESSION III Gene regulation 17 A role for Aspp2a in endoderm development in zebrafish R. Hoffmans1,2, S. Sidi1 and A. T. Look1 1 Dana Farber Cancer Institute, Boston, USA; 2Hubrecht Institute, Utrecht, Netherlands ASPP (Apoptosis Stimulating Protein of p53) is best known for its role as a transcriptional coactivator and binding partner of p53, a tumor suppressor gene that is mutated in half of all human tumors. Here we used the zebrafish to study the role of aspp in development. In zebrafish there are five aspp family members. In this work we focused on aspp2a, which is ubiquitously expressed both maternally and zygotically. aspp2a morpholino injected embryos show at 1 dpf tail defects that are not seen in control morpholino injected embryos. To characterize the function of aspp2a in the formation of the three germ layers the expression of marker genes during gastrulation were analyzed. Markers for presumptive mesoderm (ntl), ventral mesoderm/ectoderm (bmp2b) and neuroectoderm (sox2) are unaffected in the aspp2a morphants compared to uninjected controls. However, sox17 expression, which marks endodermal cells and forerunner cells, is severely reduced in aspp2a injected embryos. The liver and pancreas are endodermal derived organs and can be marked by hhex and ins. The expression of both hhex and ins was reduced in the aspp2a morpholino injected embryos compared to control injected embryos. This suggests that aspp2a loss affects endoderm formation, which in turn results in defects in liver and pancreas development. The TGFbeta/Nodal pathway is the major determinant in endoderm formation in vertebrates. Nodal signaling results in phosphorylation and activation of Smad2, which controls the expression of the transcription factors gata5, bon and mezzo. Bon and Gata5 form a complex together with Eomes and bind the cas promoter and induce cas expression. Mezzo functions redundantly with Bon and can by itself induce cas expression. The transcription factor cas regulates the expression of sox17 and foxa2. In aspp2a morphants Smad2 phosphorylation is unaffected as is the expression of bon and mezzo. However the number of foxa2 and cas expressing cells is severely reduced in aspp2a injected embryos and there is also a slight reduction in gata5 expressing cells. Epistasis analysis showed that aspp2a is epistatic to gata5 and eomes but not to mezzo and cas. These results indicate that aspp2a functions together with gata5 and eomes to induce cas expression. Current efforts focus on testing if Aspp2a binds directly to Gata5, Eomes or Bon and if Aspp2a can be found on the promoter of cas. 53 Talks 18 SESSION III Gene regulation Crumbs Complex Coordinately Regulates Neurogenesis and Neuroepithelial Polarity through Canonical and Non-canonical Notch Pathway S. Ohata1, R. Aoki1, S. Kinoshita1, S. Tsuruoka-Kinoshita1, M. Yamaguchi2, H. Tanaka1, H. Wada1, I. Masai2, and H. Okamoto1 1 RIKEN Brain Science Institute, Japan; 2Okinawa Institute of Science and Technology, Japan In the neural development, neuroepithelial cells, which are highly polarized neural stem cells, control various aspects of neural development, such as neurogenesis, guidance of neural migration, and axon guidance. They have very unique morphology with apical and basal processes that extend to the ventricular and pial surfaces, respectively. At the tip of apical processes, cellular junctions such as adherens and tight junctions are formed. For the establishment of a functional nervous system, the cooperative control of neuroepithelial functions, and morphology are crucially important. To understand the molecular mechanisms by which neuroepithelial cells coordinately regulate their functions such as neurogenesis and their morphology such as apicobasal polarity, we analyzed the zebrafish holm mutant that has a mis-sense mutation in the neuroepithelial polarity gene, epb4.1l5 (erythrocyte protein band 4.1-like 5, formerly known as mosaic eyes), which encodes a putative adaptor protein. Neuroepithelial morphology such as apicobasal polarity, adherens junctions, tight junctions, and the extension of apical processes to ventricular surface were disrupted in the holm mutant. In addition, neuroepithelial functions such as neurogenesis and guidance for tangential migration of vagus motor neurons were also affected in the holm mutant. These results suggest that epb4.1l5 is required for the cooperative control of neuroepithelial functions and morphology. To reveal how Epb4.1l5 cooperatively regulate neuroepithelial functions and morphology in the molecular level, we next analyzed potential molecules acting downstream of epb4.1l5. Crumbs family proteins had been reported to be negatively regulated by Epb4.1l5 and negatively regulate the Notch activator -Secretase in fruit fly. The holm mutant phenotype was partially phenocopied by over-expression of Crumbs2. Interestingly, Notch activity was significantly reduced in the holm mutant, and over-expression of the Notch intra-cellular domain (NICD) rescued the holm mutant. In addition, over-expression of constitutive active form of R-Ras, which had been reported to act downstream of NICD and regulate cellular polarity and adhesion, partially rescued the holm mutant, and knocking down of r-ras canceled the recovery of the holm mutant phenotypes by NICD. These results may suggest that Epb4.1l5 cooperatively regulates neuroepithelial functions and morphology through the regulation of Notch activity. Talks 54 SESSION IV Organogenesis 19 Jagged-Notch signaling patterns the facial skeleton F. Stellabotte, G. Crump Center for Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, United States The facial skeleton in all vertebrates arises from complex cellular interactions that occur between the endoderm, ectoderm, and neural crest cells of the well-conserved pharyngeal arches. Our goal is to identify the signals that are involved in patterning the facial skeleton along the dorsoventral axis. Several genes have been identified that are required for a ventral skeletal fate; however, genes responsible for dorsal identity are less clear. A ventral to dorsal gradient of the Endothelin1 (Edn1) morphogen specifies lower versus upper jaw identity by activating specifier genes, such as dlx3b, dlx5a, and dlx6a in ventral skeletal precursors. Recently, we discovered a mutation in jagged1b (jag1bb1105), which results in dorsal skeletal elements acquiring a ventral morphology. We show jag1b specifies upper jaw identity by inhibiting the expression of ventrally expressed genes. Since Jag1b is a Notch signaling ligand, we next examined which Notch receptor is involved in patterning the facial skeleton along the dorsoventral axis. Whereas jag1b is expressed in the dorsal domain of the first two pharyngeal arches, we find that notch2 is expressed in a complementary pattern in the ventral domain. In addition, reduction of Notch2 function with a morpholino (MO) resulted in dorsal-specific skeletal defects similar to that seen in jag1bb1105 mutants. Since Jagged-Notch inhibition resulted in dorsal-specific skeletal defects, we predicted that activation of Notch signaling would show the opposite phenotype. In fact, when we activated Notch signaling by misexpressing the Notch intracellular domain (NICD) during skeletal patterning stages, we saw a loss of ventral-specific gene expression and a reduction of the more ventral jaw skeleton. Since the skeletons of NICD embryos resembled the skeletons of larvae with reduced Edn1 function, we wanted to study the relationship between dorsally required Jagged-Notch signaling with the ventrally required Endothelin (Edn1) morphogen. We generated jag1bb1105 and edn1tf216b double mutants in which we find the facial skeleton is rescued. Furthermore, we wanted to identify additional DV-specific genes that control skeletal morphology, we performed fluorescent activated cell sorting (FACS) and microarray analysis of cells from transgenic zebrafish expressing GFP in different DV domains of skeletal precursors. We will present candidate genes that are expressed within discrete domains of the pharyngeal arches during patterning stages. Currently, we are testing whether these candidate genes are also regulated by Jagged-Notch signaling. Talks 55 20 SESSION IV Organogenesis Positive and negative feedback from proteoglycans during chondrocyte differentiation B. F. Eames and C. B. Kimmel Institute of Neuroscience, University of Oregon, Eugene, USA Despite the fact that skeletogenic cells immerse themselves in abundant extracellular matrix, remarkably little is known about the influence of proteoglycans on skeletal cell differentiation in vivo. Here, we reveal the effects of a loss-of-function mutation in xylosyltransferase1 (xylt1) on cartilage and bone development during zebrafish endochondral ossification. Xylosyltransferases initiate glycosaminoglycan side chain addition to core proteins during the formation of proteoglycans, such as aggrecan, which is expressed highly in cartilage. Accordingly, xylt1/zebrafish demonstrate decreased Alcian blue staining in cartilage elements undergoing endochondral ossification, although the patterning of these elements is roughly the same as wild-type siblings. At early stages of overt chondrogenesis in xylt1-/- mutants, the cartilage markers sox9b and col2a1b are expressed in similar domains as wild-type, but at decreased levels. Interestingly, xylt1-/- zebrafish also have increased and premature Alizarin red staining in perichondral bone, even though xylt1 does not appear to be expressed highly in developing perichondrium. Osteoinductive factors, such as Indian hedgehog (Ihh), are expressed in maturing chondrocytes and are known to signal to osteoblast precursors in the overlying perichondrium. In support of a hypothesis that mutant cartilages prematurely initiate chondrocyte maturation, ihh genes are expressed in xylt1-/- cartilage earlier than in wild type. In total, these data suggest that proteoglycans play a positive role during initial stages of overt chondrocyte differentiation and a negative role during chondrocyte maturation. Talks 56 SESSION IV Organogenesis 21 Mutations in components of diverse essential cellular pathways disrupt zebrafish intestinal development J.K. Heath1, A.P. Badrock1, E.L. Christie1, S.S. Markmiller1, A.C. Parslow1, Y. Rifat1, T.L. Tabone1, A.J. Trotter1, H. Verkade1,2, N.E. Hall1, A.Y.N. Ng1, E. Ober2, H.A. Field2, G.J. Lieschke3, and D.Y.R. Stainier2 1 Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Australia; 2Department of Biochemistry and Biophysics, University of California, San Francisco, USA and 3Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, Australia Our aim is to identify zebrafish genes with functions in vertebrate intestinal development and to explore their potential gf, Retinoic Acid and BMP signals. Combined depletion of cdx1a, cdx1b and cdx4 revealed that all cdx genes act redundantly during endoderm organogenesis. Thus cdx genes are responsible for providing the right positional cues along the anterior-posterior axis, placing the bipotential hepatopancreatic progenitor cells in the right embryonic environment to potentiate proper differentiation into liver and pancreas. Talks 58 SESSION IV Organogenesis Zebrafish anterior chamber development and maintenance M. Takamiya, B. Weger, U. Straehle ITG, FZK, Karlsruhe, Germany 23 Anterior chamber of the eye is a clear space filled with aqueous humour between the lens and cornea which guides the light focused on the retina, hence crucial for a normal vision. To establish the basis for understanding the origin and development of the anterior chamber at the molecular level, we focused on the cornea, a structure of neural crest origin. We established the sox10::eosFP transgenic line to follow neural crests, but also to visualize de novo sox10 transcription activity by using a unique feature of EosFP, a photo-convertible fluorescence protein; photo-conversion prior to the time lapse imaging allows one to discriminate earlier reporter expression from the later. This "pulse-chase" imaging cancelled out sox10-inactive cells to reveal a sox10-active cluster of non-ectomesenchymal neural crests in the pharyngeal arch at 18-20 hpf as a major source that contribute the anterior chamber. To gain insights into the signalling pathways involved in the anterior chamber formation, the effects of chemical inhibitors on the cornea morphology were analysed by time-lapse imaging and electron microscopy: 1) EGF and FGF signallings are required for the corneal endothelium formation; 2) planar polarity of the corneal epithelium was regulated by multiple pathways of EGF, Notch and Hh/PKA. To identify genes involved in the maintenance of the anterior chamber, we analysed gene expression profile of the adult cornea using the skin as a reference tissue, which is initially continuous with the cornea during embryonic stages and shares high anatomical similarities at the ultra-structure level. Microarray analysis presented ~165 cornea genes that are enriched in the adult cornea in comparison to the skin (P<0.01). Further RT-qPCR quantification established relative amount of the cornea genes over the skin and confirmed the results from the microarray analysis. We mapped spatial distribution of the cornea genes in the adult ocular tissues by in situ hybridisation. All together, we provide comprehensive set of gene profiling analyses, which contribute further functional analysis to examine the roles in the anterior chamber maintenance. 59 Talks 24 SESSION IV Organogenesis Renal stem cells give rise to new nephrons during zebrafish kidney regeneration C.Q. Diep1, D. Ma2 ,G. Djordjevic1, F. Bollig3, T. Ikenaga4, F. Ono4, C. Englert5, N.A. Hukriede6, R.I. Handin2, A.J. Davidson1 1 Center for Regenerative Medicine, Massachusetts General Hospital, Boston, USA; 2Hematology Division, Brigham and Women's Hospital, Boston, USA; 3Leibniz; Institute for Age Research, Fritz Lipmann Institute, Jena, Germany; 4Section on Model Synaptic Systems, NIH/NIAAA, Bethesda, USA; 5Friedrich-Schiller-University, Jena, Germany; 6Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, USA Zebrafish possess tremendous regenerative capacities and can regrow a range of organs, including the kidney. Here, we present evidence supporting the existence of a renal stem cell population in the zebrafish kidney that is involved in regenerating new nephrons (the functional unit of the kidney) in response to injury. Following gentamicin-induced kidney damage, adult fish kidneys showed increased expression of the renal transcription factor lhx1a in single mesenchymal-like cells within the kidney marrow. These cells appear to expand and coalesce into basophilic aggregates that express the transcription factor wt1b and then epithelialize into primitive nephron structures that fuse with existing nephrons. To further characterize the presumptive renal stem cell population in adult fish, we carried out transplantation experiments. Donor cells genetically tagged with a kidney-specific green fluorescence protein (GFP) transgene were injected into the circulation of unlabeled wild-type or kidney-injured recipient fish. Following transplantation we found functional donor-derived GFP+ nephrons in 47% of the recipient fish that were undergoing kidney regeneration, and in 9% of wild-type recipient fish. These findings suggest that the adult zebrafish kidney possesses a renal stem/progenitor cell population that can home back to the kidney from circulation and form new nephrons. To determine the origin of the renal stem cells, we examined the formation of the adult kidney during larval development. Single cells expressing lhx1a appeared just posterior to the swim bladder at around 10 days post-fertilization. From in vivo timecourse analyses of double transgenic larvae, these lhx1a+ cells were found to expand, initiate expression of wt1b, and differentiate into adult nephrons that fuse with the embryonic kidney. Taken together, we propose that the adult zebrafish kidney is initially formed from lhx1a-expressing renal stem cells, and these cells persist in the kidney marrow to serve as a source for de novo nephron formation in response to renal injury. Talks 60 SESSION V Emerging technologies 25 Improved genetic lineage tracing using the conditional red-to-green reporter Tg(hsp70: loxPDsRed2-loxP-EGFP) S. Hans, J. Kaslin, D. Freudenreich, M. Brand Biotec/CRTD, TU-Dresden, Dresden, Germany Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low but using transposon mediated transgenesis, we recently showed that Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2 [1]. In order to allow genetic fate mapping we previously generated a red-to-green reporter line Tg(EF1: loxP-DsRed2-loxP-EGFP). This line expresses DsRed2 under the control of the ubiquitous Xenopus Elongation Factor 1 alpha (EF1) promoter in the absence of Cre activity, but changes to EGFP after a successful recombination event. However, although we see strong expression of DsRed2 for up to 7 days, in situ analysis reveals strong ubiquitous expression only during gastrulation and mid-somitogenesis stages which limits the use of this line. To establish a faithful red-to-green reporter line we thus turned towards the zebrafish temperature-inducible hsp70 promoter which has been shown to express in a strong and ubiquitous fashion [2]. We will show that this conditional red-to-green reporter line Tg(hsp70: loxP-DsRed2-loxP-EGFP) indeed allows genetic fate mapping at all stages examined. Furthermore, in contrast to a constitutive reporter that always labels the entire lineage of a Cre expression domain, the conditional reporter line can be activated at a certain stage when the Cre expression domain has not reached its final expansion. Consequently, only a portion of the lineage will be labeled and might reveal that a certain structure is formed by the early Cre expressing cells. Hence, the conditional reporter line will facilitate fate mapping studies in the early zebrafish embryo because it takes advantage of the rapid zebrafish development that is much faster than the degradation of EGFP. 1. Hans S, Kaslin J, Freudenreich D, Brand M (2009); Temporally-Controlled Site-Specific Recombination in Zebrafish. PLoS ONE 4(2): e4640. 2. Halloran MC, Sato-Maeda M, Warren JT, Su F, Lele Z, Krone PH, Kuwada JY, Shoji W (2000); Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127(9):19531960. Talks 61 26 SESSION V Emerging technologies The zebrafish as a model system for in vivo single-molecule microscopy M. J.M. Schaaf1, W. J.A. Koopmans2, T. Meckel2, J. van Noort2, B. E. Snaar-Jagalska1, T. S. Schmidt2 and H. P. Spaink1 1 Molecular Cell Biology, Institute of Biology (IBL), Leiden University, The Netherlands; 2Physics of Life Processes, Institute of Physics (LION), Leiden University, The Netherlands Studying the dynamics of individual proteins by single-molecule microscopy has been available for several years, using laser excitation of fluorescently tagged proteins and detection by a sensitive CCD camera. However, until now this technology has only been applied to individual cells in culture. In the present study, the zebrafish was used as a model system to extend singlemolecule microscopy to the level of a living organism. As a molecule of interest we used yellow fluorescent protein (YFP) fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. In order to achieve this, a three-step approach was used. First, individual molecules were visualized in cultured (ZF4) cells using a wide-field fluorescence microscopy setup. Second, zebrafish embryos were injected with RNA encoding the fluorescently tagged protein and the injected embryos were dissociated at the sphere stage. Subsequently, imaging was performed using the dissociated primary cells. Third, imaging in living organisms was performed in epidermal cells in the skin of two-day-old zebrafish embryos, previously injected with RNA encoding the fluorescently tagged protein. A total internal reflection (TIRF) microscopy setup was used for this approach. The results of our experiments are in line with previous findings in mammalian cells and demonstrate that two populations of the YFP-tagged membrane anchor exist, which differ in the type of diffusion behavior they display. Furthermore, we demonstrate the occurrence of membrane microdomains confining the diffusion of membrane proteins. This membrane organization differs significantly between the different cell systems used in our study, illustrating the relevance of performing single-molecule microscopy in vivo. Talks 62 SESSION V Emerging technologies Transposon-based insertional mutagenesis platform D. Balciunas, D. Nagelberg, K. Gonzalez, J. Balciuniene Department of Biology, Temple University, Philadelphia, USA 27 Two key characteristics of any gene are loss of function phenotype and expression pattern. It has been extremely challenging to develop experimental approaches that simultaneously address these two characteristics in any model system. In Drosophila, the most successful and widely used approach is enhancer trap. It reveals the expression pattern of the affected gene, but is inherently non-mutagenic. In the mouse, several consortia are undertaking genome-wide insertional mutagenesis using gene traps. Due to limitations of mouse as a model system these experiments are carried out in ES cells. Thus, neither the expression pattern nor the mutant phenotype is revealed until a transgenic mouse is generated from these ES cells. We are developing transposon-based tools to simultaneously address these two characteristics (mutant phenotype and expression pattern) in the zebrafish. Our vectors are built from gene breaking transposon components known to disrupt gene expression upon integration. Two key advances were made to achieve mutagenicity and specificity of gene breaking transposons. To increase mutagenicity, fish-derived splice acceptor and transcriptional termination/ polyadenylation sequences were used. To increase specificity, the translation initiation codon was removed from the reporter. Combination of these two advances enables stringent selection for integration into genes and efficient disruption of gene expression. Our initial vectors used mRFP as the reporter for gene trapping events. One potential drawback of a fluorescent reporter is that integrations into genes expressed at very low levels may be impossible to detect. This and other considerations prompted us to replace the mRFP reporter by Gal4-VP16. To detect Gal4-VP16 expression we also added UAS:eGFP, ave also optimized our cMO synthetic procedure and investigated the relationship between cMO structure, in vitro thermodynamics, and in vivo activity. Through these studies, we have established basic principles for cMO design and successfully applied them to develop cMOs targeting other patterning genes. Our findings establish the generality of cMO technologies and illustrate the potential of these photoactivatable reagents in functional genomics research. 65 Talks 30 SESSION V Emerging technologies Chromosome painting of zebrafish chromosomes for the identification of inter-chromosomal rearrangements and marker chromosomes K. H. Brown1, J. L. Freeman2 , C. Lee1 1 Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, USA; 2School of Health Sciences, Purdue University, West Lafayette, USA Despite the importance of zebrafish as a model organism for studying vertebrate development, cell biology and cancer, the establishment of molecular cytogenetic probes to characterize the genomes of mutants and visualize specific chromosomal alterations is still lacking. Recently, we developed fluorescence in situ hybridization (FISH) probes for near-telomeric and nearcentromeric regions of all zebrafish chromosomes (Freeman et al., 2007) which has proven to be useful for identifying gross chromosomal rearrangements, but the scale at which they can identify chromosomal rearrangements, amplifications and translocations is limited. To bridge this gap, we have now developed chromosome painting probes containing combinations of DNA probes that "coat" entire chromosomes with a given fluorescent color. The development of these DNA probes permits rapid identification of inter-chromosomal rearrangements and ambiguous supernumerary marker chromosomes. We have developed chromosome-specific paint probes using pools of chromosome-specific bacterial artificial chromosomes (BACs) for each zebrafish chromosomes chosen at approximately 1 Mb intervals. Additionally, we have also identified a chromosomal translocation using two-color FISH. The establishment of this resource will enable the rapid identification of suspected chromosomal translocations in mutant strains. Future efforts to label individual chromosome-specific BACs within a specific chromosome, using a multicolor labeling approach, will eventually lead to a multi-color chromosome probe set that would enable detection of intra-chromosomal events including chromosomal inversions, deletions and duplications. Talks 66 SESSION VI Genomic workshop 31 A mesodermal gene regulatory network directed by T-domain transcription factors F.C. Wardle3, R..H. Morley1, K. Lachani2, A.C. Nelson3, D. Keefe4, M.J. Gilchrist2, P. Flicek4, J.C. Smith2 1 National Institute for Medical Research, London, UK; 2Gurdon Inst., University of Cambridge,UK; 3 Dept. Physiology, Development & Neuroscience, University of Cambridge, UK; 4European Bioinformatics Institute, Cambridge, UK Using chromatin immunoprecipitation combined with genomic microarrays and massively parallel sequencing we have identified targets of No tail a (Ntla), a zebrafish Brachyury ortholog that plays a central role in mesoderm formation. We have found that Ntla regulates a downstream network of other transcription factors and we identify an in vivo Ntla binding site that resembles the consensus T-box binding site (TBS) previously identified by in vitro studies. Using similar methods we have begun to identify targets of other T-domain factors, and using these data we describe a preliminary Gene Regulatory Network for mesoderm formation and patterning in the early zebrafish embryo. 67 Talks 32 SESSION VI Genomic workshop Automated high throughput mapping of promoter-enhancer interactions in zebrafish embryos J. Gehrig1,2, M. Reischl3, E. Kalmar1, Y. Hadzhiev1,2, M. Ferg1, A. Zaucker1,2, C. Song1, S. Schindler1, U. Liebel1 and F. Mueller1,2 1 Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, EggensteinLeopoldshafen, Germany; 2Department of Medical and Molecular Genetics, College of Medical and Dental Sciences, University of Birmingham, UK; 3Institute of Applied Computer Science, Forschungszentrum Karlsruhe, Eggenstein Leopoldshafen, Germany Zebrafish embryos offer a unique combination of high throughput capabilities and the organismal complexity of the vertebrate animal for a variety of phenotype screening applications. However, there is need for automation of imaging technologies to exploit the potential provided by the transparent embryo. Here we report the development and application of a high throughput pipeline for registering domain specificity of reporter expression in zebrafish embryos with the aim of mapping the interaction specificities between cis-regulatory modules and core promoters. Automated intelligent microscopy and custom built embryo detection and segmentation software allowed the registration of reporter activity in almost 18 thousand microinjected zebrafish embryos. Domain specificity of Venus reporter expression was evaluated by warping of individual embryo images onto a virtual two dimensional reference shape. Using the embryo imaging pipeline we demonstrate a diversity of interaction specificities between 203 enhancer-core promoter combinations. These interaction specificities underscore the importance of the core promoter sequence in cis-regulatory interactions and provide a valuable promoter resource for transgenic reporter studies such as enhancer traps. The technology described here is also suitable for the high throughput analysis of spatial distribution of a variety of fluorescence readouts in zebrafish embryos for genetic, pharmaceutical or toxicological screens. To this end we are exploring the utility of our image analysis tool in detecting gene expression phenotypes in embryos in which signalling pathways have been perturbed. Talks 68 SESSION VI Genomic workshop 33 Identifying the gene network involved in inner ear cell regeneration in the adult zebrafish S. M. Burgess1, J. Liang1, G. Renaud1, T. Wolfsberg1, and A. N. Popper2 1 National Human Genome Research Institute, Bethesda, USA; 2University of Maryland, College Park, USA Sensory hair cells of the inner ear are the mechanotransductory units in the neuroepithelia. In mammals, lost hair cells are not replaced, resulting in various permanent deficiencies in vestibuloauditory sensation. In contrast, zebrafish can replace lost hair cells with new ones throughout adulthood. Our ultimate goal is to understand the gene network requiref for inner ear hair cell regeneration in adult vertebrates. As an initial step, we identified the transcriptional response used for hair cell regeneration in zebrafish using next generation sequencing techniques for gene expression profiling. We modified a previous noise-exposure protocol, which enabled us to induce hair cell loss in the saccular epithelium of the adult zebrafish and characterize the subsequent regeneration process. We determined the inner-ear gene expression profiles at different time points during the regeneration, focusing particularly on those genes with significant increase in expression at the various time points. Here we used a new technique, Tag Profiling (Digital Gene Expression), to generate the gene expression profiles in an in-depth and high-throughput manner. Similar to SAGE in nature, the technique generates 3 million or more gene "tags" from the mRNA pool, giving us a very deep view of gene expression at each time point. We have developed and/ or utilized various bioinformatic tools for gene assignment, ontological analysis, and pathway analysis. Our analysis of the Tag Profiling data suggested new candidate genes as well as new candidate pathways involved in the hair cell regeneration. We are now working on associating the new candidate genes/pathways with various cellular events (e.g. cell division and differentiation) during hair cell regeneration to get a better understanding of the functions of those genes/pathways in the regeneration process. 69 Talks 34 SESSION VI Genomic workshop Whole-genome sequencing of a Wildtype Strain of Zebrafish for var to identify sequence that is not in the current Zv8, much of which is supported by at least one whole genome shotgun read. We have about 40Mb of such sequence, and hope to use this to improve the genome coverage. Thus we anticipate that the next release, Zv9, will be very densely covered with genetic markers, be the most complete assembly and that it is confirmed not just by sequence and clone overlaps but by extensive genetic mapping data. Talks 71 36 SESSION VI Genomic workshop High throughput screening of innate immune responses in zebrafish and carp embryos H. Spaink1, R. Dirks2, R. Carvalho2, O. Stockhammer1,2, S. He1, A. Zakrzewska1, E. SnaarJagalska1, A. Meijer1 1 Leiden University, Leiden, The Netherlands; 2ZF-screens B.V. Leiden, The Netherlands In recent years the zebrafish has been shown to be an excellent model for studying the mechanisms of the innate immune defense against pathogens. We have shown that transcriptome responses towards pathogens such as Mycobacterium marinum and Salmonella typhimurium are very similar to responses in mammalian systems. Using combinations of transcriptomic deep sequencing, morpholino knockdown and transgenic reporter fish technologies we have obtained new insights in the functions of key players of the innate immune system. These results are not only relevant to infectious diseases but also to the study of immune responses to cancer cells, for instance using xeno-transplantation assays. In this presentation we will show that such studies can also be extended to a high throughput level. For this we have applied the Copas XL biosorter (Union Biometrica) for screening disease symptoms using flow-through laser scanning profiling coupled with embryo sorting. We show that it can be highly profitable in this approach to also employ close relatives of the zebrafish for high throughput assays. For instance the common carp that can yield hundreds of thousands of embryos for each fish after in vitro fertilization can overcome limiting quantities of embryos for high throughput screening. We have obtained a shotgun sequence of the carp in order to compare results obtained with zebrafish showing that the hallmarks of innate immune responses in both fish species are extremely similar. Our results show that it is now possible to undertake chemical compound screens in carp fish the results of which can be further analyzed in transgenic zebrafish models. Talks 72 SESSION VI Genomic workshop 36 bis Amplify the impact of your research: Ensure that your data can be integrated into the electronic data stream M.A. Haendel and ZFIN staff Zebrafish Information Network, University of Oregon, Eugene, USA Publishing a manuscript was once the final step in communicating data. Now, publication is a preliminary step in a larger process that involves public databases. Public databases, such as the Zebrafish Information Network (, integrate and link your data with other data, amplifying the impact of your work on the wider research community. Public databases help researchers refine hypotheses before going to the bench, saving time and resources. Databases are only as good as their data, and those data come largely from publications. However, the quality, amount and type of information that can be captured are critically dependent upon the precision of the information available in each publication. Biocurators are trained professionals who carefully read publications and extract the most salient data for entry into a database. Authors can facilitate the curation process and ensure that their data are represented correctly and completely. Namely, authors should provide accession numbers and sequences whenever possible, check model organism databases for current gene and mutant nomenclature, and consult nomenclature committees before naming new genes. Ambiguity is the enemy - be specific! Which "shh" gene? Which fgf3 allele? Which pax2a morpholino? Which developmental stage? References to prior publications are helpful, but may not eliminate all ambiguity. As a reviewer, one can identify ambiguities and missing information before publication. The choice of journal and format is also important. Some journals do not give permission to reproduce figures in public databases, and the articles themselves may not be readily available. These issues limit the accessibility of data in public databases and should be considered when choosing a journal. Many journals now provide an open-access publication option. This is the friendliest format for a public database. Authors can help by working together with journals and biocurators to facilitate accurate data exchange and reporting. This interaction completes a new and important data feedback among authors, electronic data systems like ZFIN, and journals. 73 Talks 37 SESSION VII Cancer Zebrafish as an efficient model system to study the regulation of mitosis and chromosome instability H. Lee, K-H. Jeong, H-Y. Jeong, J-Y. Jeong, H-O. Lee, E. Choi Department of Biological Sciences, College of Natural Sciences, Seoul National University, Korea Aggressive cancers often exhibit Chromosome instability (CIN), characterized by aberrant chromosome structures and numbers. Unregulated mitosis is implicated in aneuploidy and CIN. Therefore, understanding the regulation of mitosis is not only a fundamental question in basic biology but is central to understanding cancer. Recently, anti-cancer drug development has focused on CIN and the mitotic kinases. Because established cancer cell lines harbor mutations in cell cycle control genes, development of inhibitors of mitotic kinases requires validation in an alternative in vivo system. We present data showing that zebrafish is a valuable system for studying mitosis. We propose based on this data that zebrafish can be adopted to validate the efficacy of inhibitors of mitotic kinases as anti-cancer drugs. We have specifically focused on two mitotic kinases, Plk1 and Aurora A. While Plk1 and Aurora A are both implicated in centrosome maturation and microtubule outgrowth, they have distinct roles. In zebrafish embryogenesis, loss of Plk1 or Aurora A using morpholinos resulted in growth defects followed by apoptosis. Loss of Plk1 resulted in defective microtubule outgrowth, aberrant centrosome numbers, and unstable kinetochore-microtubule attachments. Interestingly, zebrafish embryogenic cells underwent one or two more divisions before death with the noted mitotic defects, exhibiting aneuploidy. These results indicate that Plk1 is essential for progression through mitosis in multiple stages and is crucial in the genetic integrity in developing embryos. Morpholino knockdown of Aurora A displayed similar results with some distinct patterns compared with those of Plk1 morphants. A great advantage to zebrafish is the ability to analyze single cells in a living organism. Using live-cell imaging in a H2B-GFP transgenic fish we developed, we found that the loss of Plk1 resulted in a delay in mitosis for hours while normal zebrafish embryos divide in 12-17 minutes. The mitotic arrest accompanied unsegregated sister chromosome pairs, chromosome bridges, micro nuclei, and defects in centrosome numbers and maturation, all of which are hallmarks of CIN in cancer. As a result, loss of Plk1 resulted in the activation of spindle assemblyc checkpoint (SAC). Moreover, we were able to monitor the effects of Aurora A inhibitors at the single cell level with live-imaging. Taken together, we conclude that zebrafish is a valuable tool in studying the molecular mechanisms of mitosis and mitotic kinases, and zebrafish embryos will serve as an efficient model system for validating anti-cancer effects of mitotic kinase inhibitors. Talks 74 SESSION VII Cancer 38 A Mutation in Alk6b Causes Germ Cell Tumors in Zebrafish J. C. Neumann, G. L. Chandler, and J. F. Amatruda Departments of Pediatrics, Internal Medicine and Molecular Biology, Southwestern Medical Center, Dallas, USA Germ cell tumors (GCTs) affect infants, children and young adults and are increasing in incidence worldwide. GCTs arise from pluripotent germ cells and can exhibit differentiated and undifferentiated histologies, which vary in their malignant potential and response to treatment. The molecular origins of GCTs and the pathways that determine tumor cell differentiation are not known, impeding the development of new therapies. For these reasons, the treatment of GCTs has remained static since the introduction 30 years ago of cisplatin which, while effective, causes severe side effects including hearing loss, infertility and kidney damage. Currently there are no animal models of GCT, which poses a further obstacle to our understanding of the disease. We identified a zebrafish mutant line with a high incidence of GCT during a forward genetic screen to identify cancer susceptibility loci. Homozygous adult males develop tumors consisting of undifferentiated spermatogonia by 4 months of age while heterozygous males develop tumors around 7 to 9 months of age. Pedigree analysis demonstrated that the mutation is dominantly inherited. We used interval haplotype analysis of 14 affected individuals to localize the mutation to zebrafish chromosome 10. We carried out further high-resolution recombinational mapping to narrow the critical interval to a 0.6 cM interval containing three genes. We identified a premature termination codon in the type IB Bone Morphogenetic Protein Receptor, Alk6b (Activin Receptorlike Kinase 6b) in the mutant animals. Alk6b is a member of the TGF-beta/BMP superfamily of receptors. BMP signaling has diverse roles including regulation of cell proliferation, differentiation, embryonic development, germ cell specification and gonadogenesis. Misregulation of the BMP signaling pathway has been implicated in various human cancers. In agreement with a critical role for Alk6b in controlling germ cell differentiation, we find evidence of impaired BMP signal transduction in the zebrafish GCTs, as well as evidence of alterations in the expression level of BMP target genes. We are conducting experiments to further characterize the role of Alk6b in germ cell proliferation and differentiation and to determine the precise mechanisms of tumor development in the mutant zebrafish line. We have also examined activity of the BMP signaling pathway in a series of 40 clinically-annotated human GCTs of diverse histologic subtypes. In agreement with the predictions made from our zebrafish model, we find that undifferentiated GCTs such as dysgerminomas lack BMP signaling activity, whereas signaling is maintained in the differentiated subtype of Yolk Sac Tumors. These results confirm the relevance of the zebrafish model for understanding germ cell tumorigenesis, and will foster the development of improved, targeted therapy of human GCTs. 75 Talks 39 SESSION VII Cancer New Zebrafish Models of T Cell Cancer: Important Resources for Gene Discovery N. Meeker1,2, J.K. Frazer1,3, L. Rudner3, D.F. Bradley1,3, A.C.H. Smith1,3, W. Horsley3, R.W. Nipper5, S. L. Perkins4, and N. S. Trede1,3 Departments of 1Pediatrics, 2Internal Medicine, 3Oncological Sciences, and 4Pathology, Huntsman Cancer Institute, University of Utah, Salt Lake City, USA; 5Floragenex Inc., Eugene, USA The rapidly evolving field of cancer genetics is revolutionizing contemporary therapies for human malignancies. To identify new genes involved in the pathogenesis of T cell cancer, we performed a phenotypic mutagenesis screen and identified three new zebrafish models of T cell acute lymphoblastic leukemia (T-ALL). Conceptually, T-ALL genes can be grouped into three overlapping categories: 1) those predisposing to cancer; 2) those directly causing cancer; and 3) those modulating disease severity. Our new mutants are valuable resources for gene discovery in all three categories. T-ALL penetrance in the lines is incomplete, indicating that the mutated gene in each case confers a predisposition to develop disease. To identify the genetic lesions, we carried out genome-wide scans in each line using restriction site associated DNA (RAD) markers and high-throughput Solexa sequencing. Linked regions were rapidly identified using relatively small mapping populations. The search for candidate genes is ongoing. Regarding the second category, cancer-causing genes, we hypothesize that the moderate T-ALL incidence in each line represents a requirement for somatic "second hits" occurring in proto-oncogenes or tumor suppressors. To identify these genes, we are using a combination of complementary technologies including the following: micro-array transcriptome analysis to identify dysregulated genes, aCGH to identify amplified or deleted loci, and MeDIP to identify changes in DNA methylation patterns. These studies have uncovered several candidate oncogenic loci, and current progress will be presented. Finally, regarding cancer-modulating genes, serial transplantation of malignant T cells into irradiated recipients leads to a more aggressive disease with accelerated progression to death. In addition, affected zebrafish from each line show a marked reduction in tumor burden in response to irradiation treatment, but typically relapse with rapid progression to death. We hypothesize that the aggressive disease seen in both situations results from somatic mutations, either newly acquired, or already present in a subclone now evident through selective pressure. Comparison of the final and initial T-ALL clones should reveal genetic, epigenetic and genomic differences that are responsible for the more severe phenotypes. Using the technologies cited, these salient differences are currently being identified. Talks 76 SESSION VII Cancer 40 Identification of the SETDB1 histone methyltransferase as a new oncogene in melanoma L. Z. Zon1, J.J. Jane-Valbuena2, A.U. Uong1, LT. Turner1, F.F. Ferre1, W.L. Lin2, L.G. Garraway2, C.C. Ceol1, Y.H. Houvras1 1 Hematology/Oncology, HHMI/Children's Hospital Boston, USA; 2Medical Oncology, Dana Farber Cancer Institute, Boston, USA Cancer is initiated and maintained by successive genetic alterations that lead to enhanced tumor development, coupled with invasion and metastasis. In a variety of solid tumors, genomic copy number is a major mechanism of such modulation. Human melanoma is associated with copy number gain at specific genomic loci, and yet most of the genes in these intervals that participate in melanoma have not been molecularly defined. Here, we have developed a novel strategy to test large numbers of genes within these intervals for their effect on melanoma progression. This approach utilized a zebrafish strain that reliably forms melanomas due to expression of BRAFV600E, an oncogenic variant found in roughly half of all human melanomas, coupled with a loss-of-function allele of p53. When the BRAFV600E transgene and p53 mutation are put into a melanocyte-deficient mitfa mutant background, melanoma formation is completely suppressed. Injection of a rescuing mitfa minigene into BRAFV600E;p53;mitfa mutants leads to mosaic rescue of melanocytes and subsequent melanoma. Tumors arise at 3 to 4 months of age at a reproducible rate. We developed a plasmid vector called miniCoopR that harbors the mitfa minigene and a cassette with the mitfa promoter driving cDNAs of interest. This vector allows for the testing of candidate genes in regions of genome amplification in human melanoma. We examined all genes in the 1q21 region that are recurrently amplified and overexpressed in human melanoma. Our screen identified SETDB1, an H3K9 histone methyltransferase, as the sole gene in the 1q21.3 interval that cooperates with BRAFV600E;p53-/- to increase the rate of tumor formation. Human melanoma lines with amplified SETDB1 have tri-methylation of H3K9 on Western blot analysis, but lines with the lowest level of SETDB1 lacks H3K9 tri-methylation. This suggests that SETDB1 is the major H3K9 histone methyltransferase for tri-methylation in melanoma cells. Knockdown of SETDB1 in human melanoma cells decreases cell proliferation. This study demonstrates that screening candidate oncogenes using this whole animal vertebrate zebrafish system is robust, scalable and offers a unique approach to discovering oncogenes in human cancer. 77 Talks 41 SESSION VII Cancer Genetic baclines was tested in xenotransplantation experiments in zebrafish embryos. Results: We show that miR-10a expression promotes metastatic behaviour of pancreatic tumour cells and that repression of miR-10a is sufficient to inhibit invasion and metastasis formation. We further demonstrate that miR-10a is a retinoid acid target and that retinoic acid receptor (RAR) antagonists effectively repress miR-10a expression and completely block metastasis. This anti-metastatic activity can be prevented by specific knock down of HOX genes, HOXB1 and HOXB3. Interestingly, suppression of HOXB1 and HOXB3 in pancreatic cancer cells is sufficient to promote metastasis formation. Conclusions: These findings demonstrate that miR-10a is a key mediator of metastatic behaviour in pancreatic cancer which regulates metastasis via suppression of HOXB1 and HOXB3. Inhibition of miR-10a expression (with RAR antagonists) or function (with specific inhibitors) is a promising starting point for anti-metastatic therapies. 79 Talks 43 SESSION VIII Disease Models Chemical colitis models in zebrafish larvae S. Oehlers, M. Flores, K. Okuda, C. Hall, K. Crosier, P. Crosier Department of Molecular Medicine & Pathology, School of Medical Sciences, The University of Auckland, New Zealand Inflammatory bowel disease (IBD) is characterised by aberrant host-microbe interactions. Epithelial dysfunction, most commonly studied in vivo using chemically-induced colitis, is necessary and possibly sufficient to trigger the escalating inflammation characteristic of IBD. The relative contributions of genetic and environmental factors to the aetiology of colitis have, to date, mainly been studied separately. Zebrafish provide a system where these interacting factors can be investigated together in vivo. To establish doses for inducing colitis in zebrafish, larvae were exposed to standard chemicals used in murine experiments. An acute zebrafish colitis model was generated with key characteristics shared with murine models of gut inflammation. The development of inflammation was codependent on microbiota. There was a requirement for the Toll-like receptor adaptor molecule Myd88 for survival. Alteration of microbial diversity was also noted, a feature typical of colitis. Analysis of gene expression following chemical exposure demonstrated both the induction of an inflammatory state within the larval gut and changes to regional markers of intestinal epithelial cell differentiation. Increased pro-inflammatory cytokine transcription was prevented by the administration of broad-spectrum antibiotics or a non-steroidal anti-inflammatory drug. Studies of intestinal function during inflammation revealed impaired lipid metabolism and reduced intestinal vascularisation. These functional phenotypes were associated with increased intestinal cell proliferation suggesting an active damage repair response during inflammation. This study has delineated similarities between chemically-induced colitis in zebrafish larvae and human IBD. The responsiveness of the phenotypes to pharmacological modulation further demonstrates the applicability of the zebrafish to model complex disease processes. Talks 80 SESSION VIII Disease Models 44 Disruption of the Troponin Complex Leads to Loss of Sarcomeric Integrity in Skeletal Muscle M.I. Ferrante, J.E. Collins, D.A. Goulding and D.L. Stemple Wellcome Trust Sanger Institute, Cambridge, United Kingdom In myofibrils contractile filaments are organised in a highly regular fashion and are associated with several other proteins required for regulation of contraction and response to physiological stimuli. The basic unit of contraction, the sarcomere, is composed of thin filaments (Actin, Tropomyosin, Troponin, Nebulin, etc) intercalated with thick filaments (mainly Myosin), attached to the Z-disks and M-lines respectively. In striated muscle, regulation of contraction is mediated by the Troponin complex, which confers calcium dependence to the actin-myosin interactions. The three subunits of the Troponin complex are Troponin-C, which is required to bind calcium ions; Troponin-I, which is required to inhibit interaction of myosin heads with actin; and Troponin-T, which anchors the complex to the thin filament by binding to Tropomyosin. All of the subunits of the Troponin complex are implicated in human genetic diseases of the muscle, including forms of cardiomyopathy, nemaline myopathy and distal arthrogryposis. Despite the medical relevance there are few animal models available to study mechanisms underlying the different pathologies. In zebrafish, because of the ancestral teleost genome duplication, troponin genes are found in multiple copies. Genes in the same family have diversified, and in some cases show partially overlapping patterns of expression, displaying specificity to cardiac, skeletal slow or skeletal fast muscle. We have undertaken a systematic analysis of the function of sarcomeric proteins in sarcomere assembly using loss of function strategies in the zebrafish. Muscle in zebrafish larvae is easily accessible and terminal differentiation of muscle cells in the trunk is completed by 48 hours post fertilisation (hpf). To understand the role of the Troponin complex during sarcomere assembly, we have focussed on skeletal muscle isoforms of the T and I subunits. We have used antisense morpholino oligonucleotides to disrupt expression of the previously reported tnnt1, tnnt3a and tnnt3b, as well as other genes of the same family that have emerged with improved annotation of the zebrafish genome. We have also identified and characterised a mutation in the tnni2 gene, which is the first animal model of a fast-twitch muscle specific, Troponin complex subunit. We find that in the absence of functional components of the Troponin complex sarcomere assembly is impaired. Specifically, we find that disruption of the slow twitch subunit T1 prevents sarcomere formation, whereas disruption of the fast-twitch components T3 or I2 affects maintenance of sarcomere structure. Talks 81 45 SESSION VIII Disease Models Genetic analysis reveals a zebrafish model for fraser syndrome and identifies potential novel disease genes T. J. Carney1, C. Sonntag2, N. Feitosa-Martins3, M. Hammerschmidt3 1 Genes and Development, IMCB, Biopolis, Singapore; 2Muscle Development, ARMI, Monash, Australia; 3Institute for Developmental Biology, University of Cologne, Cologne, Germany Fraser Syndrome is a congenital condition characterized by syndactyly and chryptophthalmos. Approximately 50% of investigated causes are due to mutations in one of two large extracellular matrix protein encoding genes, FRAS1 and FREM2, whereas the causative genetic lesions in the other half of Fraser syndrome patients are unknown. Analysis of mouse Fras mutants suggest that embryonic blistering of the apical ectodermal ridge of the limbs underlies the syndactyly seen in Fraser patients. Here we describe the positional cloning of a number of zebrafish embryonic fin mutants, which can be grouped into two phenotypic classes. Whilst mutants of the first class display embryonic fin degeneration, the mutants of the second class display blistering of the epidermis of the fin fold. Electron microscopy demonstrates that fin blisters occur at the level of the sublamina densa of the basement membrane, as seen in mice mutant for components of the Fraser complex. Indeed, two of mutants are due to mutations in zebrafish orthologues of FRAS1 and FREM2. Critically, we identified that another fin blistering mutant is caused by mutations in the gene encoding the zebrafish orthologue of Hemicentin1. This is the first description of a role for this extracellular matrix protein in vertebrates and, along with genetic interaction data, suggests a potential involvement in the Fraser complex. Furthermore, we present biochemical and genetic interaction data demonstrating a role for the proprotein convertase FurinA in membrane shedding of Frem2 and Fras1 proteins, and identify Fibrillin2 as an essential interacting partner of Hemicentin1. Thus through identification of the genes mutated in a number of zebrafish fin mutants, we have demonstrated that zebrafish is a relevant model for human distal limb anomalies, and can serve as a useful tool for the identification of novel disease genes. Talks 82 SESSION VIII Disease Models 46 Ceylon: a zebrafish mutant with Shwachman-Diamond syndrome-like bone marrow failure N. S. Trede1,2, S. A. Hutchinson1, E. E. Locke1, D. Hu1, B. Demarest1 1 Department of Oncological Sciences and 2Department of Pediatrics, University of Utah, USA Shwachman-Diamond syndrome (SDS) is a bone marrow failure syndrome that frequently results in leukemia. SDS is also characterized by exocrine pancreas insufficiency and skeletal abnormalities. Eighty percent of patients with SDS have mutations in the SBDS gene. The SBDS protein product has been implicated in RNA metabolism but its function has not been fully elucidated. As twenty percent of SDS patients do not have mutations in the SBDS gene, mutations in other genes can result in a clinical SDS phenotype. However, to date no other genetic lesions have been identified that cause SDS. Here, we describe the zebrafish mutant ceylon (cey) that was originally identified in a screen for immunodeficiency. cey has an SDS-like phenotype characterized by skeletal abnormalities and exocrine pancreas insufficiency. Furthermore, primitive and early definitive hematopoietic stem cells (HSCs) are normal, while late HSCs are severely reduced, similar to the bone marrow failure in SDS patients. Thus, the cey lesion may represent a mutation that leads to an SDS-like syndrome in humans. Similar to cey, sbds morpholino-injected embryos exhibit defective exocrine pancreas development and dysregulated definitive hematopoiesis. However, skeletal abnormalities have not been observed in Sbds knockdown embryos. We have mapped the cey lesion to chromosome 3 and have identified candidate genes. In contrast, the zebrafish sbds gene maps to chromosome 15, and is therefore distinct from cey. We are in the process of determining if the cey gene product and Sbds interact genetically or if the cey lesion represents a new pathway that leads to SDS-like disease when mutated. Identification of the cey lesion and its potential interaction with sbds could be useful for prenatal diagnosis and reveal possible therapeutic strategies for SDS patients without a mutation in the SBDS gene. 83 Talks 47 SESSION VIII Disease Models Cellular senescence and DNA damage in a zebrafish model of Costello syndrome is linked to ubiquitin-mediated proteosomal degradation of oncogenic HRAS M. Mione, C. Santoriello, V. Anelli, G. Deflorian, and F. Pezzimenti IFOM, the Firc Institute of Molecular Oncology, Milan, Italy Some tissues are more affected than others by the expression of oncogenic Ras. This is confirmed by the preferential association of Ras mutations with certain types of cancers. We have developed several zebrafish models where oncogenic HRAS can be expressed at different levels and in different cell types, using promoters like hsp70, UAS in combination with different GAL4 driver lines. Moreover we have generated a model where HRAS is expressed in a ubiquitous and constitutive fashion. Expression of oncogenic HRAS in our models does not necessarily lead to cellular transformation, even if the oncogene is highly expressed. We therefore examined the reasons for this cell type specific "resistance" to oncogenic transformation in our model of constitutive HRAS expression (which mimics Costello syndrome, Santoriello et al., 2009). The model shows brain and heart cellular senescence, while liver and kidney are hyperproliferating. Analysis of oncogenic H-RAS post-translational modifications (PTMs) shows that in heart and brain, oncogenic HRAS is ubiquitinated and targeted for degradation. In parallel we checked HRAS ubiquitination in senescence and proliferating human fibroblasts, which have been transduced with oncogenic HRAS. Our results show that in senescent cells oncogenic HRAS is ubiquitinated, whereas in proliferating cells it is not significantly bound to ubiquitin. These data establish a link between a novel PTM of oncogenic HRAS (which presumably reduces its ability to activate downstream targets and transform) and oncogene-induced cellular senescence (a mechanism that allows cells to escape RAS induced transformation). We are now investigating the machinery involved in ras ubiquitination. Microarray data obtained in our zebrafish model of Costello syndrome show an increase of the expression of a number of ubiquitin conjugating enzymes. These are candidates for processing mutant HRAS into a less active oncogene and target it for degradation. We are investigating whether blocking proteosomal degradation restores HRAS ability to activate ERK and AKT, which is lost in zebrafish larvae constitutively expressing oncogenic ras and in our zebrafish model of Costello syndrome. Santoriello et al., Dis Model Mech. 2009 2(1-2):56-67 Talks 84 SESSION VIII Disease Models 48 A modified acetylcholine receptor d-subunit enables a null mutant to survive beyond sexual maturation J. M. Urban2, K. E. Epley1, T. Ikenaga2, F. Ono2 1 The Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, USA; 2Section on Model Synaptic Systems, Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, USA The contraction of skeletal muscle is dependent upon synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the d-subunit in zebrafish (sofa potato; sop-/-) leads to the death of embryos around 5 days post-fertilization (dpf). In sop-/- mutants, we expressed modified d-subunits, with one (d1YFP) or two yellow fluorescent protein (d2YFP) molecules fused at the intracellular loop, under the control of an a-actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop-/- fish stably expressing the d2YFP. These d2YFP/sop-/- embryos can mount escape behavior close to that of their wild type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared to wild type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ. 85 Talks 49 SESSION IX Cardiovascular system Role of notch signaling in early zebrafish cardiogenesis K. A. Nembhard and M. L. Kirby Department of Cell Biology and Department of Pediatrics, Duke University, Durham, USA Congenital heart defects account for at least 50% of deaths amongst sufferers of Alagille syndrome, a disease usually associated with a haplodeficiency of the Jagged1 or the Notch2 gene, and these genes encode members of the Notch signaling pathway. This pathway has been shown to establish subpopulations within the cardiogenic mesoderm in Drosophila melongaster and Xenopus laevis. During early mouse and chick embryogenesis, the cardiogenic mesoderm is segregated into two distinct cell populations. The first heart field is the first population to differentiate into myocardium and form the initial, non-proliferating myocardial heart tube. The second heart field is maintained in an undifferentiated state as a progenitor cell population that gradually differentiates into myocardium and smooth muscle and adds to the heart tube. Understanding how and when these two populations are formed, and how their differentiationMedicine, University of Massachusetts Medical School, Worcester, USA; 3Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, USA The aorta traverses the length of the vertebrate body to deliver oxygenated blood to the systemic arterial circulation. In zebrafish embryos, this artery is "Y"-shaped, consisting of two lateral dorsal aortae (LDA) in anterior regions of the embryo and a single dorsal aorta (DA) in the trunk. Much progress has been made in elucidating the mechanisms leading to DA formation, which has been shown to develop via vasculogenesis, when angioblasts from the lateral plate mesoderm migrate towards the midline. In contrast to this, the mechanisms and signaling molecules contributing to the formation of the branched LDA have remained elusive. In this study we show that the LDA form in a bidirectional manner from endothelial cells originating in the anterior and posterior lateral plate mesoderm. Furthermore, we show that these endothelial cells are genetically distinct. In particular, we find that expression of the chemokine (C-X-C motif) receptor 4a (cxcr4a) specifically labels endothelial cells of the anterior LDA. Accordingly, targeted inactivation of cxcr4a leads to a specific defect in anterior LDA formation. We further show that cxcl12b, a ligand for cxcr4a, is expressed in endoderm adjacent to the anterior branches of the LDA and that loss of cxcl12b phenocopies cxcr4a deficiency. Thus, our results suggest that an endoderm-derived chemokine specifically guides the development of the branched region of the aorta, while the single DA in the trunk remains unaffected. Taken together, our studies reveal an unexpected molecular diversity within arterial endothelial cells and show that this differentiation is necessary to facilitate regional patterning of the developing aorta by local extrinsic cues. Talks 88 SESSION IX Cardiovascular system MiR-1 regulates angiogenesis by modulating VEGF signaling A. J. Giraldez, Y. Mishima, and C. Stahlhut Yale University, New Haven, USA 52 Cell signaling between different tissues ensures coordinated development during organogenesis. A fundamental question in the field is to understand how these signals are modulated in vivo. MicroRNAs have recently emerged as important modulators gene expression that repress mRNA translation and accelerate target mRNA decay. However, it remains a challenge to identify their targets and dissect the functional relevance of individual miRNA-target interactions in vivo. Our previous study identified a large set of target mRNAs regulated by miRNAs during muscle development. Analysis of these targets revealed that a fraction of them were also expressed in the vasculature. Conversely, genes expressed in the vasculature were enriched for the muscle miR1 target site in their 3'UTRs compared to a control set of genes. These observations suggested the hypothesis that the muscle miR-1 might regulate the cross talk between the muscle and the vasculature during development. To test this hypothesis, we analyzed whether loss of miR-1 function affected vasculature development. Inhibition of miR-1 with two non-overlapping MOs caused an increased in the area of intersomitic vessels and the number of cells from 3 cells in wild type to 6-9 cells in miR-1 morphants. These results are reminiscent of an increase in VEGF signaling or a loss of Notch signaling during intersomitic vessel formation. To distinguish between these possibilities we performed clonal analysis and epistasis experiments. First, wild type cells labeled with the vascular marker fli1-GFP transplanted into miR-1 MO embryos show the vasculature overgrowth phenotype, suggesting that the effect of miR-1 is non-autonomous. Second, epistasis experiments using a kdrl mutant embryos and miR-1 loss of function revealed that the intersomitic overgrowth phenotype require a functional receptor. Thus the effect of miR-1 LOF is likely to act upstream of the receptor. Third, we searched the components of the VEGF pathway for miR-1 target sites. Sequence analysis and reporter studies revealed that VEGFa includes three functionally conserved miR-1 target sites in their 3'UTR able to regulate expression of a reporter mRNA. Taken together these results suggested that miR-1 regulates VEGF expression levels in the muscle thus controlling angiogenesis during intersomitic vessel formation. To test this hypothesis we blocked miR-1 function in wild type and VEGFa knock down embryos. Reducing the levels of VEGFa suppressed the miR-1 knock down phenotype. We are currently dissecting the regulation of individual target sites in VEGF using target protector MOs (TP). These TPs bind the target sites in the 3'UTR and block miRNA-mediated repression of the target mRNA. These results will be presented at the meeting. Taken together these results support a model where miR-1 regulates the potent ligand VEGFa to regulate the crosstalk between the muscle and the vasculature to modulate angiogenesis during intersomitic vessel formation. 89 Talks 53 SESSION IX Cardiovascular system The zebrafish full-of-fluid mutant identifies a secreted protein essential for lymphangiogenesis in zebrafish and humans B.M. Hogan1, M. Alders2, F.L. Bos1, 3, J. Bussmann1, M. Witte1, N.C. Chi4, H.J. Duckers3, R.C. Hennekam2 and S. Schulte-Merker1 1 Hubrecht Institute-KNAW & University Medical Centre, Utrecht, The Netherlands; 2Department of Clinical Genetics, Academic Medical Centre, Amsterdam, The Netherlands; 3Experimental Cardiology, Thoraxcenter, Erasmus University Medical Center, Rotterdam, The Netherlands; 4 Department of Medicine, University of California San Diego, Division of Cardiology, California, USA The lymphatic vascular system plays pivotal roles in fluid homeostasis, fat resorption, the immune response and cancer metastasis. Only very few factors regulating the initial aspects of lymphangiogenesis are known, such as the transcription factor Prox-1 and VEGFC, a secreted ligand that acts through Vegfr3 to direct lymphangiogenesis during development and disease. We have recently demonstrated that zebrafish possess a lymphatic system that can be both imaged in vivo as well as manipulated genetically. We have performed a forward genetic screen to identify genes required for embryonic lymphangiogenesis and identified three mutants to date. Positional cloning found one mutant, full-of-fluid, to encode a novel predicted secreted protein, Ccbe1. Ccbe1 is expressed in the somitic mesoderm along the migration pathway of lymphangioblasts, the earliest precursors of the lymphatic system, and acts non-autonomously during lymphangiogenesis. Using a series of new transgenic lines to visualize the development of the lymphatic vasculature, we found that the loss of either ccbe1 or Vegfc-Ft4 signalling leads to an identical failure of lymphangiogenesis and hemangiogenesis at the level of endothelial cell sprouting from the posterior cardinal vein. Significantly, we have recently identified a human patient population that develops lymphedema with genetic linkage to the human CCBE1 locus. We demonstrate here that these patients have molecular lesions in the human CCBE1 gene and that the equivalent mutations inhibit the function of zebrafish ccbe1 in lymphangiogenesis. These studies identify a novel conserved regulator of lymphangiogenesis in the predicted secreted protein Ccbe1 and highlight its critical role in vertebrate development and human disease. Talks 90 SESSION IX Cardiovascular system 54 Characterization of vascular mural cells during zebrafish development M. M. Santoro1,2, G. Pesce1, V. Mugoni1, D. Y. Stainier2 1 Molecular Biotechnology Center, University of Torino, Italy; 2Department of Biochemistry and Biophysics, UCSF, San Francisco, USA Development and maturation of the nascent cardiovascular system requires the recruitment of mural cells (MCs) around the vascular tree in a process called vascular myogenesis. Understanding the origins of vascular MCs has been hampered by difficulties in observing these cells in vivo and in performing defined genetic and experimental manipulations in available model organisms. Here, we investigate the origin of vascular MCs using molecular and genetic tools in zebrafish. We show that vascular MCs are present around the lateral dorsal aortae (LDA), and anterior mesenteric arteries (AMA) of developing animals and that they also contribute to the ventral aorta (VA) and outflow tract of the developing heart. Our genetic data further indicate that the vascular MCs of the trunk vessels do not arise from blood or endothelial progenitors but from other derivatives of the lateral plate mesoderm. Thus, zebrafish vascular MCs share many of the morphological, molecular and functional characteristics of the vascular smooth muscle cells and pericytes found in higher vertebrates, establishing zebrafish as a useful cellular and genetic model to study vascular myogenesis as well as tumor angiogenesis and other MCs-associated diseases. 91 Talks 55 SESSION X Hematopoiesis and immune system Both primitive and definitive hematopoiesis arise from hemogenic endothelial cells in the zebrafish embryo J.Y. Bertrand1, B. Santoso1, N. Chi2, D. Traver1 1 Biology, UCSD, La Jolla, USA; 2School of Medicine, UCSD, La Jolla, USA Development of the blood system is complex, with multiple waves of hematopoietic precursors arising in different embryonic locations. Monopotent, or primitive, precursors first give rise to embryonic macrophages or erythrocytes. Multipotent, or definitive, precursors are subsequently generated to produce the adult-type lineages. In both the zebrafish and mouse, the first definitive precursors are committed erythromyeloid progenitors (EMPs) that lack lymphoid potential. We have previously shown that zebrafish EMPs arise in the posterior blood island independently from hematopoietic stem cells (HSCs) that arise along the ventral side of the aorta. By using the CRE/LOX technology, recent reports have nicely showed that vascular endothelial cells on the ventral side of the aorta were giving rise to HSCs in the mouse embryo. Here we show similar results in the zebrafish, that HSCs are also born from hemogenic endothelial cells (ECs). We used the Flk1:CRE transgenic line. Unlike in mammals, the zebrafish flk1 promoter used here is not pan-mesodermic, so we could specifically trace the progeny of ECs in the zebrafish by crossing this CRE line to a switch-reporter line. This latter consists of the b-actin promoter driving the expression of DsRED after a STOP cassette is excised by the CRE recombinase. Double transgenic adults were sacrificed at 3 months, and in all animals (n=30), 100% of adult leukocytes were switched (DsRED+). Moreover, we could show that "switched" cells harbor long-term reconstitution when transplanted into irradiated recipients. Thus, zebrafish HSCs originate from ECs in the embryo. To corroborate these findings, we could isolate cells during their transition from EC to hematopoietic cells using the flk1:RFP and cmyb:GFP reporter lines, and show that, during the transition, the endothelial genetic program is shut down, while hematopoietic genes are turned on. As stated above, three other hematopoietic lineages arise during embryogenesis: primitive macrophages/erythrocytes and definitive EMPs. Whether or not these other lineages arise from hemogenic ECs is still unknown. To answer that question, we sorted "switched" cells at 27-30 hours post fertilization, when primitive lineages and EMPs, but not HSCs, have been generated, and characterized them. To further investigate this issue, switched adult fish have been crossed to transgenic lines that mark primitive macrophages and EMPs in order to determine if these subsets also come from flk1+ vascular cells. Altogether, preliminary data indicate that all waves of hematopoiesis might originate from hemogenic ECs. These results should give new insights on the generation of blood cells in the vertebrate embryo. Talks 92 SESSION X Hematopoiesis and immune system 56 SDF-1 Expressing Cells Establish the Hematopoietic Stem Cell (HSC) Niche and Play a Role in HSC Homing Following Adoptive Cell Transfer in a Zebrafish Model of Bone Marrow Transplant T. C. Lund1, T. J. Glass1, J. Tolar1, L. I. Zon2, B. R. Blazar1 1 University of Minnesota, Division of Pediatric Hematology/Oncology/Blood and Marrow Transplant, Minneapolis, USA; 2Children's Hospital Howard Hughes Medical Institute, Boston, USA The zebrafish, danio rerio, is increasingly becoming an important model in unraveling the development of hematopoietic cells. Recent work has also shown that myeloablative hematopoietic stem cell (HSC) transplant can be accomplished in the zebrafish. How the transplanted HSC find their way to the HSC niche and engraft is unknown in the zebrafish and currently the subject of much experimentation in the mammalian systems. In mammals the HSC niche is thought to contain various stromal cells. These cells have been known secrete stromal derived factor-1 (SDF-1), an important mediator that functions to recruit CXCR4 expressing HSCs to a supportive environment essential for fostering long-term hematopoiesis. To identify potential HSC niche-establishing cells in the zebrafish and to understand their role during marrow transplant, we created an SDF1a transgenic zebrafish by fusing the SDF1a proximal promoter to dsRed2. We have found dsRed positive cells in areas where SDF1 has previously been shown to be important in organogenesis including the heart, notochord, fins, myofibers and retinal axons. As the fish mature, we also found dsRed positive expression in the kidney tubular epithelial cells, which was maintained throughout adulthood. These cells showed in increase in numbers when fish were gamma-irradiated which correlated with an increase in SDF1a RNA expression as expected following myeloablative radiation. Finally, marrow from donor fish which was pretreated with AMD3100 (to block CXCR4) failed to home to the kidney region immediately after transplant giving an indication that the SDF1-CXCR4 axis contributes to cellular homing in the zebrafish during the transplant process as it does in mammalian systems. The creation of this tg(sdf1a:dsRed2) zebrafish line will be important in elucidating the mechanisms by which HSC interact with their niche under steady state and stress hematopoiesis conditions. In addition, this line should prove useful in the analysis of the homing process involving organogenesis and regeneration/repair of other organ systems that express SDF-1 during embryogenesis and postnatally. Talks 93 57 SESSION X Hematopoiesis and immune system Live imaging reveals that definitive haematopoietic stem cells emerge directly from haemogenic endothelial cells in zebrafish embryos M. V. Flores, E. Yi Ni Lam, K. Crosier, C. Hall, and P. Crosier Department of Molecular Medicine & Pathology, School of Medical Sciences, The University of Auckland, New Zealand The identity of the cells that give rise to the first haematopoietic stem cells (HSCs) in the developing embryo has long been controversial. HSCs have been proposed to emerge from mesodermal cells, mesenchymal progenitors, a bipotential endothelial-haematopoietic precursor (the haemangioblast) or the haemogenic endothelium. In mammalian embryos, some resolution to this debate has recently come from in vitro live imaging technology that shows evidence for the central role of haemogenic endothelial cells in the generation of primitive and definitive blood. Time-lapse microscopy revealed that bipotential haemangioblasts generate primitive blood cells through formation of a transient haemogenic endothelial stage in vitro. Similarly, the cell fates of single mouse mesodermal cells were tracked by long-term imaging to show that they can generate haemogenic endothelial cells that directly give rise to blood cells in culture. However, as these studies were performed in vitro, they still do not definitively identify the haemogenic endothelium as the origin of HSCs within the organism. Here, we provide the first demonstration of the direct emergence of definitive HSCs from the haemogenic endothelium in a living embryo. The haematopoietic program is very conserved in vertebrates. In zebrafish, as in mammals, definitive HSCs are believed to have their origins in the haemogenic endothelium. Runx1, a transcription factor essential for the development of HSCs in all vertebrates is also required for the transition from haemogenic endothelium to HSCs in mouse and zebrafish. We have generated a zebrafish Runx1 transgenic line, Tg(runx1P2:EGFP) marking definitive HSCs in the aorta-gonad-mesonephros (AGM) that later populate the pronephros and thymus. Kdr (Flk-1) is expressed in haemangioblasts and in early endothelial cells. Double Tg(runx1P2:EGFP/kdrl:nls-mCherry) transgenic lines express mCherry in the nuclei of endothelial cells, while HSCs emerging from the AGM express EGFP. During time-lapse imaging of these compound transgenic embryos over 11 hours (24-33 hours post fertilisation), all of the cells that initiate expression of EGFP, were red fluorescent mCherry-expressing endothelial cells. This demonstrates that haematopoietic cells in the AGM arise from further differentiation of endothelial cells, providing strong in vivo evidence for a `haemogenic endothelium'. Talks 94 SESSION X Hematopoiesis and immune system 58 Development of an immune-matched transplantation model to detect hematopoietic stem cell activity in zebrafish J.L.O. de Jong1, C. E. Burns1, A. Chen1, E. Pugach1, E. A. Mayhall1, A. C.H. Smith1, H. A. Feldman2, Y. Zhou1, and L. I. Zon1 1 Stem Cell Program and Division of Hematology/Oncology Children's Hospital and Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, USA; 2Clinical Research Program, Children's Hospital Boston, Boston, USA Transplantation of hematopoietic cells, tumors or any allogeneic tissue has been hindered in the zebrafish due to poor understanding of the functional major histocompatibility complex (MHC) genes and inability to immune-match donors and recipients. Despite this, the zebrafish has been used prominently for the study of hematopoiesis, due to the advantages of large scale forward and reverse screening methodologies leading to the discovery of novel genetic mechanisms. However, methodologies to assess hematopoietic stem cell (HSC) function are largely undeveloped. Here, we created a quantitative long-term hematopoietic reconstitution assay in adult zebrafish. We identified a sublethal radiation dose of 25Gy which was optimal for hematopoietic reconstitution while minimizing the mortality presumably due to radiation damage of other organs. At 3 months post-transplant, primary and secondary recipients showed multi-lineage engraftment, as measured by flow cytometric analysis of GFP-expressing donor cells. Statistical analyses of limiting dilution data suggest that at least 1 out of 65,000 nucleated zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We then defined zebrafish haplotypes at the proposed core MHC locus on chromosome 19, testing the functional significance of these haplotypes by performing matched and mismatched transplants. Utilizing a single family for sibling marrow transplants, we identified the four parental MHC haplotypes by sequencing PCR products amplified for specific genes. MHC-typed recipient fish were transplanted with whole kidney marrow cells from sibling donors, demonstrating that matching the donor and recipient MHC haplotypes at the chromosome 19 locus increases engraftment and percentage of donor chimerism in recipients compared to MHC-mismatched donors and recipients. At 3 months post-transplant, MHC-matched recipients had multilineage engraftment in 13 of 15 fish with mean donor chimerism of 47.86% (range 5.56- 93.44%) for myeloid cells and 10.51% (range 0.92-77.57%) for lymphoid cells. Engrafted animals receiving MHC-mismatched donor marrow (n= 4 of 6) had only 6.45% mean myeloid donor chimerism (range 4.58 to 8.58%) and 1.28% mean lymphoid donor chimerism (range 0.83 to 1.41%). These data represent the first assay allowing long term HSCs to be distinguished from other hematopoietic progenitors, and provide the first functional test of MHC y conserved and features all major blood cell lineages found in mammals, including erythroid, thrombocytic, myeloid and lymphoid cells. We have previously identified putative DCs in the lymphoid organs of adult zebrafish by cytochemical analyses and electron microscopy. To enable in vivo imaging, prospective isolation, and functional analyses of APCs, we have generated new transgenic zebrafish lines. Because APCs rely upon class II Major Histocompatibility complex (MHCII) genes for antigen presentation, we identified, characterized, and utilized the MHCIIDAB promoter to generate fluorescent transgenic animals. Founders were identified in which expression of the transgene is observed in all APC subtypes, including B cells, macrophages and DCs. Consistent with the role of MHCII during T cell development, expression of the transgene was also observed in thymic epithelial cells by 5 days post fertilization. Here we present the characterization of these APC-reporter lines following co-expression studies, FACS analyses, gene profiling and functional assays. The APC-reporter lines were crossed with available T cellreporter lines, allowing an unprecedented view of APC/T cell interaction in primary (thymus) and secondary (spleen) lymphoid organs. By allowing the study of APCs in their natural environment, MHCIIDAB transgenic lines should provide new insights into zebrafish immunity and the complex mechanisms that underlie APC ontogeny and functional behavior. Talks 97 61 SESSION XI Neurobiology I: patterning and behaviour Negative regulation of neurogenesis by an FGF from neurons R. Gonzalez-Quevedo1, Y. Lee2, K.D. Poss2 and D.G. Wilkinson1 1 Division of Developmental Neurobiology, National Institute for Medical Research, Mill Hill, London, UK; 2Department of Cell Biology, Duke University Medical Center, Durham, USA The precise regulation of neurogenesis is critical to generate the correct number of neurons at the appropriate location in the nervous system. This is achieved in specific regions of the vertebrate nervous system by the formation of distinct neurogenic and non-neurogenic zones. Despite the increasing progress in dissecting the mechanisms that control the temporal and spatial regulation of neurogenesis, little is known about the extrinsic signals involved in the formation of non-neurogenic zones. We have investigated how neuronal differentiation is patterned in the zebrafish hindbrain, in which neurogenesis becomes restricted to zones adjacent to boundaries. Our studies have identified a population of neural progenitors in neurogenic-free areas in segment centres. Using transgenic approaches, we have shown that FGFR signaling is required to restrict neurogenesis in the hindbrain by regulating this progenitor population. Moreover, we have identified an FGF secreted by specific neurons that maintains the non-neurogenic zones. Our findings reveal a novel mechanism in which signaling from neurons underlies the formation of a non-neurogenic zone of neural progenitor cells. Talks 98 SESSION XI Neurobiology I: patterning and behaviour 62 Segregated processing of visual inputin in the optic tectum by interlaminar inhibition revealed by calcium imaging F. Del Bene1, C. Wyart2, A. Muto1, EK. Scott1, EY. Isacoff2, H. Baier1 1 Physiology, UCSF, San Francisco, USA; 2MCB, UC Berkeley, USA The optic tectum is the major processing center for visual information in zebrafish. Axons of retinal ganglion cells (RGCs) form synaptic connections with the dendrites of tectal neurons. The retinotectal projection is organized in a topographic fashion, re-establishing a map of visual space in the brain. We have previously discovered that subtypes of RGCs terminate in four distinct layers of the tectum, one of which is further divided into at least three sublayers (Xiao et al., 2005; Xiao & Baier, 2007). Thus, the retinotectal neuropil contains six or more parallel visuotopic maps. Moreover, studies in both the tectum of fish and superior colliculus of higher vertebrates have demonstrated a size selectivity, wherein neurons respond optimof the zSARA1 endosomes with a proliferative state. We are now characterizing the zSARA1 endosome and analyzing it's cargo to clarify which signaling pathway, TGFbeta or Notch, is using the SARA endosomes to ensure the correct signaling levels across mitosis. We are also interested in knowing if zSARA1 is necessary to maintain the neuronal diversity in the Zebrafish neural tube. Talks 100 SESSION XI Neurobiology I: patterning and behaviour 64 Functional analysis of the habenula in control of fear M. Agetsuma1, H. Aizawa1, T. Aoki1, M. Takahoko1, R. Nakayama1, T. Shiraki1, M. Goto1, K. Kawakami2, S. Higashijima3 1 Lab for Devel. Gene Regulation, RIKEN Brain Science Institute, Japan;2 National Inst. of Genetics, Mishima, Japan, 3 Okazaki Institute for Integrative Bioscience, Okazaki, Japan The habenulae are part of an evolutionarily highly conserved conduction pathway within the limbic system that connects telencephalic nuclei to the monoaminergic neurons in the midbrain and hindbrain either directly or indirectly by way of the interpeduncular nucleus (IPN). In zebrafish, we showed a prominent asymmetric habenulo-interpeduncular projection caused by a prominent left-right (LR) difference in the size ratio of the medial and lateral subnuclei of the dorsal habenulae, each of which specifically projects either to the ventral or dorsal IPN targets. Furthermore, we have recently discovered that the neurons in the dorsal IPN specifically send the descending axons along the central gray in the hindbrain, and the neurons in the ventral IPN project to the raphe. Considering that the central gray is involved in instinctive defense behaviors such as freezing and that the raphe and its serotonin neurons are involved in more adaptive behaviors, we are now suspecting that the dorsal and ventral IPN may be involved in the alternative behavioral choice in the face of danger, such as whether to react in a panic or to cope with it in more sedate manners. To further investigate this hypothesis, we have established the transgenic zebrafish line expressing Gal4-VP16 specifically in the dorsal habenular subnuclei. By crossing such lines with other transgenic lines carrying the tetanus toxin gene or the nitroreductase gene under control of the target site of Gal4-VP16, we have succeeded in establishing the lines in which the neural signal transmission by way of the lateral subnucleus of the dorsal habenula is selectively impaired either constitutively or conditionally. In the fear conditioning, the manipulated fish showed extremely enhanced levels of the freezing response to the presentation of the conditioned light stimulus, suggesting the tract connecting the left-dominant lateral subnuclei of the dorsal habenula with the dorsal IPN may normally function to suppress excessive fear response. This result is especially intriguing if we take the previous report into consideration on the preferential right eye use by zebrafish when they are approaching novel objects. 101 Talks 65 SESSION XI Neurobiology I: patterning and behaviour Behavioral consequences of zebrafish epithalamic asymmetry M. E. Halpern, L. Facchin Embryology, Carnegie, Baltimore, USA We have been studying the relationship between asymmetry in the epithalamus and behaviors in larval and adult zebrafish. In >98% of wild-type zebrafish, the parapineal develops to the left of the pineal anlage and the adjacent left habenular nucleus is larger, has more dense neuropil and shows different gene expression than the right habenula. By disrupting the Nodal-signaling pathway, larvae can be produced that show a left-right (L-R) reversal of these asymmetries. Lasermediated ablation of the parapineal causes the habenular nuclei to develop more symmetrically, both with properties resembling the right habenula. Because larvae with reversed asymmetry or symmetry in this brain region are viable, we can test their behaviors as well as those of the resulting adult fish. In prior studies, we found that larvae with reversed asymmetry were indistinguishable from their siblings in a variety of es that produce portions of the paired sensory structures of the head. Most current models posit that preplacodal identity is specified by readout of a discrete level of Bmp signaling along a DV gradient. However, our studies in zebrafish support a strikingly different model: Rather than acting as a morphogen, Bmp simply establishes preplacodal competence throughout its signaling range within the nonneural ectoderm. Moreover, we find that four transcription factors co-induced by Bmp prior to gastrulation (Ap2, Ap2 Foxi1 and Gata3) are necessary and sufficient to promote preplacodal competence. Subsequently, Bmp-antagonists and Fgf secreted from dorsal tissue specify preplacodal identity within competent cells abutting the neural plate. Misexpression of Fgf8 and Chordin can activate preplacodal development anywhere within the zone of competence, leading to production of ectopic placodal derivatives on the ventral side of the embryo. Both Bmp-antagonism and Fgf are necessary, as neither signal alone is sufficient to activate preplacodal development. Knockdown of all four competence factors specifically blocks preplacodal specification without disrupting the Bmp gradient or formation of a neural-nonneural interface. Conversely, misexpression of competence factors can convert neurectoderm into preplacodal ectoderm without altering the Bmp gradient. These data support a relatively simple two-step model that traces the origins of preplacodal development to late blastula stage and resolves discrepancies in the literature regarding the role of Bmp signaling. Talks 104 SESSION XII Neurobiology II: sensory organs and regeneration 68 Repression of hedgehog signalling is required for the acquisition of dorsolateral cell fates in the zebrafish otic vesicle K. L. Hammond, F. J. M. van Eeden, and T. T. Whitfield MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, UK In zebrafish, Hedgehog signalling from ventral midline structures is both necessary and sufficient to specify posterior otic identity. A severe or complete loss of Hedgehog signalling leads to mirror symmetric double anterior ears, while severe overactivation of the Hedgehog signalling pathway gives rise to mirror symmetric double posterior ears. In contrast, in the mouse and chick otic vesicle, Hedgehog is required for dorsoventral patterning; anteroposterior effects, if present, are much less obvious. We now show, however, that while in zebrafish a loss of Hedgehog function does not affect dorsoventral and mediolateral otic patterning, an increase in Hedgehog signalling activity causes an expansion of ventromedial otic territories at the expense of dorsolateral domains. In a panel of lines carrying mutations in inhibitors of Hedgehog signalling, Hedgehog pathway activity is variably increased throughout the embryo, and dorsolateral otic structures are lost or reduced. Even a relatively modest increase in Hedgehog signalling has consequences for the ear. In the most severely affected line, the ptc1-/-; ptc2-/- double mutants, the inner ear is severely ventralised and medialised as well as displaying the previously reported double posterior character: markers of ventromedial otic domains (eya1, pax2a) are expanded at the expense of dorsolateral markers (dlx3b, tbx1). Overall these new data suggest that Hedgehog signalling must be kept tightly repressed for the correct acquisition of dorsolateral cell fates in the zebrafish otic vesicle. The role of Hedgehog in zebrafish and amniote inner ear patterning may not be as different as it at first appeared. 105 Talks 69 SESSION XII Neurobiology II: sensory organs and regeneration Regulating adult neuronal stem cell proliferation during regeneration of the light-damaged zebrafish retina D. R. Hyde, T. Bailey, S. C. Kassen, R. Thummel, F. Raycroft, C. M. Nelson Department of Biological Sciences, University of Notre Dame, USA We wish to define the mechanisms underlying regeneration of retinal neurons from an adult neuronal stem cell population. We use the lighe have identified the zebrafish Irxl1 gene and obtained the full-length cDNA of two splicing variants. Zebrafish Irxl1 gene is located on chromosome 12. It contains 7 exons and encodes a homeodomain 100 % identical to vertebrate orthologs. Transcription of z-Irxl1 was detected from 18 hpf to 5 dpf. Both isoforms were broadly expressed in adult tissues. Whole-mount in situ hybridization analysis revealed Irxl1 expression mainly in the brain and the first two pharyngeal arches. Antisense morpholino knockdown of Irxl1 resulted in deformed head and jaw in the larvae, which only survived for 5 to 7 days. The phenotype can be partially rescued by coinjection of Irxl1 cRNA. The most severe defects were observed in the craniofacial muscles and arch cartilages. This phenotype is reminiscent of a mouse Twirler mutation, to which the Irxl1 gene is linked. The promoter region of the Irxl1 gene contains several consensus Mef2 binding sites, and co-injection of Mef2ca into zebrafish embryos up-regulates the promoter activity of Irxl1. In addition, Irxl1 expression was decreased in Mef2ca mutants and morpholino-knockdown morphants. Meanwhile, myoD expression was increased in the somites of Irxl1-knockdown embryos. Co-injecting myoD promoter-driven luciferase construct with Irxl1 into zebrafish embryos revealed a dramatic inhibition of myoD promoter activity by Irxl1. These results suggest that Irxl1 is an important regulator of brain, muscle and arch morphogenesis, and its function in arch development may be mediated by Mef2ca. Moreover, Irxl1 protein may regulate muscle development through myoD-dependent pathways. 149 Posters 79 Early development and gastrulation Functional studies of muscleblind proteins in early zebrafish development K.M. Hsiao1, L.C. Tu2, C.W. Lin3 and H. Pan3 1 Department of Life Science, National Chung Cheng University, Chia-Yi, 2Institute of Medicine, 3 Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan Muscleblind (MBNL) is a family of proteins that participate in the regulation of tissue-specific alternative splicing. They bind to RNA through a conserved CCCH zinc finger domain. Three paralogs (Mbnl1, 2, and 3) have been identified in mammalian, each promotes inclusion or exclusion of specific exons on different genes. Misregulation of MBNL activity in human leads to RNA-mediated pathogenesis. To investigate the functions of Mbnl proteins in early vertebrate development, we cloned the muscleblind genes in zebrafish (zmbnl1, 2 and 3). Alternative splicing of the three zmbnl primary transcripts gives rise to at least 13 protein isoforms. These genes are broadly expressed in most adult tissues. During embryogenesis, zmbnl1 and zmbnl2 are both maternally and zygotically expressed. In contrast, zmbnl3 transcripts are not detected until late pharyngula stage. Whole-mount in situ hybridization reveals that zmbnl1 is expressed in lens, liver, otic vesicle, and muscle, while zmbnl2 is expressed in lens, olfactory epithelium, branchial arches, midbrain hindbrain boundary and swim bladder. Expression of zmbnl3 is more ubiquitous rather than specific. Knockdown of zmbnl1 and zmbnl2 results in malformation of the brain, eyes, otoliths, pharyngeal arches, heart and swim bladder. In addition, these morphants are defective in hatching and swimming behavior. In consistent with the phenotypes, the splicing patterns of several pre-mRNAs that are misregulated in cells with CUG RNA expansion, are altered in the morphant embryos. Knockdown of zmbnl3 does not result in overt phenotype. On the contrary, microinjection of zmbnl3 cRNA into the embryos resulted in defective embryos with crooked body axes and short somites. When introduced into C2C12 cells, zmbnl3 is able to inhibit cell differentiation, as evidenced by lack of cell fusion, change of splicing pattern and reduction of MHC expression. Dual-luciferase assay further reveals that zmbnl3 down-regulates myoD promoter activity in fish embryos. These data indicate that zmbnl1 and zmbnl2 are crucial for early fish development and that zmbnl3 may interfere with muscle differentiation through the MyoD-dependent pathway. Posters 150 Early development and gastrulation 80 Def6, a novel guanine nucleotide exchange factor, activates Wnt5 in the non-canonical Wnt signalling pathway K. Goudevenou1, P. Martin1, Y.J. Yeh1, P. Jones2, F. Sablitzky1 1 Institute of Genetics, School of Biology, 2School of Biomedical Sciences, The University of Nottingham, United Kingdom Gastrulation is driven by coordinated cell movements that form the embryo proper into the three germ layers: endoderm, mesoderm and ectoderm. One of the major cell movements taking place is convergence and extension (CE), where cells move toward the dorsal midline and intercalate with one another leading to the mediolateral narrowing (convergence) and anterior-posterior lengthening (extension) of the forming embryonic axis. Vertebrate CE is regulated by the noncanonical Wnt pathway, similar to the planar cell polarity (PCP) pathway that mediates the establishment of cell polarity in the plane of epithelia in Drosophila. In zebrafish, mutations in the wnt11 (Silberblick) and wnt5 (Pipetail) genes lead to defective CE movements without affecting cell fate. The two secreted glycoproteins Wnt11 and Wnt5 signal through their Frizzled receptor, Dishevelled, and Daam1 to activate the small GTPases Rho and Rac leading to the reorganization of the actin cytoskeleton. The Rho family of GTPases including Rho, Rac and Cdc42, act as molecular switches and regulate the cytoskeleton via cycling between inactive GDP-bound and active GTP-bound forms. Rho and Rac require guanine nucleotide exchange factors (GEFs) for their activation. However, the identity of the GEF involved in this pathway remains unknown, as neither Dishevelled nor Daam-1 can directly mediate the GDPGTP exchange reaction. Def6 is a novel type of GEF activating Rho GTPases through the exchange of bound GDP for GTP. We have previously shown that Def6 acts upstream of Rho, Rac and Cdc42 and also colocalizes with F-actin in mammalian cells. However, the role Def6 has in regulating cell shape, cell polarity and cell movement remain unknown. Here, we show that knock down of def6 affected CE cell movements, but not cell fate specification. Overexpression of GFP-tagged def6 protein was sufficient to rescue the def6-Morpholino-mediated phenotype indicating that the MO-induced defects were a direct result of specific def6 knockdown. To determine whether def6 functions downstream of Wnt11 and Wnt5 we attempted to rescue the Wnt11 and Wnt5 MOinduced phenotypes with def6 RNA. Although co-injection of def6 and Wnt11 MOs suggested synergism, def6 RNA did not rescue the Wnt11 knockdown. In contrast, the Wnt5-MO-induced phenotype was largely rescued by def6 RNA. Together these results suggest that def6 functions downstream of Wnt5 in the same pathway and synergistically with Wnt11 in either the same or parallel pathways. Since non-canonical Wnt signalling converges on RhoA we further tested whether RhoA acts downstream of def6.Constitutively active RhoA partially rescued the severe def6 knockdown phenotype. Our results so far show that def6 acts downstream of Wnt5 in the non-canonical Wnt signalling pathway and provide a starting point to further elucidate the role it plays in regulating CE movement during gastrulation. 151 Posters 81 Early development and gastrulation Analysis of STAT3-activity in embryonic stem cells and during early development of Oryzias latipes M. Kraussling, T. U. Wagner, M. Schartl Physiological Chemistry I, Biozentrum, University of Wuerzburg, Germany Introduction: An important aspect in stem cell research is the understanding of how the potential for self-renewal is maintained and the prevention of spontaneous differentiation in stem cells is achieved. In murine embryonic stem (mES) cells, one member of the family of signal transducers and activators of transcription proteins, STAT3, is needed to keep mES cells in the ,,stemness"status. The latent transcription factor STAT3 is activated by the leukaemia inhibitory factor (LIF) and consequently imported into the nucleus. In contrast to mES-cells, activated nuclear STAT3 is neither necessary nor sufficient to keep human embryonic stem (hES) cells in the undifferentiated status. In this work, we analyzed the localization and thereby the activity of STAT3 in embryonic stem cells of medaka fish (MES) and during early embryonic development of medaka. Material and Methods: The embryonic stem cell line MESI was obtained from blastulae of medaka fish (Oryzias latipes) and remains undifferentiated in cell culture in presence of a complex mix of growth factors. STAT3-activity in MESI-cells and early embryos of medaka fish was investigated via immunofluorescence. Results: Ectopic expression of an eGFP-tagged STAT3 resulted in dominantly cytoplasmic and thus inactive localization in MESI. Analyses in medaka embryos of blastula-stage demonstrated that STAT3 is inactive in all cells. Investigations on embryos before 1024-cell-stage revealed that STAT3 has a wave-like import into the nucleus between 64-cell-stage and 512-cell-stage with a peak at 64-cell-stage. No increase of nuclear STAT3-levels was detected before 64-cell-stage or after 512-cell-stage. Discussion and Conclusions: The assays on MESI cells and blastula-staged medaka embryos demonstrated that STAT3 is not activated in medaka embryonic stem cells. Consequently, these data indicate that activated STAT3 is not necessary for stem-cell status in medaka. Hence, the mouse-system is the only vertebrate stem cell system depending on active STAT3. Furthermore, the import-wave of STAT3 spanning 64-cell-stage to 512-cell-stage medaka embryos argues for a specific process in medaka development which is depending on nuclear presence of activated STAT3. Posters 152 Early development and gastrulation 82 Seretonergic system and the effects of neurotropic drugs at the early prenervous stages of teleost fish development E. G. Ivashkin, E. E. Voronezhskaya Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia Various neurotransmitter systems were extensively studied over the past 100 years. Most of these studies were focused on the physiological role of neurotransmitters either in cell culture or in adult organisms which contains differentiated cells and complex tissues. However, the serotonergic system, for example, is active from the very early developmental stages. Thus, serotonin (5-HT), enzymes necessary for 5-HT synthesis, transportation and degradation, as well as 5-HT receptors have been found in various animals starting from oocyte stage. It is known that neurotropic drugs that targeted on serotonergic system have an impact on early development. Despite of all existing data, the functions of neurotransmitters in morphogenetic processes are still purely understood. No data on the serotonergic system at the fish development before appearance of neurons exist. However, physiological and pharmacological properties of the serotonergic system are well known in larval and adult nervous system of zebrafish. Normal development of teleost fishes has been described in great details, and provides an opportunity to use a wide range of cytological, pharmacological and molecular techniques in a developing embryo. Taken altogether this facts makes the zebrafish embryos a favorable model to study the mechanisms of morphogenetic effects of 5-HT. We used biochemistry and laser scanning confocal microscopy to describe the distribution of 5-HT and serotonin receptors during the early development (from zigote to blastula) of two teleost fishes, zebrafish (Brachydanio rerio) and Eurasian weather loach (Misgurnus fossilis). 5-HT expression started in zigote and continued in the blastoderm. Incubation of dichorioneted eggs with 5-HT precursor increase the concentration of both 5-HT and 5-HIAA, the product of serotonin degradation. Western blot analysis with antibody against human 5HT1a receptor revealed a band with size of 44 kDa which corresponds to the zebrafish 5HT1a receptor. Utilizing the same antibody we detected a distinct punctate signal on the surface of blastomers in wholemount preparations. Pharmacological screening of serotonin-related drugs demonstrated a significant difference in induced developmental defects in two studied species which belongs to the different families from the same order Cypriniformes. 153 Posters 83 Early development and gastrulation Mutagenesis in Zebrafish by Transposon Gene Trap System S. Yang, J. Tong, Y. Han, X. Zheng, P. Xu, A. Meng Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China Zebrafish is a powerful model organism for studying vertebrate genetics and development. To identify genes involved in development and diseases, the strategies of chemical and insertional genetic screens have been carried out. Many genes essential for zebrafish development were identified by mutagenesis screening. The transposon system is an efficient insertional mutagen. We constructed a Tol2-based gene trap vector, TSBGS, which contains the reduced 5' and 3' ends of Tol2, bcl2 splicing acceptor, GFP reporter gene and the SV40 polyA signal. The transposon vector and the transposase mRNA synthesized in vitro were co-injected into fertilized eggs at one-cell stage. The injected embryos were raised and mated. In the pilot screening, 4900 injected fish were screened and 183 F1 families expressing GFP in spatial and temporal fashion were identified. Four recessive lethal mutations that are tightly linked to the transposon insertions have been identified from 121 F2 families. Beginning at 2 days post fertilization (dpf), mutants YT068 exhibit small eyes, a reduced brain, lack of jaws, branchial arches and swimming bladder, and then die at 5 dpf. YT101 mutants can be recognized at 28 hours post fertilization by the presence of hydrocephalus, edema, reduced pigmentation, reduced yolk sac extension and eyes. We have identified the interrupted genes in these mutant lines and their functions are being studied in detail. Our experience indicates that the Tol2 transposon gene trap system is applicable for mutagenesis in zebrafish though its efficiency is low. Posters 154 Early development and gastrulation 84 Allosteric Control of Alpha-Catenin in Cell Adhesion and Migration A. Schepis, W. S. Talbot and J. W. Nelson Stanford University, USA The Adherense junctions (AJs) are essential for cell-cell adhesion, and the organization of membrane domains and cell polarity. AJ organization is dynamic as the balance between assembly and disassembly plays important roles in cell migration and epithelial-mesenchymal transition during embryogenesis, wound healing and cancer. The AJs comprise members of the cadherin family, transmembrane cell adhesion proteins that bind through their cytoplasmic domain to p120 and -catenin; and -catenin in turn binds to -catenin (-cat). The textbook model of the AJ depicts a stable complex in which the AJ is bound to the actin cytoskeleton, with the -cat providing the molecular link between the actin cytoskeleton and the cadherin/ beta-catenin complex. A direct test of this model showed, however, that -cat cannot form a quaternary complex with cadherin, beta-catenin and actin. Instead -cat behaves as an allosteric protein in which monomeric -cat is able to form a ternary complex with cadherin/-catenin, but not actin, while dimeric -cat binds to and bundles actin filaments but does not interact with the cadherin/beta-catenin complex (Drees et al, 2005; Yamada et al., 2005). -cat is in dynamic equilibrium between monomer and dimer pools in the cytoplasm and monomer bound to cadherin at sites of cell-cell adhesion; it has been proposed that a local increase in -cat concentration during clustering of the cadherin/catenin complex could regulate local formation of -cat dimers in the cytoplasm. These results point towards a dynamic, rather than static role of -cat in locally regulating actin and membrane dynamics at sites of cell-cell adhesion. However, the new -cat's properties were shown by in vitro study. We chose the zebrafish gastrulation, a model system for cell adhesion, to verify the new -cat properties in vivo. We are using, together with the morpholino approach, a number of constructs that are able to mislocalized the -cat interfering with his allosteric regulation. These constructs are able to direct -cat to the mitochondria or to the plasma membrane (in a AJs independent manner). The rational of this approach is to perturb dynamically the -cat concentration of the different pools. For example, diverting the -cat to the mitochondria, we can lower the concentration of the protein available for the cytosol and plasma membrane pools. Our preliminary results shows that the morpholino caused delayed or arrested in epiboly and the embryos do not complete somitogenenis. Inducing mitochondria mislocalization of -cat causes delay in epiboly, but in contrast to the morpholino injected embryos, these embryos complete somitogenesis. However, they display morphological defects. 155 Posters 85 Early development and gastrulation Pou5f1/Oct4 contributes to dorsoventral patterning by transcriptional activation of vox expression B. Wendik, K. Lunde, B. Polok, HG. Belting, W. Driever and D. Onichtchouk University of Freiburg, Department of Developmental Biology, Freiburg, Germany Pou5f1/Oct4 in mice is required for embryonic pluripotent cell populations. In zebrafish, pou5f1/pou2 has both maternal and zygotic expression. The zygotic pou5f1/pou2 mutant spiel ohne grenzen (spg) shows neural plate pattering defects. We rescued the spg mutants by pou5f1 mRNA injection. These fish mate to produce maternal zygotic spg (MZspg) mutants, indicating that pou5f1 acts as a transcriptional activator during dorsal-ventral patterning. Overexpression of larger quantities of Pou5f1-VP16 can ventralize wild-type embryos, while overexpression of a Pou5f1-En repressor fusion protein can dorsalize. caBMPR1a can rescue dorsalization both in MZspg mutants and in wild-type embryos overexpressing Pou5f1-En. Thus, we propose a role for pou5f1 in the bmp pathway leading to ventral tissue specification. In support of this idea, via bmp2b and fgf8 expression assays, we see that MZspg mutants are dorsalized prior to 30% epiboly, and that Pou5f1-En can dorsalize wild-type embryos prior to 30% epiboly. Notably, MZspg mutants, like wild-type embryos, can be further dorsalized in response of fgf8. Microarray analysis revealed that expression of several other ventral factors, including vox, is affected in MZspg. Overexpression of Pou5f1-VP16 strongly activates vox expression in wild-type embryos by 40% epiboly, even in the presence of cycloheximide (CHX), while it hardly effects bmp2b and bmp4 expression in the presence of CHX. In silico analysis of the vox promoter reveals conserved Pou5f1 binding sites which could be confirmed by gel shift assay. Taken together, our data provide embryological and molecular evidence for the direct activation of the vox by Pou5f1 in dorsal-ventral patterning. Posters 156 Early development and gastrulation 86 Cloning, expression pattern and function analysis foxd (1,3,5) from flounder (paralichthys olivaceu) X. Tan,Y. Zhang, PJ. Zhang, Y. Xu Experimental Marine Biological Laboratory, Institute of Oceanology,Chinese Academy of Science, Qingdao, ShanDong, P.R.China Since the first Forkhead Box (Fox) gene was discovered from fruit fly, more than 100 members of this family are identified in a variety of metazoan species. All of them contained a highly conserved DNA binding domain. It had been shown genes of this family play important roles in early embryonic patterning and physiology. As an economically important fish in the Asian region, flounder has been cultured for many years, but there is little information about its skeletal muscle development and maturation. To further clarify the function of regulatory factors that are involved in muscle formation of flounder, FoxD family genes were isolated and their expression pattern and function in regulating muscle were analyzed. The flounder FoxD1FoxD3FoxD5 contain only one exon and a conserved winged helix DNA binding domain. During the early stage of embryogenesis, FoxD1 was mainly expressed in the somites, kidney progenitor cells, brain and intestine. FoxD3 was expressed in premigratory neural crest cells, somites, post otic placodes, cranial and trunk neural crest cells, and pineal gland. FoxD5 was expressed in several tissues including somites, tail bud, forebrain and otic vesicle etc. The function of Flounder FoxD1,3,5 on muscle development was conducted through overexpressing mRNA in one cell of the two-cell stage zebrafish by microinjection and analyzing the expression of Myf5 and MyoD. After flounder FoxD3 overexpressed, the two sides separated by the anterior-posterior axis of zebrafish embryo showed an asymmetrical development, the expression of MyoD and Myf5 in the paraxial mesoderm was inhibited. When Flounder FoxD1 mRNA was over expressed, MyoD expression in the somites on one side was reduced, but the expression in the adaxial cells was not affected; Myf5 expression in the presomitic mesoderm, adaxial cells and somites on one side was reduced. When FoxD5 was over expressed MyoD expression in the somites on one side was enhanced, but the expression in the adaxial cells was not affected; Myf5 expression in the somites and presomitic mesoderm on one side was also enhanced. So, FoxD1,3, 5 might regulate the muscle development by regulating the expression of MyoD and Myf5. To further understand the regulation mechanism of FoxD1,3,5, RT-PCR was performed to analyze the engogenous zebrafish FoxD1,3,5 expression. RT-PCR showed that the over-expressed Flounder FoxD3 can inhibit the expression of zebrafish endogenous FoxD3,while over-expressed Flounder FoxD1 or FoxD5 can not inhibit the expression of zebrafish endogenous FoxD1 or FoxD5.So, the regulation of FoxD1,3,5 was different from each other. This work was supported by the Natural Science Foundation of Shandong ProvinceP.R.China to Xungang Tan (Y2008E12),the National Basic Research Program of China (973 Program, No.2004CB117402)the National High Technology Research and Development Program of China (863 Program, No. 2006AA10AA402) and the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences. The author gratefully acknowledges the support of K.C.Wong Education Foundation, Hong Kong. 157 Posters 87 Early development and gastrulation The role of jnk in zebrafish early morphogenesis M. Marsal and E. Martin-Blanco Cell and Molecular Biology, Instituto de Biologia Molecular de Barcelona CSIC, Barcelona, Spain The zebrafish embryo is an ideal model system to study the molecular and cellular mechanisms implied in the expansion and fusion of epithelial sheets. At epiboly, the coordinated movement of the diferent cell layers suggests specific mechanism directing both cell polarity and cell migration. The interactions between the enveloping layer (EVL), the deep cells layer (DCs) and the yolk syncitial layer (YSL) could be directed by many signalling mechanisms.We propose JNK signalling pathway as a potential candidate to regulate the processes of epiboly and convergence and extension in the zebrafish embryo. Knock-down of zebrafish JNK leads to an epiboly phenotype, in which migration of the majority of deep cells is affected in the morphant embryos, while epibolic expansion of the superficial EVL and the YSL nuclei seems to progress at a normal rate. This suggests that JNK controls cell adhesion between the different layers. We monitor changes in cell behaviour by confocal videomicroscopy and in fixed sections with markers for both cell adhesion and cytoeskeleton. Embryos that overcome the DCs epiboly phenotype show a convergence and extension phenotype with a shorter and broader body. They also have a curled body and display circling movements. We propose that JNK could be controlling cell shape changes and motility underlying mophogenetic movements in early zebrafish embryogenesis. Posters 158 Early development and gastrulation 88 Role of the wnt/pcp pathway component trilobite/vangl2 in cell behaviors underlying convergence and extension movements I. Roszko1, J.R. Jessen2, D. Sepich1, Y. Liu1, L. Solnica-Krezel1 1 Department of Biological Sciences, Vanderbilt University, Nashville, USA, 2Department of Medicine/Division of Genetic Medicine, Vanderbilt University Medical Center, Nashville, USA The Wnt/Planar Cell Polarity (Wnt/PCP) pathway was discovered in Drosophila where it plays an essential role in the establishment of planar polarity in epithelial tissues. In these epithelia, cells are connected by tight junctions and PCP pathway components exhibit a characteristic asymmetric localization to specific membrane domains. In mesenchymal cells such tissue architecture is absent. However, in vertebrates, PCP components are necessary for polarized cell behaviors of mesenchymal cells during convergence and extension (C&E) movements, which narrow and elongate the nascent embryonic body. But, the mechanism whereby the PCP pathway regulates cell polarity in vertebrates remains a challenging open question. We investigate the role of a core PCP component, Trilobite/Vangl2 (Tri) in this process. At midgastrulation, lateral mesodermal cells are loosely packed and begin to converge slowly towards the dorsal midline by directed migration. At this stage, in tri mutants, the mesodermal cells migration is normal. As gastrulation proceeds, in wild-type embryos, cells become tightly packed and highly mediolaterally elongated, and migrate fast and along straight trajectories towards dorsal. But tri mutant cells fail to elongate and their movement is slow and less effective. We characterize this transition in cell morphology and behaviors during C&E movements. We show that endogenous Tri/Vangl2 is recruited to the cell membrane at a specific time during gastrulation, correlating with modification of cell behavior. Recent data showed that Prickle and Dishevelled (Dvl) are asymmetrically localized in mesoderm during gastrulation (Yin et al., 2008). This distribution is lost in tri mutants. We asked whether Tri/Vangl2 recruitment at the membrane results in its preferential asymmetric localization. We show that mediolateral cell polarization does not involve asymmetric Tri/Vangl2 localization. Using a Vangl2-GFP construct we perform in vivo studies to analyze the dynamic behavior of Tri/Vangl2 protein. We also investigated the relationship between Tri/Vangl2 and polarization of the microtubule cytoskeleton, by analyzing the position of microtubule organizing centers (MTOC) position in cells engaged in C&E. Whereas Dvl and Knypek/Glypican4, appear to be essential for MTOC polarization in cells engaged in C&E at late gastrulation stages, in MZtri mutants the MTOCs, are properly polarized. Based on these studies we suggest distinct roles for these PCP components in cell polarization during C&E. 159 Posters 89 Early development and gastrulation Functional Analysis of the eve1 Gene During Zebrafish Neural Development C. M. Cruz1, S. Maegawa2, E. S. Weinberg2, I. B. Dawid3, S. Wilson4, T. Kudoh1 1 School of Biosciences, University of Exeter, Devon, United Kingdom, 2Dept. Biology. University of Pennsylvania, Philadelphia, USA 3Lab. Molecular Genetics, NICHD, NIH, Bethesda, USA, 4 Dept. Anatomy and Developmental Biology, University College London, United Kingdom Eve1 is a zebrafish homologue of the even-skipped (eve) family of transcriptional repressors. Originally characterised in the fruit fly Drosophila melanogaster, where it has a crucial role in A/P polarity, eve homologues have since been identified in all vertebrate species so far studied. In vertebrates, it is expressed in the posterior during early embryonic development, and overexpression experiments have revealed its role in tail development in both fish and frog. Here, we have performed loss-of-function and early marker analyses in wild type and ichabod mutant embryos and have uncovered an important role for eve1 in both trunk and tail development. We found that eve1 activates the expression of posterior genes, such as hoxb1b, via the retinoic acid signal. In addition, we found that eve1 possesses neural inducing activity and may therefore be important both in posteriorisation as well as in the induction of neural tissue in trunk and tail. Posters 160 Early development and gastrulation 90 Characterization of the AP-1 1-adaptin in zebrafish (Danio rerio) D. Zizioli1, M. Guarienti1, R. Bresciani1, E. Monti1, G. Gariano1, C. Tobia1, A. Preti1, F. Cotelli2, P. Schu3 1 Dep. of Biomedical Science and Biotechnology University of Brescia, Italy; 2Dep.of Biology, University of Mailand, Italy; 3Center for Biochemestry, Georg August University, Gottingen, Germany Protein transport between the trans-Golgi network and endosomes is mediated by vesicles formed by the adaptor-protein complex AP-1. AP-1 consists of four subunits: 1, 1, 1, 1. In mammalia 1-adaptin subunit exists as the ubiquitously expressed 1A and the polarized epithelia-specific 1B. Mice "knock out" for the subunits 1 or 1A revealed that AP-1 complex is essential for mammalian embryonic development. We used zebrafish (Danio rerio) as a vertebrate model to explore the function of 1A and 1B subunits. In silico database analysis revealed two sequences encoding the tissue-specific 1B and the ubiquitously expressed 1A subunit, respectively. The two identified sequences share 83-85% identity to the corrisponding mammalian 1-adaptins. The genomic structure and the exon-intron boundaries of the zebrafish orthologs were determined by performing BlastN alignments of different AP-1 1 cDNAs against the ENSEMBL Genome Browser and then compared to the human and mouse genes. Zebrafish ap1m1 is located on chromosome 2 while ap1m2 is not yet located on a specific chromosome. The intron-exon structure of zebrafish ap1m1 and ap1m2 is similar to human and mouse genes: the first one is organized in 10 exons and 9 introns and the second one is organized in 11 exons and 10 introns. The RT-PCR analysis on different developmental stages revealed that 1A and 1B transcripts are already detectable at the first stages of embryogenesis, whereas high levels were observed from late somitogenesis to 72 hpf. The 1A transcript was present ubiquitously, while the 1B transcript was present in organs of endodermal derivation as expected. The whole mount in situ hybridization confirmed the results obtained by RT-PCR for both subunits. Histological sections analysis revealead that ap1m1 is expressed ubiquitously and in particular in the notochord, while ap1m2 is expressed only in organs of endodermal derivation such as such as gut, pharingeal endoderm, liver and pancreas. When the coding sequences of zebrafish ap1m1 and ap1m2 were transfected in 1-deficient mouse embryonic fibroblasts, both subunits could complement the mouse 1 deficiency and restore AP-1 vesicle formation. Depletion of 1B expression by using "translation-blocking morpholinos" (Mos ap1m2) gave rise to embryos characterized by defective formation of intestine, liver and tissues of endodermal derivation. Moreover, from 24 hpf onward the morphants showed a reduced mobility, that became more severe during the late stages of development and thereafter development ceased at 7-8 dpf. This is the first demonstration of the essential role played by 1B-adaptin in organs development of vertebrate.We are currently running "knock down" experiments in order to elucidate also the role played by 1-A adaptin during zebrafish development. Posters 161 91 Early development and gastrulation Identification and functional characterization of a novel -adaptin subunits (adaptor complex AP-1) in zebrafish (Danio rerio) M. Guarienti1, D.Zizioli1, R. Bresciani1, E. Monti1, A. Preti1, F. Cotelli2, P. Schu3 1 Dep.of Biomedical Sciences and Biothecnology University of Brescia, Italy; 2Dep.of Biology, University of Mailand, Italy; 3Center for Biochemestry, Georg August Univerisity, Gottingen, Germany In eukaryotic cells, transport of proteins between the different membrane compartments through the exocytic and endocytic pathways involves clathrin-mediated vesicles, which formation requires an adaptor protein complex (AP). The AP-1 complex mediates transport from TGN to the endocytic pathway and consists of four subunits: ring closure at around the 5 to 8 somite stage, which is earlier than trout eggs of normal size. Secondly, zebrafish egg yolks were artificially expanded by injection of chicken egg white at the blastula stage. The injected zebrafish yolk was expanded by 10 to 15% in diameter. In such embryos, the early 3-4 somites were formed before germ ring closure. They develop normally at 24h, forming the head, trunk and tail of normal shape and size. From these data we concluded that, in the evolution of teleost fish, variable yolk size has altered developmental sequence such that in eggs with large yolks, germ ring closure is prolonged according to the expansion of the yolk. Other developmental processes, such as tail bud formation and somitogenesis, occur without major dependence on yolk size. 163 Posters 93 Signaling Regulation and function of Sox6 in zebrafish skeletal myogenesis X.G. Wang1, J.F. Chai1, S. Elworthy3, W.O. Cheong1, and P.W. Ingham1,2 1 Institute of Molecular and Cell Biology, Singapore; 2Dept of Biological Sciences, National University of Singapore; 3MRC Centre for Developmental and Biomedical Genetics, and Department of Biomedical Science, University of Sheffield, UK The first muscle fibres to differentiate in the zebrafish embryo derive from the adaxial cells and are of the slow twitch fibre type, a character imparted on them by the activity of the Prdm1a protein, expression of which is induced by Hedgehog signaling. The remainder of the myogenic paraxial mesoderm does not express Prdm1a and differentiates into fast twitch muscle fibres. The sox6 gene, previously implicated in fibre type specification in the mouse, has been found to be expressed in these fast muscle progenitors and to be capable of repressing slow muscle-specific genes, such as Prox1, smyhc1 and stnnc, implying it to be a key regulator of fibre type identity. Consistent with this, sox6 is not normally expressed in slow-twitch myoblasts but is de-repressed in the adaxial cells of prdm1a mutants. Using chromatin immunoprecipitation (ChIP) with anti-Prdm1 antibody we have found that Prdm1a binds specifically to sequences near to the sox6 transcription start site. Using transgenic analysis, we have identified a sox6 genomic fragment capable of driving EGFP expression specifically in the fast twitch fibres in the trunk and head muscles. Within this fragment a 220bp conserved element was identified to be a muscle enhancer by transient expression analysis. Simultaneous morpholino knock-down of MyoD and Myf5 reveals that activation of sox6 in fast-twitch progenitors is dependent on these myogenic regulatory factors. To indentify direct targets of Sox6, we have developed an antibody against the zebrafish protein. We will report the preliminary results of ChIP on chip experiments using this antibody. Posters 164 Signaling 94 The tumor suppressor genes prdm5 and nucleophosmin1 (NPM1) cooperate in the regulation of wnt signalling during zebrafish embryogenesis G. Deflorian1,3, N. Meani2, F. Pezzimenti1,3, M. Alcalay1,2,4, M. Mione3 1 Cogentech, Consortium for Genomic Technologies, Milan, Italy; 2Department on Experimental Oncology, European Institute of Oncology, Milan, Italy; 3IFOM-FIRC, Institute for Molecular Oncology Foundation, Milan, Italy; 4Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita` degli Studi di Milano, Italy Proteins encoded by the Wnt family of genes act as signals and have been shown to play essential roles during embryogenesis. The spatial and temporal patterns of expression of the vertebrate members of the Wnt family, their receptors and effectors suggest roles for these genes in a variety of developmental processes. In all these processes Wnt signals appear to be modulated by specific sets of regulators that act at different levels of the signalling cascade. Moreover, several pathological states that may arise from altered stem cell function, such as degenerative diseses and cancer, are frequently associated with changes in Wnt/--catenin pathway activity. In previous work, we have shown that the tumor suppressor Prdm5, a transcripion factor belonging to the family of PRDM proteins, is able to modulate, in a negative way, both canonical Wnt/--catenin and planar cell polarity (PCP) Wnt pathways (Meani et al., 2009). Data obtained from in-vitro assays, indicated that human PRDM5 associates with Nucleophosmin1 (NPM1), a nucleolar protein that shuttles between the nucleus and the cytoplasm (Grisendi et al., 2006) and that acts both as proto-oncogen and as tumor suppressor. In zebrafish both genes are strongly expressed during embryogenesis. From mid somitogenesis npm1 expression localizes to domains in which various members of the Wnt family are present, whereas prdm5 remains ubiquitous. We found that the effects of prdm5 and npm1 targeted knock-down are similar and resemble an impairment of morphogenetic movements during gastrulation due to a block of PCP Wnt pathway, accompanied by a reduction of the cephalic region. In agreement with this, the overexpression of both prdm5 and npm1 causes a "dorsalized" phenotype. Functional studies using loss and gain of function approaches showed that both prdm5 and npm1 regulate dkk1 expression at early stages of development, in a way that results in repression of canonical Wnt/-catenin pathway in addition to the PCP pathway. npm1 synthetic mRNA is able to rescue the phenotype of prdm5 morphants, whereas prdm5 overexpression worsen the phenotype due to npn1 knock down, suggesting an epistatic relation between the two genes rather than simple convergence of functions. So, our results suggest that Prdm5 and Npm1 co-operate in repressing Wnt signalling during early embryogenesis in zebrafish and may as well exert their tumor suppressor activities through negative modulation of Wnt signalling. Grisendi S., Mecucci C., Falini B. and Pandolfi P.P. (2006). Nucleophosmin and Cancer. Nat. Rev. Cancer. 6; 493-505. Meani N., Pezzimenti F., Deflorian G., Mione M. and Alcalay M. (2009). The Tumor Suppressor PRDM5 Regulates Wnt Signalling at Early Stages of Zebrafish Development. PloS ONE. 4(1); e4273. 165 Posters 95 Signaling Transgenic zebrafish Tg(Nogo-B:GFP) line recapitulates the Nogo-B expression pattern in diverse tissues including the liver and intestine HW. Han1,2, YC. Chen1, BK. Wu1, CY. Chu1,2, CH. Cheng1, GD. Chen1, SP. L. Hwang3, K. Kawakami4, and CJ. Huang1,2,3 1 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan; 3Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; 4Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan In mammals, the Nogo family consists of Nogo-A, Nogo-B, and Nogo-C. Using sequences from mammalian counterparts, we cloned three zebrafish Nogo-related transcripts, termed Nogo-B, Nogo-C2, and Nogo-C1, which are generated by alternative promoter usage and alternative RNA splicing. These sequences are identical to other Nogo ligands, such as Rtn4-l/Nogo-alpha, Rtn4-n/Nogo-beta and Rtn4-m/Nogo-gamma, respectively. Whole-mount in situ hybridization was performed to demonstrate that Nogo-B mRNA was predominantly expressed in the brain, brachial arches, eyes, muscle, liver and intestine. The 4.8-kb promoter region of Nogo-B gene was isolated from a BAC clone and it was demonstrated to be functional in both cultured cells and zebrafish embryos. A transgenic zebrafish Tg(Nogo-B:GFP) line was generated using the same promoter region to drive GFP expression through Tol2-mediated transgenesis. Transgenic zebrafish Tg(Nogo-B:GFP) line recapitulates the endogenous expression pattern of Nogo-B mRNA. This line will be useful for the study of Nogo-B gene regulation during development. Posters 166 Signaling 96 The functional role of the zebrafish glucocorticoid receptor beta-isoform M.J.M. Schaaf, A. Chatzopoulou, P. Schoonheim, H.P. Spaink, Institute of Biology, Leiden University, The Netherlands Glucocorticoids are steroid hormones, which are released from the adrenal glands to regulate the body's adaptation to stress. Synthetic analogues of these hormones are widely prescribed as anti-inflammatory drugs. They exert their effects via the glucocorticoid receptor (GR). In humans, two splice variants exist: the canonical GR alpha-isoform, which acts as a ligand-activated transcription factor, and the GR beta-isoform, which has a dominant-negative effect on GRalpha. Elevated GRbeta levels have been associated with the occurrence of immue-related diseases like asthma and rheumatoid arthritis and glucocorticoid resistance in patients suffering from these diseases. Until recently, the occurrence of GRbeta had only been demonstrated in humans and therefore an animal model for studies on this receptor isoform was lacking. Surprisingly, we have found that the zebrafish GR (zGR) gene, can give rise to a GRbeta isoform. We have explored the function of the zebrafish GRbeta both in vitro and in vivo. Transient overexpression of zGRbeta in cell lines as well as microinjection of its mRNA in zebrafish embryos has shown a functional role for this isoform in the regulation of the response to glucocorticoids on a subset of GR target genes. Further genetical manipulation, like the use of splice-blocking morpholinos and the construction of a transgenic fish line overexpressing zGRbeta, is undertaken in order to explore the significance and role of the zGRbeta in various physiological conditions. 167 Posters 97 Signaling Notch signaling regulates thyroid development in zebrafish P. Porazzi1*, N. Tiso2, D. Calebiro1*, F. Benato2, T. de Filippis*, C. Fritegotto2, F. Argenton2, L. Persani1* 1 Department of Medical Sciences, University of Milan, Italy; *Laboratory of Experimental Endocrinology, IRCCS Istituto Auxologico Italiano, Milan, Italy; 2Department of Biology, University of Padua, Italy. The incidence of congenital hypothyroidism (CH) has been reported to reach 1:2400 newborns in the recent years. CH pathogenesis is largely undetermined, even if several findings support the hypothesis of a relevant genetic origin. The vertebrate thyroid gland is mainly composed of endoderm derived precursors cells. The main transcription factors and genes involved in thyroid development and differentiation have been identified, but the mechanisms underlying the early follicular cells specification are still largely unknown. A candidate pathway for thyroid formation and differentiation is represented by Notch signaling, a juxtacrine mechanism involved in several processes of cell fate decision. Mutations of Notch signaling members have been shown to affect the formation of different tissues and organs during vertebrate development. This is observed, for instance, in Alagille syndrome, a human multi-organ developmental disease. As zebrafish represents an excellent animal model to identify genes affecting thyroid gland development, in this study we aim to analyze the role of direct cell-to-cell Notch signaling in follicular cells specification in vivo. To determine whether Notch signaling plays a role in thyroid gland formation, we blocked or over-activated Notch signaling and analyzed the expression of thyroid marker genes (pax2a, nkx2.1a, tg and slc5a5) by mRNA in situ hybridization. In mib mutant embryos, which fail to activate Notch signaling, we observed an expansion in the number of thyroid cells together with a modified morphology. Coherently, embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling, showed an increased transcription of genes expressed in the thyroid. Conversely, Notch gain of function leads to the opposite phenotype. The excess of Notch signaling, obtained by intercrossing two transgenic lines: hsp70:gal4 x UAS:Notch1aintracellular domain (NICD), resulted in a strong reduction of thyroid follicular cells. Given that the pharyngeal vascular structure is seriously affected by these treatments, the observed thyroid phenotypes could result either from cell-autonomous or non cell-autonomous activities of Notch signaling. In order to define if Notch signaling plays a direct role in thyroid early dor the interaction between BR1a and STAT3. Next, we will replace the endogenous proteins with variants able to fulfill classical signal transduction roles, but are not able to interact with one-another anymore. Posters 170 Signaling 100 Characterization of expression and function of novel emilin genes in mouse and zebrafish M. Milanetto1, N. Tiso2, A. Schiavinato1, P. Braghetta1, D. Volpin1, F. Argenton2 and P. Bonaldo1 Dept. of 1Histology, Microbiology and Medical Biotechnologies, 2Biology Univ. of Padova, Italy Emilins are a family of glycoproteins of the extracellular matrix with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure and syntheny with the human ortholog. Whole-mount in situ hybridization (ISH) revealed that expression of zebrafish emilin genes is regulated in a spatio-temporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of some emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in certain morphogenetic processes (1). The most distinctive pattern is the one of emilin-3, the only component of the family lacking the C-terminal gC1q domain. While other emilin genes reveal a mesenchymal or cardiovascular expression profile, the two zebrafish genes coding for emilin-3 are abundantly expressed at early stages in notochord and floor plate, suggesting that emilin-3 may play key roles during early development (1). Emilin-3 expression does not appear to be regulated by either Hedgehog, FGF or Notch signaling, as indicated by ISH of embryos in which these molecular pathways were blocked by drug treatments. Additionally, expression of the two Emilin-3 genes appears normal in embryos mutant for Chordin (dino) or for BMP2 (swirl). We are currently investigating other relevant pathways to check whether they regulate emilin-3 expression during early development. Moreover, we are performing functional studies by microinjection of mRNA or morfolino oligonucleotides in fertilized oocytes. The studies in mice were concentrated on the gene coding for emilin-3. In the past years, targeted Emilin3 gene inactivation in murine embryonic stem (ES) cells was already undertaken in our laboratory. However, generation of Emilin3 knockout mice was unsuccessful and these previous attempts revealed that, unlike other emilins, targeted inactivation of the Emilin3 gene is particularly difficult. The frequency of homologous recombination of this gene is extremely low and transmission of the inactivated allele to the F1 generation was not reached, probably due to karyotypic instability of targeted ES cell clones. Therefore, we carried out a new Emilin3 gene targeting approach, performed by means of a large-scale experiment. We transfected R1 ES cells with a Emilin3 targeting construct and, after double positive-negative selection, 1505 clones were isolated; 995 of these individual cell clones were further expanded and investigated for identifying clones in which a correct homologous recombination event had occurred. Six positive ES clones were identified, and after additional karyotypic characterization four of them were used for generation of chimeric mice by microinjection of ES cells into host blastocysts and implant in foster females. Several chimeric mice were obtained from two independent clones, and they were bred to obtain Emilin3 null mice which are currently under phenotypic analysis. 1) Milanetto M., Tiso N., Braghetta P., Volpin D., Argenton F., Bonaldo P. (2008) Emilinsh nc mutants F. Rodrigues, D. Tosh, and R. N. Kelsh Biology and Biochemistry, University of Bath, England Signaling Whilst in vivo analysis of zebrafish mutants is widespread, the ability to study these phenotypes in vitro has been less keenly pursued. The purpose of this project is to develop culture conditions that allow for the study in vitro of the phenotypes of zebrafish neural crest (NC) mutants, as a complement to in vivo studies. The NC is responsible for several cellular lineages such as pigment cells and neurons and glia of the peripheral nervous system and is used as a model system to study developmental processes such as specification and differentiation. The study of individual NCC mutants in cell culture would open up new possibilities for testing of gene function. In the absence of published zebrafish NC cell cultures, we began with avian and mouse NCC culture conditions. Using these culture conditions, we achieved survival and differentiation of NCCs and many derivatives from a pool of disaggregated zebrafish embryos. However, application of these conditions to single NCCs or to NCCs at clonal density resulted in a failure in attachment/growth, probably due to lack of growth factors in the medium. Likewise, cultures from disaggregated single embryos also failed to thrive, highlighting the need for significant cultured cell numbers for NCC survival and differentiation. In order to solve this problem a strategy was devised where a single embryo with tranhsgenically-labelled NC was cultured in a pool of disaggregated, non-transgenic embryos. We anticipate this approach will allow us to study NCCs derived from a single embryo; in particular, it will be useful for testing the cellautonomous effects of mutant genes. For initial tests of this approach, we are using lines in which GFP is driven under the control of the sox10 promoter. This system has the limitation that tracking individual cells and their progeny is difficult since GFP expression is rapidly lost as NCCs differentiate. Consequently, we are modifying the system to use the Cre/Lox system for permanent labelling of NCCs in culture. Initial tests of a sox10:cre construct indicate that this system gives Cre expression in a sox10-like pattern. We are now attempting to generate stable sox10:cre transgenic lines. Once combined, we expect these systems will allow us to compare NCC development in cultures of NC mutants and their wild-type siblings. Posters 174 Signaling 104 Analysis of the Hedgehog signaling pathway in zebrafish H. R. Kim1, J. Richardson2, P.W. Ingham3 1 Biomedical Science, University of Sheffield, United Kingdom; 2MRC centre for Developmental Neurobiology, Kings College London, United Kingdom; 3Biomedical Science, University of Sheffield, United Kingdom Hedgehog (Hh) signaling plays a fundamental role in animal development and in postembryonic tissue homeostasis. Although mutant analysis in Drosophila and subsequently in mouse has uncovered the core components of the Hh signaling pathway there are still gaps in our understanding of this complex and unusual system. Zebrafish present an interesting model for the analysis of Hedgehog signaling, as they retain the function of pathway components that are absent from both Drosophila (eg Hip) and mammals (eg Fused). We are taking advantage of the Hh pathway mutants in zebrafish and the ease with which transgenic animals can be generated to investigate various aspects of the signaling process. In an effort to identify the functionally important domains of the Ptc proteins, we have generated Ptc fusion protein whose expression is driven by 13 kb upstream of Ptc1 translation initiation site. The efficiency of rescue of ptc1 mutants using full length Ptc1 and Ptc1 in which the C-terminus domain is removed suggests that C-terminal domain of the Ptc1 protein regulates its turnover. In previous studies, we have identified an essential role for the iguana/DZIP1 protein in Hh signaling, implicating it in the control of Gli protein activity. To investigate this function further, we have engineered 1l morphant phenotype by injection of active components of the Wnt-PCP pathway. Because several components of this pathway have been described at the base of the primary cilium, a function of Rpgrip1l could be to regulate their traffic to the cilium or their function at this specific location. To test this hypothesis, we are currently assaying the abundance and the subcellular localization of different components of this pathway in rpgrip1l morphants. Previous studies have involved the primary cilium in the balance between the Wnt- betacatenin and the Wnt-PCP pathways (reviewed in 1). The present study should allow us to better understand the function of Rpgrip1l and more generally of cilia in this process. (1) Gerdes et al., 2009. Cell 137, 32-45. (2) Vierkotten et al., 2007. Development. 134(14): 2569-77 (3) Delous et al., .2007. Nature Genet. 39(7): 875-81 179 Posters 109 Signaling Feedback Modulation of Wnt Signaling by Trophoblast Glycoprotein-like G. Weidinger1, B. Kagermeier-Schenk1, and H. Spaink2 1 Biotechnology Center, Dresden University of Technology, Germany; 2Leiden University, Netherlands Wnt/beta-catenin signaling regulates a plethora of processes during animal development, tissue homeostasis and regeneration, yet for many of its roles few target genes mediating Wnt function are known. We used inducible overexpression of Dkk1, Axin1 and Wnt8 in transgenic zebrafish lines and microarray-based transcriptional profiling to identify Wnt target genes during zebrafish embryogenesis. We found that trophoblast glycoprotein-like (tpbgl), a gene encoding a single-pass transmembrane protein with a glycosylated extracellular domain, is regulated by beta-catenin signaling in many tissues and developmental stages. Although tpbgl is conserved in vertebrates and is upregulated in a variety of carcinomas, little is known about its function. Interestingly, we found that overexpressed tpbgl potently inhibits beta-catenin signaling activated by Wnt ligands both in zebrafish embryos and cultured mammalian cells. Tpbgl acts in Wnt receiving cells, since it interferes with signal activation by Wnt-conditioned media, and upstream of the destruction complex, since it cannot interfere with pathway activation by dominant-negative Gsk3b, dominant-negative Axin1, or stabilized beta-catenin. Furthermore, tpbgl inhibits signaling induced by forced interaction of Frizzled and LRP6. Thus, tpbgl appears to interfere with beta-catenin signaling at the level of the Wnt receptor complex. Knock-down experiments in zebrafish embryos show that endogenous tpbgl is a required inhibitor of betacatenin signaling, counteracting wnt8 function in brain patterning. Intriguingly, in contrast to its role as a feedback inhibitor of the beta-catenin dependent Wnt pathway, tpbgl enhances beta-catenin independent Wnt signaling in zebrafish embryos. Thus, we suggest that tpbgl is an important regulator of Wnt receptor complex formation or activity. Posters 180 Signaling 110 PKC is required for the normal development of glutamate synapses on zebrafish Mauthner neurons DW. Ali and S.A. Patten Department of Biological Sciences, University of Alberta, Edmonton, Canada Glutamate AMPA receptors (AMPARs) are major excitatory receptors in the vertebrate CNS. Phosphorylation of AMPARs has been shown to be an important mechanism for controlling the function of these receptors in synaptic transmission and synaptic plasticity. We have previously shown that fast excitatory synaptic transmission in zebrafish Mauthner neurons (M-cells) is mediated predominantly by AMPARs. Furthermore, our recent results indicated that protein kinase C (PKC) is highly expressed in M-cells of developing zebrafish. Therefore, we sought to determine if PKC might be involved in the modulation of AMPARs. We used whole cell patch clamp electrophysiology to record AMPA miniature excitatory postsynaptic currents (AMPAR-mEPSCs) from M-cells ranging in age from 30 hour postfertilization (hpf) to 72 hpf. AMPA mEPSCs were recorded in the presence of the PKC activators, DOG and PMA. Both DOG anthis observation, lrp6 morphants also showed a Wnt11-dependent CE phenotype, which is enhanced in the antr2a/lrp6 double morphants. Both LRP6 and Antr2a localize to the plasma membrane of zebrafish embryonic cells as well as in intracellular Rab5-, and Rab11-positive endosomes. However, while LRP6 localization at the plasma membrane is mostly uniform, Antr2a appears clearly polarized. Antr2a is enriched at the sites of cell-cell contact, suggesting a possible role in regulating cell-cell interaction. Moreover, this polarization is Wnt11-dependent: embryonic cells overexpressing Wnt11 showed an increase of Antr2a at cell-cell contact sites, while in wnt11 mutant embryos (slb), Antr2a is mostly absent from the plasma membrane and accumulates intracellularly. Taken together these results indicate that Antr2a interacts with LRP6 to mediate CE movements during gastrulation presumably by regulating cell-cell interaction. Posters 182 Signaling 112 Positional cloning reveals that futile cycle encodes a maternal gene homologous to human lrmp/ jaw1 with essential roles in nuclear-centrosomal attachment and pronuclear congression RE. Lindeman and F. Pelegri Laboratory of Genetics, University of Wisconsin, Madison, USA During normal fertilization, a compacted sperm nucleus enters the egg cytoplasm bringing with it a pair of centrioles. As the sperm nucleus expands and the pronuclear envelope begins to form, the sperm-derived centrioles, along with maternally-derived centrosomal components, nucleate a sperm monoaster. The growing monoaster eventually makes contact with the female pronucleus, which then begins a rapid migration toward the male pronucleus. A close association between the sperm pronucleus and the nascent centrosome is maintained throughout pronuclear migration. After pronuclear fusion, duplicated centrosomes align on opposite sides of the newly formed zygotic nucleus in preparation for the first mitotic division. The recessive maternal-effect mutation futile cycle (fue) was previously identified as part of a gynogenesis-based screen for maternally-supplied products acting during early developmental stages in zebrafish. Embryos from homozygous mutant fue females (fue embryos) fail to undergo pronuclear migration and fusion, never form mitotic spindles, and consequently fail to properly segregate chromosomes. Interestingly, in the majority of mutant embryos, cytokinesis proceeds normally during early cell divisions even though DNA is absent from most dividing cells. Here we show that fue embryos develop a normal monoaster, but manifest abnormal nuclear membrane dynamics and morphology shortly after fertilization. Additionally, the close apposition of male pronucleus and centrosome appears to be lost shortly after fertilization. Positional cloning allowed us to narrow down the location of the fue mutation to a 56 kb region on linkage group four that contains two genes. Both genes were sequenced in their entirely and a single missense mutation was detected in the longest transcript of lymphoid restricted membrane protein (lrmp). Very little is known about the molecular function of lrmp in any system, and the maternal variant found in zebrafish appears to be significantly larger than variants described in adult tissues of other species. Analysis of lrmp transcript localization in wild-type embryos by in situ hybridization and gamma-tubulin antibody staining showed that lrmp mRNA colocalizes with centrosomes as early as 15 mpf, prior to pronuclear fusion. During prophase, lrmp is associated with nuclear membranes and centrosomes, and is later found on the spindle during mitosis. This distinct localization pattern is completely lost in fue embryos, where the transcript appears to be spread diffusely throughout the blastodisc. Our results suggest that recruitment of lrmp transcript to centrosomes and/or the nuclear periphery may be critical for proper function and distribution of these organelles during early development, possibly by providing a highly localized source of Lrmp protein. 183 Posters 113 Regulation of Chemokine Signaling by the microRNA miR-430 A. A. Staton and A. J. Giraldez Department of Genetics, Yale University, New Haven, USA Signaling MicroRNAs shape gene expression by regulating mRNA degradation and translation. To study the role of miRNAs during zebrafish development, we used embryos deficient in the miRNA processing enzyme dicer (maternal and zygotic dicer, MZdicer). These mutants are depleted of mature microRNAs. Our laboratory has shown that one miRNA in particular, miR-430, is ubiquitously expressed in early development and plays important roles in embryo morphogenesis. Analysis of the cell migration phenotype in MZdicer mutants revealed that primordial germ cell migration was affected in the absence of miRNAs. Because chemokine signaling guides germ cell migration, we assayed whether any components of this pathway are regulated by miRNAs. Migrating cells express the receptor CXCR4 and follow a gradient of the secreted ligand SDF1. CXCR7 is believed to be a non-signaling receptor responsible for refining SDF1 protein expression. Our analysis revealed that (i) SDF1, CXCR4 and CXCR7 have putative miR-430 target sites in their 3'UTRs, (ii) loss of dicer function enhances the expression of the GFP-3'UTR reporters for all three genes and (iii) mutation of the putative miR-430 target site abolished GFP-reporter repression in wild type embryos. Taken together, these results indicate that SDF1a, CXCR7a, and CXCR7b mRNAs are likely regulated by miR-430 in vivo. To assess the physiological significance of this regulation for germ cell migration, we used Target Protector morpholinos. These target protectors are complementary to the miRNA target site and have been shown to specifically interfere with the individual miRNA-target interactions. This approach allowed us to investigate the functional relevance of miR-430-mediated regulation of each individual target. Interestingly, protection of SDF1a affected germ cell migration to the germ line. We are currently analyzing the regulation of the antagonist CXCR7, and these results will be presented at the meeting. Our results support a model in which miR-430-mediated repression is acting at the transcript level to modulate and refine the expression patterns of these potent regulatory molecules, thereby promoting precise and accurate migration. Posters 184 Signaling 114 Notum-homologue is an element of the negative feedback loop regulating Wnt/beta-catenin signaling J. Topczewski, G. Parker Flowers, and J. M. Topczewska Northwestern University Feinberg School of Medicine, Department of Pediatrics, Children's Memorial Research Center, Chicago, USA Wnts, a large family of secreted proteins, are crucial for numerous processes in the developing embryo. Proper development requires tight regulation of Wnt signaling both intracellularly and extracellularly. Glypicans are a class of heparan sulfate proteoglycans linked to the cell surface via a glycosophosphotidylinositol (gpi) anchor that serve as co-receptors for ligands of several signaling pathways including Wnts, in both invertebrates and vertebrates. A secreted alpha/betahydrolase called Notum was found to antagonize the signaling of the prototypical Drosophila Wnt, Wingless, by shedding the glypican Dally and Dally-like from the cell-surface. Biochemical work has demonstrated that a mammalian Notum homologue can similarly induce the release of several glypicans by gpi cleavage. We have identified multiple zebrafish notum homologues. We determined that Wnt/beta-catenin signaling regulates the expression of one of these homologues called notum3. Overexpression of Notum3 demonstrates that it can function as a potent inhibitor of Wnt/beta-catenin. Specifically, Notum3 antagonizes the effects of Wnt8 overexpression and can synergize with the well-established Wnt/beta-catenin inhibitor Dkk1 to suppress posterior body formation. We have created a stable transgenic line, Tg(hsp70:notum3), to allow inducible overexpression of Notum3. Ectopic expression of Notum3 induces stage-dependent perturbations of development consistent with Wnt/beta-catenin inhibition. In particular, we have shown through loss and gain of function studies that Notum3 functions in the patterning of the dorsal neural tube. We propose that Notum3 acts to restrict the spread of Wnts signaling from the roof plate. Our analysis of Notum3 overexpression phenotypes suggests that Notum3 has a limited set of targets. We have found no evidence that Notum3 inhibits the function of Glypican4 (Knypek) in Wnt/PCP signaling or the activity of other gpi anchored proteins. Together these data suggest that Notum3 functions as part of an autoregulatory loop by which Wnt signaling is restricted and that there is an unexplored specific requirement for glypicans for proper Wnt/beta-catenin activity during vertebrate development. 185 Posters 115 Signaling Role of WNT/-CATENIN signalling during segmentation of the presomitic mesoderm J. M. Topczewska, A. Lacrosse, G. Parker Flowers, J. Topczewski Department of Pediatrics, Northwestern University Feinberg School of Medicine, Children's Memorial Research Center, Chicago USA The segmentation clock controls the periodic formation of somites from the presomitic mesoderm (PSM); it involves Notch, Wnt and FGF signaling pathways. Nrarpa (Notch regulated ankyrin repeat protein a) is both a target and an inhibitor of Notch signaling. We propose that a fast turnover of Nrarpa protein limits the duration of the inhibitory effect and sustains the oscillation of Notch activity in the PSM. Furthermore, we provide evidence that Wnt/-catenin signaling positively regulates nrarpa transcription. By transiently decreasing Wnt activity, we established that Wnt determines the area of the PSM that is responsive to Notch signaling. In summary, canonical Wnt signaling is required to maintain the identity of PSM cells; Wnt initiates periodicity of Notch activity by controlling the expression of nrarpa, and permits cells to respond to the Notch oscillator. Therefore, Wnt/-catenin signaling underlies segmentation of the PSM in the zebrafish. Posters 186 Signaling 116 The signaling component Inka links cytoskeletal dynamics to pattern formation T. T. Luo1, S-K. Hong2, Y. Xu3, T. D. Sargent4 1,3,4 Program in Genomics of Differentiation, Lab of Molecular Genetics, National Institute of Child Health and Human Development; 2Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, U.S.A Complex and linked inductive interactions pattern the early embryo across the animal kingdom, including the zebrafish. At the end of gastrulation in vertebrates, the neural crest (NC) is induced by interactions among neuroectoderm, the epidermis and paraxial mesoderm at the neural plate border. This is accompanied by the expression of numerous transcription factors, among which is the transcriptional activator protein AP2a (TFAP2a). We and others have shown that TFAP2a is a critical element in the induction, migration and differentiation of cranial NC cells into skeletal and other tissues in the embryonic head. To further understand this function we carried out a microarray-based screen for TFAP2a-target genes expressed in NC, and identified several, including a novel adhesion molecule, the protocadherin PCNS, an unconventional myosin MyoX, and a novel protein we named Inka (formerly Inca). Inka proteins interact physically with p21-activated kinase 4 (PAK4), an effector of Rho-class of small GTPase, regulate cytoskeletal dynamics including stress fibers formation and microtubule modifications, subsequently change cell migration. These effects correlate with the inhibition of PAK4, in both kinase-dependent and kinase-independent manners. There are three inka genes in zebrafish: inka1a, inka1b and inka2. Each has a unique expression pattern. The inka1a gene is zygotically expressed predominantly in the early ectoderm and later in neural crest (NC), and loss of function by MO knockdowns showed that it is required for terminal differentiation of cranial NC into head cartilage in both fish and frog embryos. The inka1b gene is expressed maternvided into distinct populations that presage segment domains of the mature nephron. An examination of renal progenitors in RA-deficient embryos found that RA was requisite for specifying the rostral domain. When RA is absent, the IM renal progenitors adopt a caudal identity and the resulting nephron is comprised solely of distal segment fates. Conversely, in embryos treated with exogenous RA, the IM renal progenitors adopt a rostral identity and the resulting nephron is comprised entirely of proximal segment fates. To delineate the molecular pathways downstream of RA, we are performing morpholino-mediated knockdown of genes unique to the various renal progenitor groups. This strategy has discovered that the Iroquois transcription factor Irx3b is needed to confer the identity of the first distal tubule segment and prevent a corresponding expansion of proximal fates. Interestingly, this phenotype recapitulates a recently reported role for Irx3 in the amphibian pronephros, thus providing evidence that Irx3 function in nephrogenesis is conserved. Furthermore, embryos doubly-deficient for RA and Irx3b develop nephrons which express markers of the second distal segment. This suggests that in the absence of signals from RA and Irx3b, the default fate of renal progenitors is to acquire a distal segment identity. Taken together, our analysis suggests a model whereby the IM is initially patterned into two broad domains by RA, followed by sequential and dynamic refinement into two proximal and two distal tubule segments by later-acting transcription factors like Irx3b. These studies establish a powerful framework to assemble the molecular networks that drive nephron formation during vertebrate embryogenesis, and will likely provide useful insights into both normal and defective kidney development and disease. 193 Posters 123 Organogenesis Identification and functional analysis of the promoter region in the torafugu myosin heavy chain gene, MYHM5, involved in cardiac and superficial slow muscle specific expressions S. Kinoshita, Y. Sugano, Y. Ono and S. Watabe Faculty of Agricultural and Life Sciences, The University of Tokyo, Japan Myosin heavy chain gene (MYH) encodes a large subunit of myosin, the major contractile and structural muscular protein. In vertebrate genome, MYH forms multiple gene family, and tissuespecific and developmental stage-specific expressions of each MYH lead to the formation of different muscle-fiber types such as slow, fast and cardiac ones. However, the details of molecular mechanisms involved in such complex expression patterns of MYHs are still unknown. In this study, we examined transcriptional regulation in the expression of MYHM5 from torafugu Takifugu rubripes, having the smallest genome size among vertebrates, in zebrafish Danio rerio where various transgenic approaches are possible. MYHM5 is one of the torafugu MYHs identified from the genome database by in silico screening (Ikeda et al., 2007). In situ hybridization and RT-PCR previously showed its specific expression in torafugu embryos and adult slow and cardiac muscles. In this study, the GFP reporter vector carrying the -4kb sequence from the start codon of MYHM5 was constructed and injected into zebrafish embryos. The expression of the GFP was observed in cardiac and myotomal skeletal muscles of transgenic embryos at 1-2 days post fertilization. Myotomal skeletal muscle of zebrafish embryos consists of fast muscle, superficial slow muscle and medial fast fibers in addition to muscle pioneer cells. GFP-expressing cells were not stained with anti-engrailed antibody, suggesting that they are not medial fast or muscle pioneer cells. All GFP-expressing cells were stained with F59 antibody specific to slow muscle myosin but not with F310 antibody specific to fast muscle myosin, indicating that these correspond to superficial slow muscle fibers. In the differentiation of superficial slow muscle fibers, hedgehog signaling has been reported to be a key regulatory factor. The GFP expression by MYHM5 promoter was apparently reduced in embryos treated with cyclopamine, a hedgehog signaling inhibitor, as compared to that in the control. These results indicate that the -4kb sequence from the start codon of MYHM5 is sufficient for gene expression specific to superficial slow and cardiac muscles, and its regulatory activity is conserved among different fish species. Smyhc1 encodes an MYH isoform expressed in superficial slow muscle in zebrafish embryos (Elworthy et al., 2008). In this study, -5kb upstream sequence from the start codon of torafugu MYHM5 and zebrafish smyhc1 were compared by LAGAN program, yielding one conserved region of 62b. Then we created deletion mutants in the -4kb region of MYHM5 and examined their functions. Unexpectedly, the reporter construct lacking the conserved region induced GFP expression in slow and cardiac muscles with the same activity as that having the intact -4kb. On the other hand, the deletion of -4k~-2k, -4k~-1.5k and -4k~-1kb decreased progressively the rate of GFP expression, suggesting that not a single sequence but several regions distributed from -1kb to -4kb are involved in the transcriptional regulation of MYHM5. Posters 194 Organogenesis 124 Retinoic acid acts upstream of WT1A and FOXC1A to specify podocytes from the intermediate mesoderm A. Davidson, Z. Kostun, M. Grimaldi, R. Wingert, R. Selleck, L. O'Brien. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, USA The nephron is the functional unit of the kidney and filters the blood via a size-selective sieve that contains specialized epithelial cells called podocytes. The developmental pathways governing podocyte formation during renal development are poorly understood. Using zebrafish as a model, with its simple two-nephron pronephric kidney, we have identified a number of key developmental pathways that are involved in podocyte formation. Retinoic acid (RA) was found to be essential during a small window at the end of gastrulation for the induction of podocytes from the intermediate mesoderm. Expression of the transcription factor Wilms' Tumor Suppressor-1a (wt1a), implicated in podocyte differentiation, is dependent on RA signaling. Knockdown of wt1a led to an early reduction in podocyte progenitor number followed by a progressive decline between 24-48 hours post-fertilization. A similar phenotype was observed following knockdown of the forkhead transcription factor foxc1a, which is also dependent upon RA signaling for its expression in podocyte progenitors. Double wt1a/foxc1a knockdown led to a complete loss of podocyte specification suggesting that these transcription factors act in a partially redundant fashion, downstream of RA, to induce podocyte cell fate from the intermediate mesoderm. Taken together, these data provide a better understanding of how podocytes arise during kidney development and may provide insights into the molecular mechanisms that cause renal birth defects and disease. 195 Posters 125 Organogenesis Functional analysis of zebrafish connexin41.8 for pattern formation M. Watanabe1 and S. Kondo1,2 1 Graduate School of Frontier Biosciences, Osaka University, Japan; 2Graduate School of Science, Nagoya University, Japan One of the most beautiful features seen in nature is skin pattern of animals. We have proposed that pigment cells, melanophore and xanthophore behave like reaction-diffusion wave on fish body, consequently the pigment cells make stripe pattern. However molecular mechanism of the pattern formation remains unclear. leopard, a pigment pattern mutant of zebrafish is one of the good models for the pattern study. This mutant has spot pattern in spite of stripe pattern, and this phenotype is caused by the mutation on connexi41.8 gene. Connexin is a component of gap-junctions that mediate intercellular communication. Null mutant of this gene, namely allele leot1, shows clear spot pattern. Dominant negative mutations in this gene induce variations of pattern in zebrafish. Allele leotq270, which has I202F amino acid substitution, shows severer phenotype; smaller spots than that of allele leot1 and reduced nund analyzed the number and size of muscle fibers. As a result, our histological examinations revealed clear evidence of marked hyperplasia, but not hypertrophy, in the skeletal muscle of transgenic medaka. Each of the muscle fibers in transgenic fish was smaller than that in wild-type, resulting in no apparent alteration of the whole muscle mass. In conclusion, phenotypic analysis of the transgenic medaka revealed that exogenous expression of a dominant-negative form MSTN (C315Y-MSTN) induces hyperplasia of skeletal muscle, but not hypertrophy. Our results suggest that endogenous MSTN has an inhibitory effect on muscle cell proliferation in medaka. 199 Posters 129 Organogenesis Characterisation of neural stem cells in medaka fish midbrain A. Alunni, J-M Hermel, A. Heuze, F. Jamen, F. Bourrat, J-S. Joly U1126 INRA group, UPR2197 DEPSN, Institut Fessard, CNRS, Gif Sur Yvette, France Very few adult neural stem cells have been described in detail and their diversity thus remains undocumented. Adult neurogenesis in mammals is restricted to the forebrain. Neuronal production in teleost occurs throughout life-span in many regions of the brain. Whether nonmammalian progenitors have similar characteristics to the mammalian neural stem cells remains unknown. To this aim, we use an ideal model found in the midbrain of the medaka fish Oryzias latipes, the optic tectum (OT). We previously demonstratedthat this cortical structure of the dorsal midbrain grows by successive additions of open rings of progenitor cells originating from a population of actively dividing progenitors located at the periphery of the organ. To characterize further this region, we performed two long pulses of nucleoside analogs (IdU and CldU) at different intervals in juveniles and adult fish. This strategy lead to the identification of a subset of long-term label retaining cells that are likely to be the OT stem cells. They are located in the marginalmost regions of the proliferation zones (PZ) of the adult brain but are not detected in the juvenile brain. We also analyzed the expression patterns of the pluripotency-associated markers Sox2, Musashi 1 and Bmi1. These genes are expressed in the PZ of the adult OT, indicating that the core machinery for neural pluripotency is conserved throughout vertebrate evolution. Moreover, the adult gene expression patterns differ considerably from those at embryonic stages. This provides evidence that a molecular mechanism specifies an adult stem cell niche. Fish tectum thus represents a new powerful model to study the diversity of neural stem cells. Posters 200 Organogenesis 130 The molecular structures and expression patterns of zebrafish troponin I genes CY. Fu, H-C. Lee and H-J. Tsai Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan Troponin I (TnnI), a constituent of the troponin complex located on the thin filament, provides a calciumsensitive switch for striated muscle contraction. Cardiac TnnI is, therefore, a highly sensitive and specific marker of myocardial injury in acute coronary syndromes. The TnnI gene, which has been identified in birds and mammals, encodes the isoforms expressed in cardiac muscle, fast skeletal muscle and slow skeletal muscle. However, very little is known about the TnnI gene in lower vertebrates. Here, we cloned and characterized the molecular structures and expression patterns of three types of zebrafish tnni genes: tnni1, tnni2 and tnni-HC (heart and craniofacial). Based on the unrooted radial gene tree analysis of the TnnI gene among vertebrates, the zebrafish TnnI1 and TnnI2 we cloned were homologous of the slow muscle TnnI1 and fast muscle TnnI2 of other vertebrates, respectively. In addition, reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization demonstrated that zebrafish tnni1 and tnni2 transcripts were not detectable in the somites until 16 h post-fertilization (hpf), after which they were identified as slow- and fast-muscle-specific, respectively. Interestingly, tnni-HC, a novel tnni isoform of zebrafish was expressed exclusively in heart during early cardiogenesis at 16 hpf, but then extended its expression in craniofacial muscle after 48 hpf. Thus, using zebrafish as our system model, it is suggested that the results, as noted above, may provide more insight into the molecular structure and expression patterns of the lower vertebrate TnnI gene. 201 Posters 131 Organogenesis Preliminary study on the role of sialidase neu3.1 and neu4 in zebrafish development D. Gotti, M. Manzoni, G. Paganini, *N. Tiso, G. Borsani, A. Preti, E. Monti, R. Bresciani Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy; *Department of Biology, University of Padova, Italy. Sialidases or neuraminidases are a family of enzymes that catalyze the removal of terminally linked sialic acid residues from glycoproteins and glycolipids. Thus, sialidases represent the initial and essential step for the degradation of sialoglycoconjugates. Four different mammalian sialidases have been described: lysosomal (NEU1), cytoplasmic (NEU2), plasma membraneassociated (NEU3) and intracellular membrane-associated (NEU4) sialidase. Mammalian sialidases are implicated in lysosomal catabolism, as well as in regulation of important cellular events including cell differentiation, growth, adhesion, and apoptosis. We recently cloned and characterized seven different genes of zebrafish (Danio rerio) related to human sialidases. Two of them, neu1 and neu4, represent the orthologs of the mammalian sialidases NEU1 and NEU4, respectively. The remaining genes, i.e. neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5, are all more closely related to mammalian sialidase NEU3. Here we focus on neu3.1 and neu4. RT-PCR and in situ hybridization experiments demonstrate that neu3.1 and neu4 are expressed already at early development stages and show temporal and spatially localized expression areas corresponding to gut and lens, respectively. In order to test the role of neu3.1 and neu4 in zebrafish development we performed gene inactivation experiments using splice-inhibiting Morpholinos (MO). neu3.1 knock-down causes significant phenotypic alteration. Morphants show head and eye development retardation, trunk and tail abnormal curvature and defective somites patterning. We now plan to characterize the morphant phenotype observed by in situ hybridization experiments using different markers. Knock-down of neu4 by injecting 1 ng of MO results in a spectrum of phenotypes ranging from brain edema, heart edema, shortening of the main body axis, misshaped somites. Moreover, at 24 hpf, a significant impairment of the region where digestive system will develop is observed. Higher injection amounts gave rise to much stronger phenotypic alterations but also caused severe mortality at 24 hpf, suggesting that a complete depletion of neu4 is lethal. We are currently characterizing the phenotypes observed by whole-mount in situ hybridization of injected embryos for the expression of different marker genes. mRNA rescue experiments are presently ongoing in order to confirm the specificity of the phenotypic alterations observed. Posters 202 Organogenesis 132 Characterization of vsp, an interactor of arms/kidins220 expresses in the ventral region of zebrafish eye M. Andreazzoli1, G. Gestri2, B. D'Orsi1, M. Barilari1, A. Iervolino1, E. Landi1, M. Tsang3, T. Kudoh4, G. Barsacchi1, S.W. Wilson2, I.B. Dawid5, L. Dente1 1 Laboratorio di Biologia Cellulare e dello Sviluppo, Universita' di Pisa, Italy; 2Department of Anatomy and Developmental Biology University College London, UK; 3Department of Microbiology and Molecular Genetics, University of Pittsburgh, USA; 4School of Biosciences, University of Exeter, UK; 5Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, USA Looking for genes involved in eye development, we selected vsp in the course of a large scale in situ screening. vsp encodes a protein containing two PDZ domains and a RING-finger domain and displays specific expression in the developing eye. vsp expression initiate in the ventral region of the optic primordia at the 10-somite stage. As eye morphogenesis proceeds, vsp expression becomes restricted to the ventral retina and to the initial tract of the optic stalk. Outside the optic regions, vsp is expressed in rhombomere 1 and in specific areas of the trunk possibly corresponding to ganglia. We recently found that vsp specifically interacts with the protein ARMS/kidins220, a target of neurotrophin and ephrins. These data, together with the absence of vsp expression in the ventral eye region of noi mutants, suggest that this gene might be involved in controlling axon outgrowth of retinal ganglion cells. 203 Posters 133 Unc119b Regulates Ciliogenesis and Left-Right Axis Determination D. V. French and D. B. Pilgrim Dept. of Biological Sciences, University of Alberta, Edmonton, Canada Organogenesis The Unc-119 protein is required for normal nervous system development in metazoans, although its molecular role remains unclear. C. elegans mutants in unc-119 have abnormalities of dye uptake and the distal tips of cilia are not formed properly. In mammalian systems, as well as work done in C. elegans in our lab, Unc-119 protein family members have been shown to interact Arl family members, which are involved in ciliogenesis. We therefore tested whether Unc119b has a role in ciliogenesis in zebrafish. Morpholino (MO)-mediated knockdown of Unc119b produces animals with a curved body, a phenotype reminiscent to other cilia mutants. Unc119b also displays a cilia phenotype in the pronephric duct similar to an arl13b mutant, and genetic epistasis experiments indicate that unc119b acts either upstream or parallel of arl13b. In addition, Unc119b morphant hearts are unlooped or displayed randomized looping indicating a role in the establishment of left-right asymmetry. The pineal gland, as marked by crx, fails to migrate in Unc119b morphants, which is a left-right dependent event. These results are consistent with Unc119b acting as a ciliogenesis gene. Analysis of cilia at Kupffer's vesicle demonstrates shorter and fewer cilia in Unc119b morphants. In addition to defects in ciliogenesis, Unc119b have hydocephalus and an open neural tube phenotype. Injection of Rhodamine-dextran into the hindbrain reveals that the midbrainhindbrain boundary does not close in Unc119b morphants. Further analysis reveals that atonal homologue1a (atoh1a) is excluded from the midbrain-hindbrain boundary, reminiscent of Fgf mutants. This result coupled with the pineal gland and Kupffer's vesicle phenotypes suggests Unc119b interacts with the Fgf signalling pathway, a hypothesis we are currently testing. Altogether, this work provides a novel role for Unc119b in ciliogenesis and determination of left-right asymmetry. Posters 204 Organogenesis 134 Modelling arrhythmogenic right ventricular cardiomyopathy in zebrafish M. A. Moriarty1, E. D. Martin1, L. Byrnes2, M. Grealy1 1 Department of Pharmacology and Therapeutics, National University of Ireland, Galway, Ireland; 2 Department of Biochemistry, National University of Ireland, Galway, Ireland Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a congenital heart disease associated with ventricular arrhythmias and sudden death in young people. Several proteins have been implicated in ARVC including the desmosomal armadillo proteins plakophilin 2 and plakoglobin. These are integral to maintaining tissue integrity and strength, and are particularly important in tissues which undergo routine mechanical stress such as the skin and heart. Loss of plakophilin 2 and plakoglobin in mice leads to cardiac instability and embryonic lethality. We have previously shown that loss of plakoglobin in zebrafish is a model for ARVC (Martin et al., 2009). Knockdown of zebrafish plakophilin 2 or plakoglobin by morpholino antisense technology has resulted in morphant embryos with specific cardiovascular defects - pericardial oedema, enlarged hearts, reduced heart beat. This is accompanied by stage-dependent anterior neural and tail defects. Structural analysis of plakoglobin morphants using transmission electron microscopy (TEM) showed altered adhesion and cellular morphology in their cardiac valves along with reduced numbers of junctions in the heart as a whole. Initial TEM studies in plakophilin 2 morphants also show a decrease in junction numbers along with altered junctional morphology. These data show the importance of desmosomal armadillo proteins in zebrafish cardiac development and the usefulness of zebrafish as a model for ARVC. Martin, E.D., Moriarty, M.A., Byrnes, L., Grealy, M. (2009) `Plakoglobin has both structural and signaling roles in zebrafish development.' Developmental Biology 327 205 Posters 135 Organogenesis The Transcription Factor Six1a Plays an Essential Role in the Craniofacial Myogenesis of Zebrafish CY. Lin, W-T. Chen, H-C. Lee, P-H. Yang, H-J. Yang and H-J. Tsai Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan Transcription factor Six1a plays important roles in morphogenesis, organogenesis, and cell differentiation. However, the role of Six1a during zebrafish cranial muscle development is still unclear. Here, we demonstrated that Six1a was required for sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscle development. Although Six1a was also necessary for myod and myogenin expression in head muscles, it did not affect myf5 expression in cranial muscles that originate from head mesoderm. Overexpression of myod enabled embryos to rescue all the defects in cranial muscles induced by injection of six1a-morpholino (MO), suggesting that myod is directly downstream of six1a in controlling craniofacial myogenesis. However, overexpression of six1a was unable to rescue arch muscle defects in the tbx1- and myf5-morphants, suggesting that six1a is only involved in myogenic maintenance, not its initiation, during arch muscle myogenesis. Although the craniofacial muscle defects caused by pax3-MO phenocopied those induced by six1a-MO, injection of six1a, myod or myf5 mRNA did not rescue the cranial muscle defects in pax3 morphants, suggesting that six1a and pax3 do not function in the same regulatory network. Therefore, we proposed four putative regulatory pathways to understand how six1a distinctly interacts with either myf5 or myod during zebrafish craniofacial muscle development. Posters 206 Organogenesis 136 Zebrafish as a model to study of thyroid development E. Maquet, R. Opitz and S. Costagliola IRIBHM, ULB, Brussels, Belgium The thyroid is an endocrine gland composed of follicular cells that develops from a primordium bud derived from the ventral floor of the anterior pharyngeal endoderm. After a migration process, the primordium loses contact with the pharynx, relocates in the hypopharyngeal mesenchyme and adopts its final bilobed shape in the neck area of mice and man or forms an elongated strand of tissue along the ventral aorta in zebrafish. At this stage of development, the thyroid becomes fully differentiated, expresses thyroid specific genes and produces thyroid hormones. In human, congenital hypothyroidism (CH) is a frequent congenital disease. In 85% of the cases, CH is the result of thyroid dysgenesis which comprises agenesis, hypoplasia and/or ectopy of this gland in newborn humans. To date, only a minor proportion of familial CH cases have been associated with mutations located in transcription factors known to be involved in thyroid development, such as NKX-2.1, PAX8 or FOXE1. This suggests that other, still unknown genes might play an important role during thyroid organogenesis. To identify novel genes involved in the regulation of thyroid development and differentiation, we combined LongSAGE with Solexa deep sequencing to analyze the transcriptome of thyroid primordium, surrounding mesenchyme and pharyngeal endoderm in E11.5 mouse embryos. This approach proved successful to quantitatively and qualitatively characterize gene expression at an early stage of thyroid development. Subsequent application of various bioinformatical approaches yielded more than 1300 candidates genes potentially involved in early thyroid developmesubunit, atp1a1, is required for zebrafish brain ventricle inflation (Lowery and Sive 2005). Here we demonstrate that Atp1a1 is necessary for epithelial integrity early in development, and for brain ventricle inflation after neurulation. In addition, we show that the beta subunit atp1b3a is strongly expressed in neuroepithelium surrounding the forebrain and midbrain ventricles, and that it synergizes with Atp1a1 during ventricle inflation. Dose response assays show that forebrain brain ventricle area is highly sensitive to the amount of functional Atp1a1. Preliminary data suggest that the gamma subunits, FXYD1, 5 and 6 are essential for brain ventricle inflation. In sum, these data show that the Na+K+ATPase plays different roles before and after neurulation, and that the correct balance of subunits is necessary to direct brain ventricle size, results with implications for the understanding the etiology of hydrocephalus. 213 Posters 143 Organogenesis X-ray imaging of 3D structures of teeth and skeletal elements in zebrafish and medaka, and their motions during respiration and feeding K. Hatta1, K. Fujita1, T. Yamamoto1, K. Uesugi2, S. Nakayama1, P.K. Moly1 1 Grad. Sch. of Life Sci. Univ. of Hyogo, 2JASRI/SPring-8 Japan X-ray micro tomography at SPring-8 synchrotron was used to analyze three-dimensional structures of skeletal elements in adult and larval zebrafish and medaka. Adult zebrafish has relatively large ~24 pharyngeal teeth only at the ventral side. Right and left ventral teeth were found to face each other towards midline. In contrast, adult medaka has more than 1000 pharyngeal teeth distributed both at ventral and dorsal sides. Dorsal teeth and ventral teeth are facing each other. Medaka also has teeth on upper and lower jaws. We then performed live X-ray imaging of the skeletal elements to study their motions during respiration and feeding. Each skeletal element was found to move periodically with different phase during respiration. Functions of pharyngeal teeth in fish have been poorly understood. We hope to elucidate their roles by comparing these two teleosts established as model vertebrates that appear to have teeth with different anatomical characteristics. Posters 214 Organogenesis 144 Gene expression profiling of zebrafish embryonic hearts identifies molecular networks involved in early cardiac morphogenesis F. Priller1, M.R. Huska2, M. Andrade2, S. Abdelilah-Seyfried1 1 Cardiovascular Research, Max Delbrueck Center, Berlin, Germany; 2Computational Biology, Max Delbrueck Center, Berlin, Germany The multi-chambered hearts of vertebrates initially arise from two lateral populations of cardiac precursor cells (CPCs), which are subject to morphogenetic rearrangements in the course of cardiogenesis. In zebrafish, heart tube formation involves the asymmetric involution of the right posterior heart field and extensive migratory processes within distal portions of the heart field. Although a couple of signaling pathways have been implicated in these processes based on loss- or gain-of-function phenotypic analyses, the exact genetic and cellular mechanisms that underlie CPC behavior remain to be unraveled. Here, we make use of whole transcriptome gene expression profiling on highly purified embryonic heart tissue to identify novel and key regulators of heart tube formation. Out of all annotated zebrafish genes expressed in the heart, we find hundreds that are associated with particular morphogenetic or physiological processes. These include extra- intracellular signal transduction, specification or patterning of the heart, ion homeostasis and myogenesis. Further, we are particularly interested in genes that regulate migratory behavior, cell polarity or adhesion, as these genes are likely to be involved in morphogenetic processes. Comparison of myocardial gene expression to a reference sample of whole embryos lacking heart tissue at the stage of cardiac field involution reveals that about one quarter of all genes that fall within those categories are specifically up-regulated in the heart and likely contribute to its unique features. Based on our findings, we have now started to verify prime candidates and to elucidate their function in cardiogenesis. In addition, we now employ transcriptional profiling to assess the impact of known signaling pathways on CPC gene expression. Thereby we hope to elucidate their specific requirements during heart development. 215 Posters 145 Organogenesis Characterization and functional analysis of gluconeogenesis during zebrafish development P. Gut, M. Porte, D. Stainier UCSF, San Francisco, USA The steadily rising obesity prevalence in western civilization has lead to a pandemic increase in insulin resistance as part of the Metabolic Syndrome and Type 2 Diabetes Mellitus (T2DM). Insulin resistance in hepatocytes induces elevated glucose output from the liver and contributes to hyperglycemia in T2DM. In order to characterize zebrafish as a model for studies on the regulation of glucose metabolism and insulin resistance, we carried out in-situ hybridization expression studies for three key enzymes of the gluconeogenic pathway: Glucose-6-phospatase (encoded by g6pc), fructose1,6-bisphosphatase (encoded by fbp1) and Phosphoenolpyruvate-carboxykinase (encoded by pck1). In the early embryo pck1 is strongly expressed in the yolk syncytial layer. Considering the higher energy density of lipids compared to glycogen, this early pck1 expression suggests a conversion from abundant yolk lipids to glucose "on the spot" prior to the functional differentiation of gluconeogenic organs. At 48 hpf we detect expression of fbp1 and g6pc in the developing liver whereas pck1 is initiated later in liver and intestine, between 72 and 96 hpf. In addition to the onset of pck1, g6pc and fbp1 show a strong increase around this time point when larvae have hatched and start to swim. Energy demand and lactate production of the swimming fish is likely to contribute to the logic of this time course of gene expression. Using pharmacological and genetic studies we want to test this hypothesis and gain insights into the regulation of glucose production. To study the requirement of gluconeogenesis for the developing embryo, we are conducting Morpholino-mediated gene knock down analyses targeted against g6pc and pck1. Glucose6-phosphatase functions at the end point of the gluconeogenic program where it is important for the release of glucose into the blood stream. Preliminary results indicate a largely normal development, but decreased liver size in injected embryos compared to controls. Pck1 is the most upstream enzyme of the pathway that converts oxaloacetate to phosphoenolpyruvate. Since oxaloacetate is derived from lactate, pyruvate or glucogenic amino acids, we expect to see a phenotype that is specific to impaired utilization of those metabolites, but not of the entire gluconeogenic program. Posters 216 Organogenesis 146 Defects in ribosome biogenesis underpin the morphological characteristics of a zebrafish development mutant, setebos, with eye, intestinal and craniofacial abnormalities A.P. Badrock1, H. Verkade1,2, A.J. Trotter1, Y. Rifat1, A.Y.N. Ng1, E. Ober2, H.A. Field2, R.D. Hannan3, G. J. Lieschke4, D. Y. R. Stainier2 and J. K. Heath1 1 Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Australia, 2Department of Biochemistry and Biophysics, University of California, San Francisco, U.S.A, 3Research Division, Peter MacCallum Cancer Centre, East Melbourne, Australia, 4Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, Australia Ribosome biogenesis is the most energy consuming process in growing cells. As yet, the mechanisms that link ribosome biogenesis to the regulation of cell growth and proliferation are incompletely understood. In mammals, disruption of ribosome biogenesis leads to `nucleolar stress' resulting in apoptosis or cell cycle arrest through activation of the tumour suppressor protein, p53. The ribosomal protein L11 has been shown to be a key player in this process, highlighting the need to understand extra-ribosomal functions of ribosomal proteins. seteboss453 is a zebrafish mutant that was identified in the Liverplus screen designed to identify genes involved in endodermal organ morphogenesis. At 5 days post fertilisation the intestinal epithelium in setebos is hypoplastic compared to wildtype embryos. The liver, pancreas and eye are also markedly under-developed and craniofacial cartilage development is severely disrupted. The responsible mutation in setebos is a premature stop codon in nucleolar protein 8 (nol8), a gene that has been shown to be required for ribosomal RNA (rRNA) processing in yeast. We have employed metabolic labeling experiments alongside Northern blot and bioanalyzer analyses to demonstrate defective rRNA processing in setebos embryos. This disruption culminates in reduced levels of mature 28S rRNA species. Thus, Nol8 function is conserved from yeast to vertebrates. In setebos embryos, the mRNA level of the N-terminally truncated isoform of p53, 113p53, is specifically and markedly upregulated in response to impaired rRNA processing, and the mRNA levels of the cell cycle arrest genes, p21 and cyclinG1 are also elevated. We are using setebos embryos to dissect the role of p53 and its various isoforms in the cellular response to impaired ribosome biogenesis, and to explore the consequences of compromising this important cellular process on tumour formation. 217 Posters 147 Organogenesis Nkcc1/Slc12a2 is required for the regulation of endolymph in the otic vesicle and volume of the swim bladder in the zebrafish larva T. T. Whitfield and L. Abbas MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, UK Endolymph is the specialised extracellular fluid present inside the inner ear. In mammals, disruptions to endolymph homeostasis can result in either collapse or distension of the endolymphatic compartment in the cochlea, and concomitant hearing loss. The zebrafish little ears (lte) mutant shows a collapse of the otic vesicle in the larva, apparently due to a loss of endolymphatic fluid in the ear, together with an over-inflation of the swim bladder. Mutant larvae display signs of abnormal vestibular function by circling and swimming upside down. The two available alleles of lte are homozygous lethal: mutant larvae fail to thrive beyond 6 days post fertilisation. Patterning of the otic vesicle is apparently normal. However, expression of several genes thought to play a role in endolymph production is downregulated, including the sodium-potassium-chloride cotransporter gene nkcc1 (slc12a2) and several Na+/K+-ATPase channel subunit genes. We have cloned the lte mutations and show that they correspond to lesions in nkcc1. Each allele has a point mutation that disrupts splicing, leading to frame shifts in the coding region, and predicting the generation of truncated products. Endolymph collapse in the lte/nkcc1 mutant shows distinct parallels to that seen in mouse Nkcc1 mutants, validating the zebrafish as a model for the study of endolymph disorders. The collapse in ear volume can be ameliorated in the to27d allele by injection of a morpholino to block splicing at the new site, forcing use of the original site. This exemplifies the use of morpholinos as potential therapeutic agents for genetic disease. Posters 218 Organogenesis 148 Cellular and genetic interactions underlying choroid fissure formation and closure G. Gestri and S. Wilson Anatomy, UCL, London, UK The choroid fissure is a transient opening on the ventral side of the optic cup through which blood vessels enter and retinal axons leave the developing eye. Failure of the choroid fissure to close results in ocular colobomas, a family of common ocular pathogeneses that can cause severe visual impairment. Despite this fact, virtually nothing is known about the genetic mechanisms and cell movements that underlie choroid fissure closure. Recent studies by us and others indicate that mesenchymal cells outside the retina play a critical role in choroid fissure closure. Although the importance of these periocular mesenchymal cells (POM) is established, we do not know how they function. We hypothesize that there is signaling between these cells and the retina to coordinate the timing of closure. In our ongoing research we are exploring the identity of the POM cells required for choroid fissure fusion, determining when and how they function and elucidating the molecular pathways that mediate fusion. We are collaborating with the laboratories of William Harris and Brian Link. 219 Posters 149 Organogenesis Soul-2, a heme binding protein-coding gene in kidney development F. Langellotto1,2,, A.E. Fortunato1,3,, L. Castaldo2, P. Sordino1 1 Lab. of Cellular and Developmental Biology, Stazione Zoologica Anton Dohrn, Naples, Italy; 2 Dep. of Biological Functions, Structures and Technology, Faculty of Veterinary Medicine, University of Naples Federico II, Italy; 3Dep. of Cellular and Developmental Biology, University of Palermo, Italy Besides iron is a crucial element for body homeostasis, being mainly incorporated in heme and acting as cofactor in many proteins and enzymes, our knowledge of iron genetics during embryonic development is still fragmentary. During kidney differentiation, abnormal iron metabolism contributes to the development of anemia in chronic kidney disease patients. We are studying the SOUL/p22HBP family of heme binding proteins, generic tetrapyrrole-binding factors with high affinity for porphyrins and heme. SOUL proteins may act as cytosolic buffer against cell intoxication by iron products, or promote necrotic cell death. We identified four zebrafish Soul orthologs, as candidate factors for heme biosynthesis and iron homeostasis. We focused our attention on soul2, a gene that is expressed in collective ducts and distal segment of the pronephric ducts from eighteen somites stage until 96 hours post fertilization (hpf). Preliminary results by morpholino-driven knockdown support a dual role in patterning and differentiation during pronephric duct development. Kidney of soul2 morphant embryos displays thickening of the distal tubules, as shown by histology and in vivo imaging, as well as altered expression patterns of regionalization and differentiation markers. We are further characterizing additional phenotypic aspects, including a mechanistic link with heme oxygenase 1 (HO-1), a regulator of sodium uptake in the kidney and a rate-limiting enzyme in the degradation of heme, and with Nitric Oxide (NO) signaling, which is required for proper HO-1 activity. Posters 220 Organogenesis 150 Uncovering new genes involved in zebrafish thyroid development I. Porreca1,2, H. Fagman2, M. De Felice1,2, P. Sordino1, R. Di Lauro3 1 Lab. of Cellular and Developmental Biology, Stazione Zoologica Anton Dohrn, Naples, Italy; 2 Biogem, Institute of Genetic Research "Gaetano Salvatore", Ariano Irpino, Italy; 3Dep. of Biology and Cellular and Molecular Pathology, University of Naples Federico II, Italy The thyroid is an endocrine gland that produces the thyroid hormones involved in many functions in vertebrate and invertebrate organisms. Thyroid organogenesis is a very complex developmental process that, through the combination of migration of thyroid precursor cells, their organization into follicular structures and functional differentiation, leads to the formation of an organ able to produce specific hormones. In mammals, alteration of these mechanisms might be responsible of congenital hypothyroidism (CH), the most frequent inherited endocrine disease. The genetic basis of CH is known only in few cases. Thyroid formation in fish is comparable to mammals at both ontogenetic and molecular levels: the thyroid develops from the endodermal tissue, at the midline of the pharyngeal floor, and its formation is subdivided into few successive steps. The genetic programme involved in thyroid differentiation, including the activity of transcription factors such as Nkx2.1a, Pax8 and Hhex, is conserved with respect to expression patterns and functions. To gain further insights on the genetic machinery implicated in normal thyroid development, and therefore in CH disease, an LMC (Laser Capture Microdissection)-based oligonucleotide microarray analysis was performed in mouse to identify genes differentially expressed during the gland organogenesis. This approach highlighted a list of genes that are enriched in mouse E10.5 thyroid. We are now using the zebrafish as a simple model to perform functional analysis on the fish counterparts of these genes. Surfing the zebrafish genome for the homologs of 74 murine genes, we found 89 orthologs/paralogs of 56 mouse counterparts, while 18 mouse genes did not show zebrafish orthologous matches. To date, we have analyzed the expression patterns of 20 genes, finding new factors that are expressed in the developing thyroid gland of zebrafish. In addition, some genes show expression in the regions surrounding the gland primordium raising the possibility of contamination from these tissues during the LMC procedure. One of the genes expressed in zebrafish thyroid primordium is an anti-apoptotic factor. We found that this gene is expressed in thyroid from 30hpf. Interestingly its expression disappears early in both Nkx2.1a and Hhex morphant embryos when the thyroid primordium is still present. These results lend support to a hypothetical implication of anti-apoptotic processes in the conservation of thyroid structure. In the future we will advance in the characterization of the transcriptional profiles of zebrafish orthologues, with the aim to provide new hypotheses on the genetic and cellular bases of specification and differentiation of thyroid follicles. In this frame, the role of thyroid-expressed genes will be addressed by loss-of-function approaches, whereas knock-down embryos will be characterized by WMIHC (Tg, T4), WMISH (hhex, nk2.1a, pax8, pax2.1, tg) and TUNEL in wildtype, mutant (no isthmus, hands off) and transgenic (Tg(nk2.1a:YFP) backgrounds. 221 Posters 151 Organogenesis Sec24d transports extracellular matrix proteins during zebrafish skeletal morphogenesis E. W. Knapik1, S. Sarmah1, A. Barrallo-Gimeno2, J. Topczewski3, L. Solnica-Krezel3 1 Division of Genetic Medicine, Vanderbilt University Medical Center, Nashville, USA; 2 Developmental Biology, Institute Biology I, University of Freiburg, Germany; 3Department of Biological Sciences, Vanderbilt University, Nashville, USA, Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Cargo binding mechanisms have been studied in vitro by biochemical and cellular biology approaches using model proteins, however, the cargo specificity and the role of individual transport adaptors during vertebrate development are largely unknown. Cargo adaptor Sec24D is an integral component of the COPII complexes. We have positionaly cloned zebrafish mutation bulldog and shown that Sec24D is essential for secretion of cartilage matrix by mature chondrocytes, whereas transport of extracellular matrix proteins critical for migration of craniofacial primordia and pre-chondrogenic condensations proceeds normally. Consequently, chondroblasts fail to change shape and intercalate, thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. We demonstrate that the arrest in cartilage development stems from matrix deficits, and not a lack of membrane bound receptors since b1-integrin and cell adhesion molecules are transported in Sec24D-independent fashion. Furthermore, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes visualized by electron microscopy and NBD6ceramide staining appear functional. The backlog of proteins in the ER leads to upregulation of BiP and activation of the ER stress response machinery. We found that knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than bulldog/sec24d, indicating that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. This study presents the first developmental prospective on the Sec24D cargo specificity during development. Our findings establish Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects. Posters 222 Neurobiology I: Patterning 152 Modulators of calcium signaling induce developmental brain defects and behavioral changes R. Creton1, J. A. Kreiling1, and R. M. Colwill2 Brown University, 1MCB Department, 2Psychology Department, Providence, Rhode Island, USA Pharmaceuticals that modulate calcium signaling are successfully used for treating high blood pressure, heart arrhythmias, angina pectoris, and migraine. However, along with their widespread use, comes an increased risk for exposure during embryonic development. Human embryos may inadvertently be exposed by maternal use of medicine during early pregnancy or by trace concentrations of pharmaceuticals in the environment and drinking water. These exposures are troubling, as studies in animal model systems have shown that subtle calcium manipulations during embryonic development can induce specific brain defects. The potential risk for human brain development is difficult to evaluate because of the large number of pharmaceuticals that can affect calcium signaling either directly or indirectly, a lack of information on the sensitive developmental times, and the potentially pleiotropic effects on brain development and behavior. We use zebrafish as a model system to examine how modulators of calcium signaling affect brain development and behavior. We found that zebrafish embryos are sensitive to modulation of calcium signaling during gastrulation. Exposures during this time affect the development of laterality in the brain and asymmetry in behavior. The underlying calcium-sensitive mechanisms include convergence extension during gastrulation and the formation of Kupffer's vesicle. Further information on the developmental mechanisms, windows of sensitivity, and dose-response relationship is essential for risk assessment of pharmaceuticals that modulate calcium signaling. 223 Posters 153 Neurobiology I: Patterning Hypocretin interacts with melatonin in regulating sleep in zebrafish L.A. Appelbaum1, G.W. Wang2, G.M. Maro3, W.M. Marin1, T.Y. Yokogawa1, K.K. Kawakami4, E.M. Mignot1, P.M. Mourrain1 1 Department of Psychiatry and Behavioral Sciences, Stanford University, Palo Alto, USA; 2 Molecular and Cellular Physiology, Stanford University, Palo Alto, USA; 3Biological Sciences, Stanford University, Palo Alto, USA; 4Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Japan In mammals, hypocretin/orexin (HCRT) is strongly wake-promoting and HCRT deficiency causes narcolepsy. This indicates that a major function of the HCRT system is to consolidate wakefulness. In addition to fragmented wake however, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in zebrafish HCRT receptor mutant (hcrtr-/-). This suggests that HCRT regulates not only wake but also sleep consolidation, and that the later phenotype is conserved in zebrafish. To investigate potential mediators of sleep consolidation in zebrafish, we characterized HCRT neurons in this species. We found that, like in mammals, zebrafish HCRT neurons express vesicular glutamate transporter vglut2 but not the glutamic acid decarboxylase gad67 indicating conservation of the glutamatergic phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed clear parallel with established mammalian HCRT neuroanatomy. We also found significant projections to the pineal gland, a brain structure expressing hcrtr mRNA. As pineal melatonin is a major sleep promoting hormone in diurnal zebrafish and is released in the dark, we further characterized melatonin response in hcrtr-/- larvae and adults. Interestingly, a low dose of melatonin increases sleep amount and sleep consolidation in hcrtr-/- fish but not in their wildtype siblingsy EE2 exposure during early life. Whilst unexposed males showed no preference in courting exposed or unexposed females, the unexposed females had significantly higher reproductive success compared with exposed females, and they were dominant in the colonies in most cases. Unexposed females spawned fewer eggs when courted by males exposed to the 10ng/L EE2, compared with those exposed to 3ng/L EE2. These results strongly suggest that exposure to EE2 during early life subsequently impacts on behaviour and social interactions in spawning groups and alters reproductive capability and outcome. Posters 228 Neurobiology I: Patterning 158 The neurogenic niches in the adult zebrafish telencephalon: proliferation characteristics, cellular composition and regulation by fgf and bmp signaling J. Ganz, J. Kaslin, H. Grandel, D. Freudenreich, S. Hochmann, M. Brand Biotechnology Center and Center for Regenerative Therapies Dresden, University of Technology Dresden, Germany In contrast to mammals, adult neurogenesis is a widespread lifelong process in the central nervous system of non-mammalian vertebrates (Kaslin et al., 2008). We have previously shown that distinct neurogenic niches are present in different brain parts along the whole rostral-caudal axis of the adult zebrafish brain (Grandel et al., 2006, Kaslin et al., 2009). These observations raise several key questions: How are the different neurogenic niches organized, and which cell types and signals regulate neurogenesis in the different neurogenic niches? By analyzing the cellular and molecular composition of neurogenic niches in the adult zebrafish telencephalon we show that the four different niches in the dorsal and ventral telencephalon display distinct proliferation characteristics, cellular arrangements and glia domains. We demonstrate that, in contrast to the common view, a major part of the progenitor pool in the zebrafish does not display typical radial glia characteristics. Furthermore, by comparing the neurogenic niches between mouse and zebrafish we reveal that the niches in the adult zebrafish telencephalon display differences for example in the spatial arrangement of transit-amplifying cells and the migration characteristics of the neuroblasts in the ventral telencephalon. However, the niches in zebrafish are similar to mouse in their molecular composition. Using gain and loss of function approaches, we demonstrate important functions of the Fgf and Bmp signaling pathways in regulating adult neurogenic niche activity. Surprisingly, we find distinct requirements for Fgf and Bmp signaling, depending on the individual telencephalic proliferation zone considered. These findings highlight the importance of studying adult neural stem cell regulation in the context of their adult anatomical setting in-vivo. Kaslin J, Ganz J, Geffarth M, Grandel H, Hans S, Brand M. (2009). Stem cells in the adult zebrafish cerebellum: initiation and maintenance of a novel stem cell niche, Journal of Neuroscience, in press Kaslin J, Ganz J, Brand M. (2008). Proliferation, neurogenesis and regeneration in the nonmammalian vertebrate brain. Philos Trans R Soc Lond B Biol Sci. 29;363 (1489):101-122. Grandel H, Kaslin J, Ganz J, Wenzel I, Brand M. (2006). Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate. Dev Biol. 295:26377. 229 Posters 159 Neurobiology I: Patterning Netrin Signaling is Required for Development of an Identified Zebrafish Motoneuron L.A. Hale, J.S. Eisen Institute of Neuroscience, University of Oregon, Eugene, USA CaP and VaP, identified zebrafish motoneurons, initially form an equivalence pair but later VaP typically dies. CaPs are present in all spinal hemisegments and extend long axons that innervate ventral muscle. VaPs are present in about half of the spinal hemisegments and extend short axons that stop at the muscle pioneers (MPs), identified slow muscle fibers that divide dorsal and ventral muscle. Previous work showed that both CaP and the MPs are required for VaP formation. Here we identify Netrin 1a (Ntn1a) as an MP-derived molecular signal necessary for VaP development. We also identify Deleted in colorectal carcinoma (Dcc) as a Netrin receptor that mediates the ability of Ntn1a to cause VaP axons to stop at the MPs. Netrin is a secreted ligand with roles in axon guidance and cell survival. Zebrafish ntn1a is expressed in MPs at the time of contact by CaP and VaP growth cones. Knocking down Ntn1a using morpholinos (MOs), followed by interval observations of dye-labeled presumptive CaPs and VaPs, revealed that Ntn1a is required for development of VaP but not of CaP. In ntn1aMO injected embryos, presumptive VaP axons extended beyond the MPs and the motoneurons survived longer than wildtype VaPs. Thus, Ntn1a may be the MP-derived signal preventing VaP axons from extending into ventral muscle. We also examined whether Dcc mediates the ability of Ntn1a to cause VaP axons to stop at the MPs. dcc is expressed in CaP and VaP throughout axon outgrowth. MO-mediated Dcc knockdown revealed that, like Ntn1a, Dcc is required for development of VaP but not of CaP. In dcc-MO injected embryos, presumptive VaP axons extended beyond the MPs and VaPs survived beyond their typical time of death. Collectively, the dcc-MO and ntn1a-MO experimental results suggest that Netrin signaling is required for VaP development. Posters 230 Neurobiology I: Patterning 160 Dissecting the mechanism of action of histone deacetylase 1 in epigenetic control of neural progenitor fate V.T. Cunliffe1, M.R. Harrison1, A.S. Georgiou1, E.G. Lightman1, A.J. Buckle1, H.P. Spaink2 1 MRC Centre for Developmental & Biomedical Genetics and Department of Biomedical Science, University of Sheffield, UK, and 2Institute of Biology, Leiden University, The Netherlands The transformation of neural progenitors into differentiated neurons and myelinating glia is under stringent epigenetic control. Our studies have revealed critical functions for Histone Deacetylase 1 (Hdac1) in regulating the pace and pattern of both neurogenesis and oligodendrogenesis. Our results further show that Hdac1 promotes expression of proneural genes throughout the CNS by a mechanism that antagonises the Notch pathway-mediated maintenance of neural progenitors. These observations identify a regulatory role for Hdac1 in promoting commitment of neural progenitors and differentiation of post-mitotic neurons via expression of proneural genes, but the precise molecular mechanisms of Hdac1 action in neurogenesis remain unclear. Consistent with its known in vitro deacetylase activity, we find that Hdac1 regulates steady-state levels of histone acetylation during embryogenesis and specifically promotes histone methylation, which suggests that Hdac1 may repress genes by limiting histone acetylation in the vicinity of target genes. To dissect further the mechanism of action of Hdac1 in neurogenesis, we performed a genome-wide expression analysis of the hdac1 mutant phenotype and identified several Hdac1-regulated, potential direct targets of Hdac1 in the developing CNS. We are now investigating the binding of Hdac1 to target genes in chromatin, and analysing the functions of Hdac1-regulated genes that are implicated as downstream targets of Hdac1 activity. We also used the Hdac1-regulated genes as markers to carry out a comparative analysis of the phenotypes of hdac1 mutants and embryos lacking the functions of other chromatin regulators that are likely to interact with Hdac1 in the developing CNS. The results of these experiments indicate that Hdac1 may promote neural development as a component of multiple distinct transcriptional silencing complexes. Taken together, our results also suggest that Hdac1 may regulate genes that control the behaviour of ventricular zone neural progenitors as well as other genes that influence the behaviour of differentiating, post-mitotic cells in the mantle region. 231 Posters 161 Neurobiology I: Patterning Gdf6a is required for initiation of dorsal-ventral retinal patterning and lens development C. R. French, T. Erickson, D. V. French, D. B. Pilgrim, ent of Biology, Leuven, Belgium Correct wiring of neuronal circuits in the developing brain relies on the precise spatial positioning of neurons and their axons. The optic circuit, which contains the axons of retinal ganglion cells (RGCs), is a powerful model system to study axon guidance, midline crossing and formation of topographic neuronal maps. Recent loss/gain-of-function studies in different animal models revealed several attractive and repulsive guidance cues that regulate the proper formation of the visual pathway. Recently matrix metalloproteinases (MMPs) have been shown to regulate migration and survival of neurons, axon guidance and myelination in the developing brain. There is also substantial evidence that MMPs participate in the development of retinotectal projections. However, the nature and working mechanisms of these MMPs/TIMPs (tissue inhibitors of metalloproteinases) in retinotectal pathfinding remain completely unidentified. We characterized the expression of several MMPs and TIMPs, identified in zebrafish, in the brain of zebrafish embryos at various time points during development of the retinotectal system by in situ hybridization. Especially zMMP2, zMMP14a, zMMP14b and zTIMP2 show an extensive expression pattern in the developing brain. Cross-sections of these in situ stainings revealed that these MMPs are present in the developing optic tectum / pathway. Knockdown of MMP14a, using a specific MMP14a morpholino, and subsequent immunostainings with the axonal marker anti-acetylated tubulin, at 5dpf (days post fertilization) showed a significant reduction in the size of the optic tectum (p<0.05). Similar results were obtained with the broad-spectrum MMP inhibitor EDTA. They confirm our hypothesis and show that MMPs indeed play a role in retinotectal axonal pathfinding. Altogether, these novel findings reveal that MMPs are expressed in the zebrafish brain during development of the visual pathways and that axon pathfinding in the retinotectal system of zebrafish can be disrupted by blockade of MMP signaling. Our results might have implications for future strategies interfering with neural circuit development and/or regeneration. Posters 236 Neurobiology I: Patterning 166 Function of the Lhx genes in thalamic development of the zebrafish D. Peukert1, A. Lumsden2, S. Scholpp3 1 Institute for Toxicology and Genetics, Research centre Karlsruhe (Helmholtz-Gemeinschaft), Germany; 2Centre for Developmental Neurobiology, MRC, London, UK; 3Institute for Toxicology and Genetics, Research centre Karlsruhe (Helmholtz-Gemeinschaft), Germany The thalamus is the major relay station in the forebrain and connects the sensory organs with higher brain parts. In mammals the flow of information is organized in a stereotypic manner: sensory organs project towards specific thalamic nuclei from which subsequently target a specific area in the cortex. Although much simpler, this basic principal can be observed within the nonmammalian lineage. Interestingly, the developmental process of regionalization of the thalamus and formation of its functional units: the nuclei are unknown. Data from mouse and Xenopus show that the LIM homeodomain genes are expressed in the different thalamic compartments. Here, we elucidate the parcellation process in detail by using zebrafish as a model organism. First, we mapped the expression pattern of lhx2 and lhx9 relative to well-known markers of the anterior ZLI border and the posterior located pretectum (Scholpp et al., 2006). We find that lhx2 and lhx9 mark the pool of thalamic postmitotic projection neurons, whereas the interneuron population does not express lhx2 and lhx9. Furthermore, we could show that lhx2 and lhx9 are expressed in distinct but partially overlapping domains within the thalamic projection neurons, suggesting a crucial role during the further parcellation process of this population into the final nuclei. To explore this further we will study the function of these LIM homeodomain factors, which will give us a greater insight into thalamic development in vertebrates. 23Morales, A. Roberts* and K. Lewis Physiology Development & Neuroscience Department, Cambridge University, UK *School of Biological Sciences, University of Bristol, UK As yet it is far from understood how neurons acquire specificity, form a given neural network and participate in different chemical and physical stimuli. The simple anatomy of Danio rerio and Xenopus make them ideal models for the study of neural formation and gene expression, as well as for physiological studies. With this in mind, we aim to develop a series of transgenic lines with specifically labelled cell types in both animal models, focusing on different populations of spinal cord interneurons. These will be used as genetic tools to study the neural circuit formation during development. As a proof of principle, we made a construct from sequence upstream of HuC with a GateWay TOL2 system and successfully generated transgenics that labelled interneurons and Rohon Beard cells in both species. In order to label specific interneuron populations we are using a bioinformatic approach to identify conserved non-coding elements, as a means of isolating putative cis-elements, which are then used to generate reporter constructs. Our analyses of genes such as evx1, evx2, isl1, chx10 and lbx1, have resulted in reporter constructs that are expressed in specific cell types. These will be invaluable tools that will enable us to relate the specific gene expression of spinal cord cells, their particular morphology and function in both species. Posters 246 Neurobiology I: Patterning 176 Endocytosis and neurogenesis in the zebrafish spinal cord S. Abke and M. Gonzalez-Gaitan University of Geneva, Switzerland For now almost 20 years a new view of signaling has emerged, a view raising the possibility that signal transduction does not exclusively happen at the plasma membrane but also on early endosomes. After this finding for the EGF signaling pathway, during the years, more and more evidence suggested a role for endocytosis in signaling for NGF-, Notch- and also TGFsignaling. In our lab one main focus lies on a TGF- signaling adaptor protein called SARA, which stands for Smad anchor for receptor activation. Upon ligand binding, the heteromeric receptor complex is internalized into early endosomes and signaling is induced after a phosphorylation event, which propagates the signal to intracellular signaling mediators. This is where SARA comes into play: next to other functional domains it has a FYVE domain by the means of which it binds to the phosphatidylinositol-3-phosphate (PI3P) on the early endosome. An interesting feature of these SARA positive endosomes, studied in our lab mainly in drosophila, is that they either segregate symmetrically in symmetric cell division or asymmetric in asymmetric cell division. Studies in zebrafish SARA show that it can segregate asymmetrically during cell division in the spinal cord and that knock-down by morpholinos leads to a neurogenic phenotype. My interest lies on exploring the role of trafficking in neurogenesis with SARA being the center of my attention. Several small GTPases, the Rabs, mark the different compartments in the trafficking pathway. I will use live imaging taking advantage of Rab GFP fusion proteins to study their behavior live and during cell division as well as a morpholino knock-down approach to analyze their function in neurogenesis and their influence on the signaling in which SARA is involved. Moreover, drugs and dominant-negative or dominant-active forms of these small GTPases can be used to interfere with trafficking at precise steps of the pathway and to block endocytosis in a more general way. This study will not only allow us to understand the cellular basis of our phenotype but also understand how cells exploit the traffic machinery to regulate key events such as: Neurogenesis and directional signaling between cells. 247 Posters 177 Neurobiology I: Patterning Localization of Urotensin 1 and Urocortin 3 neurons in the embryonic zebrafish nervous system L. Braeutigam1, J. Hillmer2, I. Soll2, G. Hauptmann2 1 Biosci. and Nut., Karolinska Institutet, Stockholm, Sweden; 2School of Life Sciences, Soedertoerns Hoegskola, Stockholm, Sweden The body's response to stress is comprised of a variety of physiological changes and behavioral patterns including alterations in locomotion. Locomotion is dependent on specific neuronal circuits in the brainstem including reticulospinal neurons and the Mauthner cell. These circuits are modulated by the neurotransmitters dopamine, noradrenaline and serotonin in all vertebrates. CRH family peptides are known to interact with these aminergic brain circuits and thereby changing locomotor activity. To achieve a deeper understanding of the connection between the stress system and locomotion, we analyzed the spatial transcript distribution of two CRH family peptides, Urotensin 1 (UTS1) and Urocortin 3 (UCN3) by whole-mount in situ hybridization and immunohistochemistry during zebrafish embryogenesis. We indeed found that UTS1 and UCN3 are expressed in key neuronal sites regulating locomotor behavior, suggesting the interrelation of the stress response and locomotion via these neuropeptides. With our detailed data on the embryonic expression of UTS1 and UCN3 mRNA, we can now dissect the function of these CRH family peptides in stress induced locomotion. Posters 248 Neurobiology I: Patterning 178 Homeostatic synaptic plasticity in the developing spinal cord and its behavioural correlates L.D. Knogler and P. Drapeau Pathology and Cellular Biology, Universite de Montreal, Canada Objectives: Behaviors are generated through populations of nerve cells, wired into circuits that form a neural network. There are homeostatic mechanisms in place to modify synaptic strength in response to changes in activity, one of the best documented being synaptic scaling, a uniform increase or decrease in the size of miniature excitatory post-synaptic currents (mEPSCs). There is little understanding of the relationship synaptic between scaling and behaviour, so our goal is to investigate synaptic scaling in the developing Danio rerio (zebrafish) spinal cord and its effects on early locomotor behaviours. Materials and methods: Wild-type embryos were injected between 17 hours post-fertilization (hpf) and 48hpf with either TTX (voltage-gated Na+ channel blocker), AP-5 (NMDAR blocker), CNQX (AMPAR blocker), TNF (molecule known to increase mEPSC amplitude during acute application), or vehicle as a control. Whole-cell patch clamp recordings of mEPSCs from spinal neurons and muscle fibres were made at 4dpf. Behavioural analyses of spontaneous coiling (1728hpf), touch response (30hpf), and swimming (2-4dpf) were also performed. Results: Blocking activity with TTX and CNQX for 48hrs (2-4dpf) resulted in a significant, multiplicative increase in glutamatergic mEPSC size, scaling mEPSC amplitudes up by ~1.4. Current threshold data shows that increased mEPSC amplitudes in CNQX-treated fish cannot be attributed to changes in intrinsic excitability of neurons. AP-5 treated fish showed no significant changes. Injection of TNF resulted in significantly scaled down (decreased) glutamatergic mEPSC amplitudes after as little as 24hrs. Other parameters including mEPSC frequency, kinetics, inter-event interval, and AMPA:NMDA ratio are also described. Behaviourally, TTX treatment resulted in immotile embryos, whereas AP-5 and CNQX-injected fish could swim. Further analyses of stereotypical swimming behaviour suggest differences between experimental conditions that may be correlated with synaptic scaling. Muscle recordings show that CNQXtreated embryos have trends towards increased muscle activity, showing a tendency towards longer burst durations in swimming episodes than control. Conclusions: These results support a role for synaptic scaling in vivo as a means of maintaining homeostasis during development. Glutamatergic mEPSCs scale up multiplicatively in response to activity blockade to strengthen existing synapses. Our observations of muscle fibre and swimming activity following chronic blockade of synaptic activity provide new ev of transmembrane proteins that belong both to the LRR (leucine-rich repeat) and Ig superfamilies. Expression of AMIGO1 could be induced by RAGE (receptor for advanced glycation end products) ligation with amphoterin [Juha K.P., 2003]. The expression pattern of AMIGO1 in the mice embryo has been detected by whole mount in situ hybridization and found to be mainly expressed in the central nervous system during early development stage [Shunsaku H, 2009]. But except for its potential roles in the apoptosis of granula neurons and mediating fasciculation of neurites in vitro [Tomio O, 2003], few functions have been so far found. In order to explore AMIGO1 roles during early development stage, we have managed to knock down its expression in the zygote by the introduction of specific morpholino oligos via microinjection. The inhibition has been confirmed both by western-blot and whole mount immunostaining in 3dpf (days past fertilization) and 5dpf injected larvae. AMIGO1 knock-down larval brains usually show development defects at the areas where AMIGO1 is normally expressed. Tyrosine hydroxylase (TH) immunohistochemistry for labeling zebrafish catecholaminergic system shows a prominent decrease and disordered organization in TH-immunoreactive neuronal fiber projections in the hindbrain. TH cell cluster formation and distribution in the morphants' telencephalon, midbrain and eyes are also impaired, which could be detected from the very beginning of development. Furthermore, the swimming behavior test shows a disorganized pattern of swimming in addition to decreased distance in AMIGO1 5dpf morphants, which is an expected consequence of the catecholaminergic system deterioration. The AMIGO1 knock-down morphant phenotypes have been rescued successfully by co-injection of AMIGO1 mRNA together with morpholino oligos. The 5dpf co-injected larvae even show hyperactive effects in the behavioral test. Our study shows an important role of AMIGO1 in the developing zebrafish larval brain and corresponding functions in vertebrate catecholaminergic system development. 253 Posters 183 Neurobiology I: Patterning Mechanisms controlling polarized dendrite formation of Purkinje cells in the zebrafish cerebellum K. Tanabe, YK. Bae, S. Kani, T. Shimizu, M. Hibi Laboratory for Vertebrate Axis Formation, RIKEN Center for Developmental Biology, Kobe, Japan Although neurons display remarkable diversity of dendritic morphology, mechanisms that pattern specific shapes of dendrites are still to be understood. Among processes of dendritic patterning, such as dendritic growth or branching, we focus on the mechanism that controls the number of primary dendrites (dendrites extending directly from a cell body) as the initial process for dendritic patterning. Purkinje cells in the cerebellum give us a good model system for studying this issue since they exhibit characteristic polarized morphology having a single primary dendrite. Taking advantages of zebrafish for live-imaging experiments, we are studying how Purkinje cells acquire the highly polarized morphology and the mechanisms that control the process. We established transgenic fish that express membrane-targeted Venus specifically in Purkinje cells and observed their morphologies at single cell resolutions. We found that zebrafish Purkinje cells showed different dendritic morphologies according to the somal location along anteroposterior and midio-lateral axes. In spite of the morphological diversity, all Purkinje cells have a single primary dendrite as observed in mammalian cerebellum. Time lapse observations for the morphological changes of Purkinje cells revealed that they initially had multiple neurites and only one of them was finally selected as the future primary dendrite to form the mature polarized morphology. We found that Golgi apparatus was exclusively localized at the root of primary dendrite in Purkinje cells and Golgi localization preceded morphological polarization, suggesting that localized Golgi regulates the polarized dendrite formation. We also found that Purkinje cells retained ation. Hence, we are currently performing local bmp2b/bmprI gain and loss of function experiments in the diencephalic anlage and perspective telencephalon to directly analyse the potential of BMP signalling to induce early telencephalic markers. Posters 260 Neurobiology I: Patterning 190 The role of Dynein cytoplasmic 1 heavy chain 1 in peripheral nerve myelination M. Langworthy, B. Appel Pediatrics, University of Colorado Denver, Aurora, USA The formation of myelin sheaths around axons is required for rapid salutatory conduction of action potentials in the central and peripheral nervous systems (CNS, PNS). In the PNS, myelin is formed by Schwann cells, glial cells produced from neural crest that migrate to and ensheath target axons. The failure to form or maintain peripheral myelin can result in muscle weakness or paralysis, motor neuron degeneration, an inability to sense pain or change in temperature and neuropathic pain. To investigate the mechanisms that control the formation of the myelin sheath around axons, our lab analyzes mutations that result in myelin deficits. Surprisingly, we found that larvae homozygous for an insertional mutation in the dynein cytoplasmic 1 heavy chain 1 gene (dync1h1hi3684Tg) have myelin deficits in both the CNSA and PNS. In particular, dync1h1 mutants have very few cells that express myelin genes. Nevertheless, Schwann cells are p resent at motor roots of mutant embryos and larvae, suggesting that dync1h1 function is necessary for a late step in myelination. Loss of Dync1h1 function in neurons and disruption of axon transport has previously been linked to peripheral nerve disease in humans and mice. To determine if zebrafish dync1h1 function is required in axons or Schwann cells for myelination, we initiated a genetic mosaic analysis. Our preliminary data indicate that dync1h1 function is necessary in Schwann cells for axon wrapping and myelination. Therefore, our data reveal a potential new role for Dynein in peripheral nerve development and maintenance and raise the possibility that disruption of Dynein function in both axons and Schwann cells can contribute to peripheral nerve disorders. 261 Posters 191 Neurobiology I: Patterning Semaphorin-neuropilin interactions elicit aberrant spinal cord exit by rohon-beard central axons N. S. Asuri and M. C. Halloran Departments of Zoology and Anatomy, and Genetics Training Program, University of WisconsinMadison, USA Rohon-Beard (RB) sensory neurons reside in the spinal cord and extend two types of axons, central and peripheral, that display distinctly different behaviors. The central axons extend longitudinally within the spinal cord and fasciculate with one another. In contrast, the peripheral axon, which arises as a branch directly off the central axon, exits the spinal cord and extends to the skin. Peripheral axons within the skin repel one another on contact. We are interested in understanding the mechanisms controlling these different behaviors. Perhaps the most notable difference between the RB axons is that the central axons remain in the CNS while the peripheral axons exit to the periphery. Previous work in our lab showed that knockdown of Sema3d, a guidance signal expressed in the spinal cord roof plate, caused a reduction in peripheral RB axons. Furthermore, ectopically expressed Sema3d repelled peripheral axons but did not affect central axons. These results suggest that Sema3d may act to repel the peripheral axons out of the spinal cord. We are investigating the roles of potential Semaphorin receptors in the guidance of RB axons. We drove expression of a Neuropilin 1a-GFP fusion construct in RB cells, initially to examine its localization. Interestingly, we found that exogenous Nrp1a-GFP leads to errors in central RB axon pathfinding. The most remarkable effect is that some central axons exit the spinal cord and grow into the skin. In other cells, the central axons grow ventrally and take aberrant pathways within the spinal cord. To test whether this effect is caused by a particular Semaphorin, we are examining potentialing study in our lab has helped to reveal the neural plate origins of cells that give raise to these diencephalic subterritories. This study demonstrated that very little mixing occurs between distinct cell populations. Surprisingly, it was also found that cells of the prospective prethalamus are already functionally specified and irreplaceable as early as the bud stage (Staudt and Houart, 2007). In an effort to unravel the genetic components of this specification, we have isolated and RNA-profiled cells of the diencephalic anlage at bud stage and compared their transcriptome to this of the presumptive midbrain. Currently, we are analysing the list of diencephalic enriched transcripts and verifying the data. We have started to allocate gene expression patterns to the cell populations defined by fate-mapping, identify areas of restricted pathway activities and assess gene function for specific candidates. Staudt, N. and Houart, C. (2007) The prethalamus is established during gastrulation and influences diencephalic regionalization. PLoS Biol. 5(4):e69. Posters 264 Neurobiology I: Patterning 194 Role of the dlx genes in GABAergic interneuron and forebrain development of zebrafish R. MacDonald, M. Debiais-Thibaud, and M. Ekker Center for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Canada The expression of the Dlx homeobox genes is closely linked with interneurons expressing gammaaminobutyric acid (GABA) in the forebrain. These neurons provide inhibitory signals to the cortex and hippocampus which are necessary for information processing. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and will migrate tangentially to the overlying cortex. Single Dlx mutant mice show subtle phenotypes if any at all. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, due to cross-regulatory mechanisms via their regulatory elements. The forebrain phenotype of Dlx5/Dlx6 double mutant mice can not be characterized due to exencephaly. Ectopic expression of Dlx is sufficient to induce expression of glutamic acid decarboxylase (Gad), the enzyme necessary for GABA production. Therefore, the Dlx genes play an important role in the differentiation and migration of GABAergic interneurons of mice. In zebrafish the role of the dlx genes in GABAergic interneuron development is unknown. To investigate this issue, we have generated transgenic zebrafish lines with three dlx enhancers which are active in the forebrain and tested the role of the dlx transcription factors in brain development by Morpholino knockdown. Co-expression of reporter transgenes and gad is observed in most cells of the zebrafish forebrain starting at approximately 48 hpf. Morpholino knockdown of the dlx genes causes a reduction in the activity of dlx regulatory elements and interferes with proper gad expression in the zebrafish forebrain. Also, the migration of gadexpressing neurons from the ventral telencephalon to the pallium appears dlx-dependant. This implies an interconnected dlx cascade potentially crucial for the proper expression of dlx genes throughout forebrain development, comparable to the one described in the mouse. These results implicate the dlx genes in an evolutionarily conserved pathway controlling GABAergic interneuron differentiation and migration in vertebrates. This work is supported by CIHR MOP14460 and NSERC. 265 Posters 195 Neurobiology I: Patterning Spatial and temporal regulation of robo2 splice variants MY. Law and C-B. Chien Neuroscience Program, Department of Neurobiology & Anatomy, University of Utah, Salt Lake City, USA Alternative splicing is actively induced during neuronal development, contributing to proteomic diversity, and has been implicated in cell-fate determination, axon guidance, and tissue patterning (Li et al., 2007). Axon guidance receptors ta submission) to express NpHR broadly or in specific subpopulations of neurons. Applying light through thin optic fibers positioned above the animal's head enabled us to target small groups of cells and to simultaneously test the effect of their silencing on behavior. With this technique, we localized cells in the caudal hindbrain that control forward swimming and determined the kinetics of the swim command in hindbrain and spinal cord by a combination of silencing with NpHR and activation with the light-gated cation channel channelrhodopsin-2 (ChR2). The NpHR-assisted "reversible spinalization" revealed that the central pattern generators in the spinal cord were under tight temporal control of the hindbrain throughout the studied swimming behavior. Furthermore, we used visual behavioral assays (optokinetic response) to identify a cell population in close proximity to the cranial nucleus VI that is required and sufficient for the generation of fast eye movements (saccades) in zebrafish larvae. Bilateral stimulation of NpHR suppressed saccades completely but left slow pursuit eye movements unaffected. Unilateral stimulation blocked saccades of both eyes in the direction of the stimulated side, but not in the other direction. Conversely, ChR2-mediated activation of this neuronal population elicited fullblown saccades in both eyes. In an unrelated forward-genetic screen (Muto et al., PLoS Genet. 2005), our group had identified a sodium-channel mutant, didy, with a specific deficit in sustaining saccades. The saccade deficit in mutants could be rescued by locally activating ChR2. Together our studies introduce an optogenetic toolkit for precise loss-of-function and gain-of-function analysis of neural circuits and behavior. 273 Posters 203 Neurobiology II: Sensory organs Fgf signaling is necessary for pharyngeal taste bud receptor cell formation M. Kapsimali and F. M. Rosa Genetique Moleculaire du Developpement, INSERM U784, Ecole Normale Superieure, Paris, France Taste buds are the vertebrate sensory organs of taste composed of distinct cell types including taste sensory receptor, presynaptic and support cells. They are located in a patterned manner in the vertebrate oropharynx and depending on the species can be harboured in epithelial structures, the papillae. Although much progress has been achieved in understanding the molecular mechanisms underlying papillae formation, little is known about the development of pharyngeal specific taste bud cell types (taste receptor, presynaptic, supportive) per se. To start elucidating the mechanisms underlying specific taste bud cell type formation, we first characterized the zebrafish taste buds and found that are composed of distinct cell types and located in a pattern manner in the oropharyngeal epithelium. Fgf8 and Fgf receptors are expressed in the pharyngeal epithelium and their loss of function in the ace mutant and embryos with pharmacologically compromised Fgf receptor function, respectively, showed that Fgf signaling is necessary for taste bud formation. To dissect the role of Fgf signaling in taste bud cell type formation, we specifically blocked Fgf receptor signaling within the pharyngeal epithelium. We show for the first time that Fgf signaling is critically required within the pharyngeal epithelium for taste receptor cell formation. Posters 274 Neurobiology II: Sensory organs 204 Wnt/b-catenin signaling regulates morphogenesis of the lateral line T. Piotrowski, A. Aman, R. Crosbie, MT. Nguyen University of Utah, Dept. of Neurobiology and Anatomy, Salt Lake City, USA The lateral line is an excellent model for studying fundamental developmental mechanisms, such as stem cell regulation, cell migration, cell fate specification and patterning in vivo. The lateral line system develops from a migrating placode (primordium) that sequentially deposits prosensory organs from the trailing edge of the primordium at fairly regular intervals. We have recently described the cell-cell signaling events that occur between cells in the leading and trailing zones of the migratinhat underlies an early motor behavior in the zebrafish embryo. This behavior, the touch response, consists in coiling of the trunk of the embryo in response to a tactile stimulus. Rohon-Beard sensory neurons transduce the stimulus and pass the signal on to commissural interneurons (CoPAs) through glutamatergic synapses. To understand how the synapses between RB cells and CoPA interneurons form during development, we characterized the timeline of recruitment of synaptic components by immunolabeling and live imaging techniques using pre- and postsynaptic markers. We examined synaptic labeling between 16 to 25 hours post fertilization (hpf), a developmental timeframe that straddles the time of initiation of the touch response at 21hpf. Synaptic vesicle precursors labeled with SV2 were equally distributed throughout RB axons at all time points examined and this distribution was confirmed by imaging of fluorescently tagged VAMP2. In contrast, synapsins 1 and 2, proteins that tether synaptic vesicles to the cytoskeleton, localized at contacts with CoPA cells after 21 hpf. On the postsynaptic side, immunoreactivity for the proteins PSD-95, PSD-93, and SAP97 were first seen at 19hpf. This time line of synaptic recruitment reveals a sequential assembly of both pre- and postsynaptic components at a synapse that is critical for a sensory-motor behavior. This is the first description of protein recruitment to a precisely defined population of glutamatergic synapses in an intact vertebrate. 279 Posters 209 Neurobiology II: Sensory organs Metabotropic glutamate receptors in the zebrafish retina M. Haug, Y-Y. Huang, M. Gesemann, S.C.F. Neuhauss Institute of Zoology, University of Zurich, Switzerland Background: Metabotropic glutamate receptors (mGluRs) have been identified at all synapses of the vertebrate retina, where they likely regulate neurotransmitter release. The only example of an mGluR functioning in direct synaptic transmission is mGluR6, which is expressed on ON bipolar cell dendrites and is found to mediate the ON response in the mammalian retina. As a first step to elucidate the functional role of mGluRs in the teleost retina, we set out to clone all members of this family and determine their respective expression pattern in the developing and adult zebrafish retina. Results and Conclusion: With its division into three subgroups, the phylogeny of the mGluR family is comparable to humans and mice. For most members (mGluR1, 2, 5, 6, and 8) we detected for each mammalian mGluR ortholog two paralogues. Those likely arose in a teleost specific whole genome duplication event. RNA in situ hybridization experiments revealed a unique expression pattern for all mGluRs in 5 day old zebrafish, supporting the distinct roles of the different mGluRs in the retina. Different expression patterns between two paralogues, as found for example for mGluR1a, and -1b, suggest a distinct subfunctionalisation of both gene paralogues. Due to the specific function in the mammalian retina, we focused on the two mGluR6 paralogues. In contrast to mammals, where mGluR6 is expressed in bipolar cells, we located both genes predominantly in retinal ganglion cells. This surprising result was supported by coexpression with gnao (coding for Go proteins), the G-protein which is thought to conduct the mGluR6 signalling pathway. Since gnao is also expressed in inner retinal cells, we deem it likely that the mGluR6 paralogues are expressed below detection level in bipolar cells. We are in the process of generating antibodies to identify the synaptic localization of the mGluR6 paralogues. In the future, knockdown experiments will determine the function of these genes in vision. Posters 280 Neurobiology II: Sensory organs 210 A gfp-based genetic screen reveals mutations affect ciliogenesis in photoreceptor cells Y. Omori1, J. Malicki2, T. Furukawa1 1 Osaka Bioscience Institute, Osaka, Japan; 2Harvard Medical School, Boston, USA A photoreceptor cell develops a photo-sensitive outer segment on the apical side of the cell body. A cilium with a microtubule ax zpr1 appears to be reduced overall in raf mutants. However, staining of newborn rod photoreceptors in the peripheral retina is normal. Preliminary light microscopy data suggests a morphological defect in raf photoreceptors and retinal pigment epithelium (RPE). Conclusions: We have identified and partially characterised a novel zebrafish model of inherited blindness. Preliminary results suggest that specific defects in raf outer retina morphology underlie loss of visual function. 289 Posters 219 Neurobiology II: Sensory organs Differentiation and maintenance of zebrafish photoreceptor polarity M. Luz and E. Knust Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Photoreceptor cells have a unique morphology, essential for their highly specialized function. Polarity in mature photoreceptor cells is particularly evident in their apical surface, which is subdivided into the outer segment, the connecting cilium and the inner segment. In Drosophila, the Crumbs protein complex has been shown to control the differentiation of the apical surface in epithelial and photoreceptor cells. The core components are the transmembrane protein Crumbs and the scaffolding proteins Stardust, DPATJ and DLin-7. Defects in individual components lead to disintegration of embryonic epithelia, morphogenetic defects in photoreceptor cells and lightdependent retinal degeneration. The zebrafish genome encodes five crumbs genes, a single stardust gene and three lin7 genes. Mutations in one of the crumbs orthologues, oko meduzy, and in the stardust orthologue nagie oko affect the polarized structure of the neuroepithelium. In Drosophila, absence of crumbs leads to a reduction in the length of the stalk membrane, a portion of the apical membrane which corresponds to the inner segment of vertebrate photoreceptor cells. In zebrafish, morpholinoinduced loss of crb2b function has been shown to affect the differentiation of apical characteristics in photoreceptor and kidney epithelial cells. We are currently performing a detailed analysis of the development of photoreceptor polarity at different stages, both in fixed tissue and by in vivo imaging. In addition, we are screening for mutations in crb genes by TILLING. We will report on the results, which can provide insights on the cell biological function of the Crumbs protein complex in the differentiation and maintenance of apico-basal polarity of photoreceptor cells. Posters 290 Neurobiology II: Sensory organs 220 Arrestin availability in cone photoreceptors modulates opsin inactivation S.L. Renninger, M. Gesemann, S.C. Neuhauss Institute of Zoology, University of Zurich, Switzerland Human vision is governed mainly by cone photoreceptor function. Not only are cones responsible for color vision; they also enable the retina to rapidly signal changes in illumination even in presence of high ambient light levels (photopic vision). However, little is known about the mechanisms shaping the time course of the cone response, avoiding photobleaching and ensuring high temporal resolution. In contrast, rod photoreceptors are highly sensitive to light and, therefore, responsible for vision at dim light (scotopic) conditions. The initial step in shutting-off rod signal transduction is comprised by the phosphorylation of activated rhodopsin. Subsequent binding of the arrestin protein to phosphorylated rhodopsin is essential for the complete inactivation the photopigment. Recent studies have shown that phosphorylation of the photopigment opsin is also a crucial step for proper cone response inactivation - suggesting a quenching mechanism similar to the one in rods. Here, we used the cone-dominated retina of zebrafish larvae to genetically dissect the mechanism of photopigment inactivation in cone photoreceptors, and, in particular, to study the impact of arrestin on quenching cone signal transduction. We identified two cone arrestins and verified their cell type specificity by mRNA and protein expression analysis. Morpholino-mediated knock down of the arrestins and subsequent electroreal Biology, University of Wuerzburg, Germany, 2Institute of Zoology, University of Innsbruck, Austria, 3Department of Biochemistry and Biophysics, University of California, San Francisco, USA, 4Department of Biology, National University of Singapore, Singapore. The Popeye domain containing (Popdc) gene family is predominantly expressed in heart and skeletal muscle. In mice loss of Popdc1 and Popdc2 affected sinus node function in the postnatal mouse heart. In this study, we therefore asked whether the requirement of Popdc genes for cardiac pacemaking is evolutionary conserved. We addressed this possibility using an antisense morpholino-based approach in zebrafish embryos. Both, Popdc1 and Popdc2 morphants developed pericardial edema and aberrant tail muscle development. In Popdc2 morphants we observed irregular ventricular contractions with 2:1 and 3:1 atrio/ventricular rhythm while sinus rhythm and atrial contractility appeared normal. Recordings of calcium transients using an indicator transgenic line and selective plane illumination microscopy revealed the presence of an AV block in Popdc2 morphants. Surprisingly, Popdc1 morphants did not develop an AV block but showed an overall reduced beating rate indicative of a sinus bradycardia. Thus, Popdc1 and Popdc2 have overlapping functions, however also differential requirements of these genes in cardiac conduction tissue development became apparent. This work was funded by grants from the COB 2006, EMBO ASTF 96-2007, FP6 project LSHMCT-2005-018630 `Heart Repair ` and the DFG-funded graduate college 1048 ,,Organogenesis". 307 Posters 237 Cardiovascular system Zebrafish models of cardiac development and disease D. Bournele 1, D. Stainier2 and D. Beis1 1 Developmental biology, biomedical research foundation, Academy of Athens, Greece; 2 Biochemistry and biophysics, University of California, San Francisco, USA Cardiac valves derive from endocardial cells and function throughout the life of vertebrates to prevent retrograde blood flow. These cells undergo elaborate morphogenesis and several signaling pathways converge to orchestrate valve development. We have published a detailed description of the cellular architecture of the zebrafish heart during stages of atrioventricular valve development, using gfp transgenic lines, immunofluorescence and confocal microscopy. We showed that the differentiation of the atrioventricular canal involves endocardial-myocardial interactions and that it relies on heart function. We also showed that Notch and calcineurin signaling are required for the regulation of the initial stages of zebrafish atrioventricular endocardial cushion formation. In mammals, endocardial cushion development has been linked to the etiology of atrial and ventricular septal defects and of atrioventricular canal defects. Zebrafish offers the possibility of forward genetics and study of gene function at the cellular level. The long-term aim of our efforts is to find out how heart valves form and function throughout the life of vertebrates. To that effect, we take advantage of the mutants identified during a large-scale forward genetic screen in the UCSF (Stainier and Baier labs). These mutant lines exhibit a retrograde blood flow through the heart as an indication of defective cardiac valve development. In one of these lines (s411), we observed blood regurgitation at the atrioventricular canal as a result of outflow tract stenosis at 72 hours post fertilization (hpf), similar to an aortic stenosis phenotype in humans. Homozygous mutant embryos fail to develop a functional outflow tract and the circulation, normally initiated, stops finally at 55 hours post fertilization. The mutant embryos appear morphologically wild type at 72 hpf and have normal myocardial function, but exhibit pericardial edema due to an outflow tract stenosis. In parallel, we use confocal microscopy to analyze at cellular resolution the valve development in these mutants and we showed that the atrioventricular canal is lined by a single layer of cuboidal endocardial cells. In addition, we have shown that our mutants have, compared to wild type embryos, less elastin-positive cells residing at the outflow tract. We are currently working on identifying and cloning the mutated gene responsible for the phenotype and elucidating how it functions into the developmental pathways that control cardiac valve formation. The mutation maps on linkage group 15 (48cM on MGH). The latest data on the phenotype, mapping and function of this gene will be presented during the meeting. Posters 308 Cardiovascular system 238 Regulation of Sphingosine-1-phosphate (S1P) signaling during zebrafish cardiovascular development C. Detzer1, K. Scholich2 and B. Jungblut1 1 Max-Planck-Institute for Heart- and Lung Research, Bad Nauheim, Germany; 2Institute for Clinical Pharmacology/ZAFES, Johann Wolfgang Goethe University, Frankfurt am Main, Germany Sphingosine-1-phosphate (S1P) is a lipid mediator derived from sphingosine and plays an important role in developmental processes including angiogenesis, vascular maturation, heart development and immunity. S1P is a ligand of five G-protein coupled S1P receptors (S1P1-S1P5) which are also known as ,,Endothelial Differentiation Genes". These receptors are differently expressed and coupled to various G-proteins, so numerous downstream signals can be regulated. The S1P ligand is produced by the phosphorylation of sphingosine, catalyzed by sphingosine kinases (SpK). S1P is degraded by the S1P phosphatases (SPPs) and by the S1P lyase (SPL). The S1P phosphatases dephosphorylate S1P back to the precursor sphingosine which can then be N-acylated to form ceramide that is also a precursor of S1P. SpKs, SPPs and SPL are essential regulators for maintenance of the S1P rheostat inside the cell, which is very important, because ceramide- and sphingosine lead to growth arrest and cell death whereas S1P is needed for growth and cell proliferation. The S1P lyase degrades S1P to phosphoethanolamine and hexadecanal. Tight control of local levels of S1P is important for heart morphogenesis (Wendler and Rivkees, 2006). In addition SPL knock-out mice displayed histopathologic lesions in the heart by increased interstitial cellularity and vacuolation in the myocardium. S1P level, were elevated in SPL knockout mice (Vogel et al., 2009). To investigate the role of SPL in zebrafish heart morphogenesis we analyze its expression pattern by in-situ hybridization and its function by morpholino mediated knock-down. In addition we determine lipid concentrations of ceramide, sphingosine and S1P by mass spectrometry. Morpholino knock-down led to a phenotype in the heart, which was reproducible and dose dependent. Vogel P, Donoviel MS, Read R, Hansen GM, Hazlewood J, et al. (2009) Incomplete inhibition of sphingosine 1-phosphate lyase modulates immune system function yet prevents early lethality and non-lymphoid lesions. PLoS ONE 4(1):e4112 Wendler C.C., Rivkees S.A. (2006) Sphingosine-1-phosphate inhibits cell migration and endothelial to mesenchymal cell transformation during cardiac development. Developmental Biol. 291, 264-277 309 Posters 239 Cardiovascular system The role of atp binding domain protein 4 in heart development of zebrafish S. Hundt, D. Y.R. Stainier*, and B. Jungblut Max-Planck-Institute for Heart- and Lung Research, Bad Nauheim, Germany; *Department of Biochemistry and Biophysics, University of California, San Francisco, USA The embryonic development of the heart is a highly complex process. Starting from a linear heart tube very early in development the heart undergoes many changes. The mature heart is a result of ballooning, looping, and the formation of the atrio-ventricular and the sinoatrial junctions. It is known that specific patterns of proliferation and differentiation as well as specific changes in cell shape and cell configuration are essential for the normal function of the heart. Understanding the genetic control of these cellular processes during heart development could help in better understanding congenital heart diseases. In a genetic screen for heart morphogenesis mutants (Beis et al, e confocal analysis performed 2 hours after transplantation showed circulating hCD34+ cells in developing vessels and no evidence of vascular occlusions. Further, at 12 hours after injection , some hCD34+ cells had incorporated into the vessel wall. One day after transplantation, hCD34+- injected embryos exhibited altered blood vessels sprouting in the growing tail vasculature as well as enhanced and ectopic angiogenesis at the level of the subintestinal vein; these results raised the possibility that hCD34+ cells may synthesize angiogenic factors. In agreement with this hypothesis it was found that hCD34+ cell injection into the zebrafish blastula rescued the vascular phenotype caused by Vegfc knock-down. On the contrary, hCD14- cells injected into the embryo do not seem to affect vascular development. Conclusions: The present study demonstrates the evolutionary conservation of hCD34+ cells differentiation mechanisms toward the endothelial lineage and their angiogenic properties in the zebrafish embryo. 313 Posters 243 Cardiovascular system Chemical Inhibitors of Developmental Angiogenesis in the Eye B. Kennedy1, Y. Alvarez1, N. Waghorne1, O. Astudillo-Fernandez1, L. Jensen2, S. McLoughlin2, Y. Cai1 1 UCD SBBS & UCD Conway Institute, University College Dublin, Dublin, Ireland 2Institution for Microbiology Tumor and Cell Biolog, Karolinska Institute, Stockholm, Sweden Retinal neovascularisation is a pathological hallmark of debilitating forms of blindness including diabetic retinopathy and retinopathy of prematurity. In order to identify lead drugs that inhibit ocular neovascularisation, we performed a chemical screen for inhibitors of developmental angiogenesis of the hyaloid vasculature in zebrafish larvae. Data will be presented on the screening, validation and characterisation of some of the lead drugs uncovered. Primary drug hits are identified as those that induce defects in developmental angiogenesis of the hyaloid at 5 dpf without inducing gross developmental abnormalities. Validation involves confirming a dose- and time-dependent response on hyaloid angiogenesis. Drugs that pass these stages are then screened for their ability to inhibit ectopic angiogenesis in a mutant model and regenerative angiogenesis in adult fin clips. Tertiary characterisation looks at the effect of the selected drugs on visual behaviour and retinal morphology. To date we have screened over 1500 drugs and identified 2 that inhibit angiogenesis in the eye without affecting visual morphology or function. Posters 314 Cardiovascular system 244 Dll4 suppresses Vegfc/Flt4 signalling in the developing zebrafish arterial system B. M. Hogan1, R. Herpers1,2, M. Witte1, H. Duckers2, K. Alitalo3, S. Schulte-Merker1 1 Hubrecht Institute-KNAW & University Medical Centre, Utrecht, The Netherlands; 2Experimental Cardiology, Thoraxcenter, Erasmus University Medical Center, Rotterdam, The Netherlands; 3 Molecular Cancer Biology Laboratory, Department of Pathology, Haartman Institute, Biomedicum Helsinki, University of Helsinki, Finland Hemangiogenesis and lymphangiogenesis, the development of blood or lymphatic vessels from pre-existing vessels, are intimately linked processes that share many common molecular regulators. To orchestrate the development of these two distinct vascular systems in the vertebrate embryo, the function of many reiteratively acting molecular pathways is expected to be regulated with exquisite precision. We performed a forward genetic screen in zebrafish to identify mutants that fail to form embryonic lymphatic vessels and isolated the mutant expando. Positional cloning identified expando as a flt4 signalling deficient mutant but arterial hemangiogenesis occurred normally in these mutants. Recent data have demonstrated that Flt4 signalling is required for hemangiogenesis and that Dll4 limits angiogenic potential by limiting Flt4 function in developing vessels. To test whether flt4 acts downstream of dll4 in zebrafish, we knocked down dll4 in the flt4 mutant background and found that the hyper-branching phenotype observed s more sensitive to lack of Gremlin 2. Consistent with this idea, our results show that injection of gremlin 2 mRNA in zebrafish embryos expands the atrial territory and restricts ventricular development. Our results also show that treatment of differentiating mouse embryonic stem cells with Gremlin 2 stimulates expression of atrial genes. Gremlin 2 induces atrial-specific transcription factors, whereas it blocks expression of suppressors of atrial differentiation. Our results suggest that Gremlin 2 controls the activity of signaling pathways that promote atrial fate leading to novel strategies for differentiation of pure populations of stem cell-derived atrial cardiomyocytes. This knowledge may also impact our treatment of degenerative atrial diseases. Posters 320 Cardiovascular system 250 The Netrin receptors Unc5b and DCC are required for parachordal vessel formation during vascular development A.H. Lim1, A. Suli1, D.Y. Li2 and C-B. Chien1 1 Dept of Neurobiology and Anatomy, 2Dept of Oncological Sciences, University of Utah, USA Recent evidence has shown that axon guidance molecules play an important role in vascular development. In particular, we have shown (Wilson et al., 2006) that Netrin1a is required for the formation of the zebrafish parachordal vessel (PAV). Here we address the identity of the cells and receptors that receive this Netrin1a signal. Zebrafish trunk vasculature development begins with the formation of the dorsal aorta (DA) and posterior cardinal vein (PCV). The PCV gives rise at ~32hpf to the secondary sprouts, which grow dorsally until they reach the horizontal myoseptum (HMS), then grow laterally into the superficial muscle, and then turn anteriorly and posteriorly to give rise to the PAV. Prior to and during PAV formation, Netrin1a is expressed by the muscle pioneers, which lie along the HMS. Knocking down netrin1a or blocking muscle pioneer formation both prevent formation of the PAV (Suli et al., submitted). When we use morpholinos to knock down the known Netrin receptors Unc5b or DCC, the PAV also fails to form. In these morphants as well as Netrin1a morphants, secondary sprouts form from the PCV, but they fail to turn at the HMS to form the PAV. Thus, Netrin1a from the muscle pioneers acts to promote PAV formation, likely mediated through Unc5b and DCC. However, we are unable to detect unc5b or dcc mRNA in secondary sprouts by in situ hybridization, raising the question of whether these receptors act autonomously in endothelial cells, or perhaps elsewhere. It has often been hypothesized that axons might guide endothelial cells, and netrin signaling through DCC and/or Unc5 homologs is known to be important in axon guidance for several neuronal types. In fact, the HMS is contacted by axons of the primary motor neurons. In particular, the Rostral primary motor neuron (RoP) develops shortly before the PAV, and courses along the HMS just as the PAV does. We are now testing whether Unc5b and DCC act in endothelial cells, or whether instead they may guide motor axons which are necessary for formation of the PAV. 321 Posters 251 Cardiovascular system Characterization of tbx5b, a novel paralog of tbx5a, during zebrafish cardiac development S. Park, T. Camarata, J. Topczewski, H-G. Simon Department of Pediatrics, Children's Memorial Research Center, Northwestern University Chicago, USA T-box proteins (tbx) are an evolutionarily conserved family of transcription factors comprised of 19 members in zebrafish Danio rerio. Gene paralogs (homologous genes that have evolved by duplication) have been reported in the zebrafish genome. Among the T-box family members, no tail a/b, tbx2a/b, and tbx3a/b represent examples of gene duplication. Here we present the cloning and characterization of tbx5b, a novel paralog to zebrafish tbx5a. The tbx5a heartstrings (hst) mutation results in severe cardiac abnormalities of the zebrafish two-chambered heart and absence of pectoral fins; and similarly, Tbx5 in the mammalian four-chambered heart is implicated in congenital heart disease. Our studies show that like tbx5a and its mammalian orthologs Tbx5 (mouse)/TBX5 (human), tbx5b expression is critical for heart development. First, based on amino acid sequence, tbx5b is significantly diverged from tbx5a, while retaining the highest homology in the DNA-binding T-domain, indicating that tbx5b retained its function as a transcription factor. tbx5b expression is dynamic in the heart tube and dorsal eye, paralleling but not identical to the expression pattern of tbx5a. Surprisingly, however, tbx5b lacks expression in the fin bud, suggesting a divergence in function between the two genes. Embryonic heart tubes of tbx5b anti-sense morpholino-treated embryos do not undergo looping morphogenesis, a critical step in shaping the vertebrate heart. Moreover, tbx5b morphants demonstrate misregulation of cardiac valve markers, bmp4 and versican. We tested the hypothesis that simultaneous loss of both zebrafish tbx5 paralogs would result in compound cardiac defects. By knocking down tbx5b on an hst-/- background, we found that a linear heart tube does form, but with attenuated expression of the cardiac marker, cmlc2 at 48hpf. In addition to a comparative assessment of their roles in development, we also examined the property of tbx5b protein to shuttle between the nuclear and cytoplasmic compartment, a feature previously demonstrated in our laboratory for tbx5a. Indeed, like tbx5a, tbx5b localizes to the cytoskeleton through direct contact with the actin-binding PDZ-LIM protein pdlim7. In summary, the zebrafish paralogs tbx5a and tbx5b have conserved sequence homology and functional properties. Our comparative analysis has identified similar and divergent roles for tbx5a and tbx5b in zebrafish development. These studies may lend further insight to the evolutionarily conserved role of T-box proteins in development across species, and to the functional consequence of TBX5 mutations in human genetic disease. Thus the identification of zebrafish tbx5b affords an opportunity to examine the evolution of gene function in relation to embryonic development. Posters 322 Cardiovascular system 252 VE-Cadherin is required for endothelial rearrangements during ISV Formation H-G. Belting, Y. Blum, L. Herwig, A. Krudewig, E. Ellertsdottir and M. Affolter Dept. of Cell Biology, Biozentrum/Uni Basel, Switzerland The intersegmental blood vessels (ISVs) of the zebrafish embryo serve as a paradigm to study sprouting angiogenesis in vivo. We have previously shown that during ISV formation endothelial cells (ECs) divide extensively during sprout outgrowth. Furthermore, by mosaic labelling experiments and examination of junctional proteins, we have shown that ISVs are multicellular tubes containing an extracellular lumen. To examine the role of VE-Cadherin (VE-Cad) during ISV formation we have performed morpholino knockdown experiments. VE-Cad morphants display multiple cardiovascular defects. Initial sprouting of the ISV appears normal but they become disrupted during subsequent stages. Analysis of junctional proteins and in vivo time-lapse analyses indicate that in the absence of VECad, ECs fail to perform cellular rearrangements, which are required for proper vessel formation. Taken together our findings suggest that VE-Cad is required for migratory behavior of ECs that occurs during vessel assembly. We propose a revised model of ISV formation, in which "scaffold formation" and "vessel assembly" participate as two distinct morphogenetic events and only the latter depends on VE-Cad function. 323 Posters 253 Cardiovascular system Hand2 Ensures a Proper Environment for Cardiac Fusion Through Control of Fibronectin Levels Z. Garavito-Aguilar and D. Yelon Skirball Institute, NYU School of Medicine, New York, USA The vertebrate heart results from the fusion of two separate populations of cardiac precursors in the early embryo. How these populations come together, how they interact with their environment, and how their fusion is regulated is poorly understood. Here, we focus on the role of Hand2, a bHLH transcription factor and an essential regulator of heart d immunity. 325 Posters 255 Hematopoiesis and immunology Watching thymopoiesis in stable transgenic zebrafish embryos I. Hess, M. Schorpp, D. Diekhoff, T. Boehm Max-Planck-Institute of Immunobiology, Freiburg, Germany In zebrafish, the thymic anlage develops from the pharyngeal endoderm as a bilateral structure, first detectable at 48 hpf. It is colonized by lymphocyte progenitor cells, which differentiate into mature T cells. The thymus provides a special microenvironment for the interaction of lymphoid and stromal cells that is required to establish a self-tolerant repertoire of T cells. Lymphocyte development, comprising the homing to the thymus and their intrathymic differentiation is directed by a complex network of different transcription factors. One of these key factors is ikaros. In zebrafish, ikaros expression is first detected at 24 hpf in the intermediate cell mass (ICM), the first hematopoietic structure detectable in the embryo. By 54 hpf and at later stages it is expressed in the thymus together with other marker genes characteristic of lymphoid differentiation, like rag1. Another important factor is foxn1, a regulator of thymic epithelial cell (TEC) differentiation. In zebrafish its expression is detectable from 48 hpf onwards. In order to study the process of thymopoiesis in vivo, we generated ikaros::eGFP and foxn1::mCherry transgenic lines using modified BAC-DNA. These lines allow us to follow the development of the thymic epithelial anlage and the homing of prothymocytes to the thymus in long-term confocal time-lapse imaging. Using the ikaros::eGFP line, we describe the temporospatial characteristics of the migratory pattern of hematopoietic cells from the ICM, the AGM (aorta-gonad mesonephros) and the CHT (caudal hematopoietic tissue) to the thymic anlage. Using the foxn1::mCherry line, we describe the temporo-spatial development of the thymic anlage. We also use these transgenic lines to characterize the factors influencing thymus development and T cell homing in vertebrates. The former is investigated by introducing various mutations affecting thymopoiesis, the latter by functional interference with gene-specific antisense morpholinos. Of special interest to us is the process of thymus homing. We show that injection of morpholinos (MOs) directed against foxn1 leads to aberrant migration of hematopoietic progenitor cells, indicating that the differentiation of TECs and the interaction of stromal and lymphoid cells is important for normal immigration of prothymocytes into the thymus and subsequent T cell development. We also show, that ccl25a/cxcl12a morphants show normal numbers of lymphoid progenitors but their thymus homing is impaired, indicating that these chemokines play an important role in thymus homing. Posters 326 Hematopoiesis and immunology 256 LPS enhanced neutrophil recruitment in a zebrafish embryo injury model is p38 MAP kinase dependent H.B. Taylor1, S.B. Brown2, J.R. Lamb3, M.J. Dallman3 1 Division of Cell and Molecular Biology/CIR, Imperial College London/University of Edinburgh, London/Edinburgh, UK; 2CIR, University of Edinburgh, UK; 3Division of Cell and Molecular Biology, Imperial College London, London, UK Recruitment of leukocytes is an important component of the complex inflammatory response to local injury and infection where the mechanisms of recruitment remain incompletely understood. Here we use a zebrafish embryo injury model to study and image this process. LPS was found to enhance neutrophil recruitment to a tail wound via the activation of p38 MAPK but was independent of zTLR4a and zTLR4b. zTLR4a and zTLR4b were differentially expressed in embryo and adult tissue. Zebrafish myelomonocytes did not display morphological activation when challenged with LPS but adult whole kidney marrow derived cells did respond to LPS stimulation in a dose-dependent manner with increased expression of TNFa and TNFb cytokine. Fish functional and transcriptional responses to LPS and other PAMP challenges are relatively unknown. The results presented in this study complement recent findings that suggest LPS recognition and TLR4 signalling in fish are different from the mammalian system. This model allows in vivo investigation of the mechanisms of LPS recognition in fish and evaluation of other modulators of leukocyte recruitment and the acute inflammatory process independent of TLR4 activation. 327 Posters 257 Hematopoiesis and immunology Transcriptome responses and receptor signalling during salmonella infection of zebrafish embryos A. H. Meijer1, O. W. Stockhammer1, A. Zakrzewska1, Z. Hegedus2, H. P. Spaink1 1 Institute of Biology, Leiden University, Leiden, The Netherlands; 2ZenonBio Ltd. and Biological Research Center, Szeged, Hungary Due to the clear separation of innate immunity from adaptive responses, the externally developing zebrafish embryo represents a useful in vivo model for identification of innate host determinants of the response to bacterial infection. We used microarrays and novel deep sequencing technology to perform a detailed time-course transcriptome profiling study and gene ontology analysis of the embryonic innate immune response to infection with two model Salmonella strains that elicit either a lethal infection or an attenuated response. The transcriptional response to infection with both the lethal strain and the avirulent LPS O-antigen mutant showed clear conservation with host responses detected in other vertebrate models and human cells, including induction of genes encoding cell surface receptors, signalling intermediates, transcription factors and inflammatory mediators. Furthermore, our study led to the identification of a large set of novel immune response genes and infection markers, the future functional characterization of which will support vertebrate genome annotation. Although microarrays and deep sequencing identified similar functional groups of genes, the unbiased nature of deep sequencing provided insights that microarray analysis could not have achieved, such as infection-dependent transcript isoform switching, expression of novel splice products not present in the current transcript and EST databases, and a high level of antisense transcription. Our transcriptome profiling study revealed specific regulation of Toll-like receptor 5 and downstream signalling components. Using knockdown analysis we showed that zebrafish TLR5 is required for the recognition of Salmonella flagellin, which is the first demonstration of a conserved TLR ligand specificity in this model. We also showed that zebrafish embryos use both MyD88-dependent and -independent signalling pathways and identified novel immune response targets of TLR5 and MyD88 signaling. Posters 328 Hematopoiesis and immunology 258 Using zebrafish to investigate the molecular control of hypoxic signalling during inflammation P. Elks1,2, F. van Eeden1,2, M.K.B Whyte2, S. Walmsley2, S. Renshaw1,2 1 MRC Centre for Developmental and Biomedical Genetics, University of Sheffield Medical School, UK; 2Academic Unit of Respiratory Medicine, University of Sheffield Medical School, UK Diseases of neutrophilic inflammation are an increasing problem in the developed world, and remain largely untreatable. The genetic mechanisms by which neutrophil function is regulated remain poorly understood, and a greater knowledge of these mechanisms may lead to the identification of therapeutic targets. Hypoxia is a profound stimulus prolonging neutrophil functional lifespan. The hypoxia genetic pathway regulates the stability of the hypoxia inducible factor (HIF) protein in an oxygen dependent manner. In normoxia, HIF is phosphorylated by hydroxylases and targeted for degradation. In hypoxia, the hydroxylases are inactivated and HIF is stabilised allowing transcription and activation of downstream targets. However, the HIF pathway can be activated by inflammatory stimuli independently of hypoxia. Neutrophils were visualised in the zebrafish using a transgenic line in which green fluorescent protein (GFP) is driven by the expression of the neutrophil specific gene myeloperoxidase. The hypoxia pathway was upregulated in these embryos by the addition of dimethyloxaloylglycine (DMOG) or by outcrossing to vhl or fih null TILLING mutants. Tail transection induces a selflimiting neutrophilic inflammatory response that can be observed using fluorescent microscopy techniques. Activation of the hypoxic signalling pathway with DMOG was found to significantly delay the resolution of inflammation. This delay in resolution was not observed in vhl-/- or fih-/mutant embryos. Neutrophil behaviour was investigated using a novel spinning disk confocal microscopy timelapse approach. The velocity and meandering index of neutrophils was not found to significantly differ after the upregulation of the hypoxia pathway by DMOG or in the TILLING mutants. Using molecular biology techniques, dominant active and dominant negative forms of zebrafish hif1 have been cloned. Initial studies have indicated that these successfully upregulate or downregulate the HIF pathway respectively, by microinjection into a phd3:GFP transgenic line. PHD3 (prolyl hydroxylase 3) is a known downstream target of HIF1 and thus the Tg(phd3:GFP) line acts as a readout of the hypoxia pathway. These mutant forms of hif1 require further characterisation before specifically expressing them in neutrophils to observe their effects on neutrophil behaviour and the resolution of inflammation. These studies highlight the potential of the zebrafish model to aid in our molecular dissection of the hypoxic signalling apparatus during in vivo physiological processes such as inflammation resolution. Posters 329 259 Hematopoiesis and immunology Zebrafish Mast Cells Demonstrate Conserved Innate and Adaptive Immune Responses S. Da'as1, E.M Teh1, J.T. Dobson 1,2 ,D.S. Neuberg4, J. Marshall2, T-J. Lin1,2,3, J. Berman1,2,3 1 IWK Health Centre, Departments of 2Microbiology and Immunology and 3Pediatrics, Dalhousie University, Halifax, Canada, 4Department of Biostatistics and Computational Biology, DanaFarber Cancer Institute, Boston, USA Mast cells (MCs) are multifunctional immune cells derived from hematopoietic stem cells that complete maturation where they take up residence, namely in tissues exposed to the external environment. These anatomic locations position them to play a critical primary regulatory role in eliciting both innate and adaptive immune responses. The zebrafish has emerged as a powerful new model system for studying infection and immunity owing to conserved cell biology, and ease of manipulation and phenotypic analysis due to ex-utero embryonic development. We were the first (Dobson et al., Blood 2008) to identify MCs in zebrafish gills and intestine and carboxypeptidase A5 (cpa5) as a developmental marker of both embryonic progenitors and mature MCs. Intraperitoneal injection of compound 48/80, a MC activator, resulted in MC degranulation and elevated plasma tryptase levels, which can be reduced following treatment with the MC stabilizing agent, ketotifen. Similarly, infection with heat-inactivated A. salmonicida or the fungal wall constituent, zymosan, resulted in zebrafish MC degranulation, increased plasma tryptase levels and interestingly, eosinophil recruitment. These results suggest MCs participate in innate immune responses to pathogens, which may be mediated through well-conserved Toll-like receptors. Mammalian MCs are better known for adaptive immune responses mediated through IgE/FcRI signaling. We are characterizing an analogous pathway in the zebrafish. Structurally, we have demonstrated positive immunostaining in zebrafish MCs for a rabbit-derived polyclonal antibody that specifically targets the gamma subunit of the human high affinity IgE receptor. Using whole mount RNA in situ hybridization on 7 day embryos, we subsequently showed colocalization of zebrafish homologues of IgE receptor subunits with expression of cpa5, suggesting the presence of an IgE-like receptor on zebrafish MCs. Functionally, zebrafish MCs sensitized with mouse anti-DNP IgE followed by injection of DNP-BSA responded by degranulation and leukotriene production. Our studies reveal conserved MC function in zebrafish innate and adaptive immune reponses, effectively establishing the zebrafish as a novel organism for evaluating vertebrate MC activity. Ultimately, we will be able to exploit the zebrafish system as an in vivo platform for high-throughput screening of potential MC stabilizing/inhibiting agents, with a goal of identifying new effective therapies for allergic, inflammatory, and malignant MC diseases. Posters 330 Hematopoiesis and immunology 260 Notch signaling is required for mast cell development in the zebrafish S. Da'as,1 L. Rygier,2,3 A. Ferrando4, J.N. Berman1,2,3 1 IWK Health Centre, Departments of 2Biology and 3Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada, 4Institute for Cancer Genetics, Columbia University Irving Cancer Research Center, New York, USA The molecular pathways regulating mast cell (MC) development in vertebrates remain to be elucidated. The Notch signaling pathway is highly conserved in all metazoans and has been implicated in regulating hematopoietic stem cell induction and lineage cell fate decisions. Notch receptors and their ligands are expressed in a number of hematopoietic cells, including MCs. We were the first to identify zebrafish MC equivalents (Dobson et al., Blood 2008) and examine vertebrate MC transcriptional regulation in vivo. These studies demonstrated the significance of carboxypeptidase A 5 (cpa5) as a zebrafish MC-specific marker. We have now shown that the zebrafish Notch signaling mutant, mind bomb, displays profound loss of cpa5 expression. Similarly, wild type zebrafish embryos treated with Compound E, (Cpd E), a -secretase inhibitor that inhibits Notch signaling, show a similar phenotype. We previously identified pu.1 and gata2 as essential transcription factors for early MC development. Wild type zebrafish embryos treated at 50 M Cpd E show decreased gata2 expression, but wild type pu.1 and gata1 expression, suggesting a particular sensitivity of the MC lineage to Notch pathway inhibition mediated through gata2. At 75 M Cpd E, we observed severe reductions in gata2, pu.1, gata1, and mpo, suggesting a broader dose dependent role for Notch pathway signaling in both myeloid and erythroid lineage development. Our data suggest that Notch signaling is a critical pathway necessary for MC development in the zebrafish. Reciprocal experiments overexpressing notch mRNA in wild type embryos and rescue experiments overexpressing pu.1, gata-2 and other transcription factors in embryos in which the Notch pathway is absent (mind bomb and Cpd E treated) are underway to determine the specific signaling cascade downstream of Notch. A Notch green fluorescent protein (GFP) reporter zebrafish line has been generated in the laboratory, which will enable the tracking of Notch signaling in developing and migrating MCs. Moreover, a second transgenic line generated in the laboratory expressing the human CKIT D816V mutation found in the preleukemic condition, systemic mastocytosis, will be employed to test Notch pathway inhibitors in this condition. Parallel studies in a human mastocytosis cell line are also being undertaken. These studies promise key insight into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model for identifying novel therapeutic strategies in MC diseases. 331 Posters 261 Hematopoiesis and immunology ENU-mutagenesis in zebrafish identifies a specific requirement for Lsm8 in thymus development N. Iwanami, M. Schorpp, and T. Boehm Max-Planck-Institute of Immunobiology, Freiburg, Germany Proper development of T lymphocytes in the thymus is necessary for the establishment of acquired immune systems in vertebrates. Understanding the mechanisms for T lymphocyte and thymus development is crucial for controlling immunodeficiency syndromes and autoimmune diseases. However, the relevant molecular pathways are not fully understood. We have previously established a panel of ENU-mutagenized zebrafish lines with abnormal accumulation of rag1expressing immature T-cells in the thymus. At the time of this writing, we have identified 10 mutations among 41 lines. Here we report on one of the mutants, WW18/10. Homozygotic WW18/10 mutants have no apparent developmental abnormality including craniofacial structures, but exhibit a reduction of immature T-cells in the embryonic thymus. Positional cloning identified a nonsense mutation in the Lsm8 gene. Lsm8 encodes a member of Sm-like proteins associated with U6 snRNA in the spliceosome. Possible functions of Lsm8 will be discussed. Posters 332 Hematopoiesis and immunology 262 Characterizing zebrafish defensins N. L. Reynolds1, D. Macmillan2, C. A. Semple1, E.E. Patton3, J. R. Dorin1 1 Institute for Genetics and Moecular Medicine, MRC Human Genetics Unit, Western General Hospital, Crewe Road South, Edinburgh, UK 2Department of Chemistry, University College London,UK 3Institute for Genetics and Molecular Medicine, Edinburgh Cancer Research Centre, Western General Hospital, Crewe Road South, Edinburgh, UK The -defensins make up one of the largest groups of antimicrobial peptides and are important innate immune components in a variety of species throughout the animal and plant kingdoms. Although the -defensins were first identified for their broad-spectrum anti-microbial activities, vertebrate -defensins also have well-documented roles in the chemoattraction of adaptive immune cells such as immature dendritic cells, macrophages and T cells. This provides an important link between the innate and adaptive immune systems. The receptor through which beta-defensins chemoattract macrophages is as yet unknown. This work builds on the initial reporting of three zebrafish -defensin-like genes (Zou et al, 2007) and aims to assign functionality to these genes by analysing their expression, function and inducibility in both embryos and adult fish. This has been achieved using a combination of morpholino knockdowns, in-situ hybridisations and quantitative RT-PCR, in addition to in vitro anti-microbial assays. In addition, the zebrafish system was exploited to further define the reported interactions between Human -defensin 3 (HBD3) and the Mc1r receptor (Candille et al, 2007). Zebrafish are able to background adapt by dispersing the pigment (melanin) within their melanophores in order to appear darker in colour. This process can be observed under the microscope and is controlled by interactions between -MSH and Mc1r. This system has been exploited using freshly prepared, cultured zebrafish melanophores to directly observe the effects that a panel of human -defensins have on melanophore pigment dispersal both before and after the morpholino knockdown of Mc1r. Overall, this work describes the novel use of the zebrafish model to investigate the function of both intrinsic and mammalian -defensins. This also represents the development of a novel, valuable disease model for the study of -defensins in this research group. Candille SI et al (2007) A -defensin mutation causes black coat color in domestic dogs. Science 318(5855) :1418-23 Zou J. et al (2007) Discovery of multiple beta-defensin like homologues in teleost fish. Mol Immunol 44(4) : 638-47 333 Posters 263 Hematopoiesis and immunology Specific combinations of CRFB chains constitute receptors for the virus induced IFNS in zebrafish D. Aggad1, M. Mazel1, P. Boudinot3, O. J. Hamming4, R. Hartmann4, S. Kotenko5, P. Herbomel2, G. Lutfalla1, J-P. Levraud2 1 CNRS, Montpellier, France; 2Institut Pasteur, Paris, France; 3INRA, Jouy-en-Josas, France; 4Aarhus University, Denmark; 5New Jersey Medical School, Newark, USA The number of fish proteins identified as cytokines by sequence homology has grown tremendously, but the identity of their receptors is difficult to predict. The receptors of class II helical cytokines (interferons and IL-10-related cytokines) are heterodimers of CRFB transmembrane proteins, of which at least 13 members exist in the zebrafish genome. We have previously identified functionally the receptor of the first viro-induced interferon identified in the zebrafish (IFN1, also known as IFN1 or IFNL1) as a dimer of CRFB1 and CRFB5. Since then, the number of fish viro-induced interferons has expanded, with two distinct classes defined according to the number of conserved cystein pairs. Zebrafish IFN1 belongs to class I; the more recently described IFN2 and IFN3 belong to class II. While these three genes sit on a single chromosomic cluster, we have identified on a different chromosome a fourth sequence, encoding a class I member: IFN4. By overexpression studies or injection of recombinant proteins, we found that these four IFNs induce the expression of antiviral genes such as viperin and increase the resistance of zebrafish larvae to a viral challenge, with IFN1 appearing the most effective while IFN4 has only marginal potency. Using CRFB gain- and loss of function experiments, we found that not all IFNs bind to the same receptor; the two class I IFNs signal through the previously described receptor, while the two class II IFNs rely on a different dimer. Both receptor complexes include a common short chain, associated to a specific long chain. Posters 334 Hematopoiesis and immunology 264 TIF1gamma regulates the erythroid and myeloid lineage output from haematopoietic progenitors R. Monteiro and R. Patient MHU, WIMM, University of Oxford, UK During vertebrate development, primitive haematopoiesis is followed by a definitive wave that generates haematopoietic stem cells (HSCs), which maintain haematopoiesis throughout adulthood. HSCs arise in association with the ventral wall of the dorsal aorta. From 2dpf onwards in the zebrafish, they migrate to the caudal haematopoietic tissue (CHT, the foetal liver equivalent), where they co-exist with locally arising erythromyeloid progenitors, and finally to the thymus and to the kidney (the bone marrow equivalent). At present, little is known about how the differentiation potential of HSCs in the different niches is regulated. To address this, we made use of the zebrafish mutant moonshine (mon). mon harbours a mutation in the nuclear RING domain ubiquitin ligase, transcriptional intermediate factor 1 (TIF1 or Trim33), a factor essential for primitive red cell survival. Because mon mutants are devoid of primitive red cells, later waves of haematopoiesis can be studied without the interference of circulating primitive cells. At 4dpf, cells expressing erythroid and myeloid markers are found in the CHT. Although erythroid markers are also expressed in the CHT of mon mutants, no recovery of circulating red blood cells was observed, suggesting that these cells are unable to complete their maturation in the absence of TIF1. Thus, TIF1 is required for the survival or differentiation of the erythroid cells arising de novo in the CHT. Surprisingly, TIF1-deficient embryos showed a dramatic increase in expression of myeloid markers in the CHT, suggesting TIF1 is required to restrict myeloid cell fate in this haematopoietic niche. We have thus uncovered a novel role for TIF1 in regulating the erythroid and myeloid differentiation output from haematopoietic progenitor cells residing in the CHT niche. 335 Posters 265 Hematopoiesis and immunology Live imaging demand-driven haematopoiesis during infection C. Hall, M. Vega Flores, A. Chien, E. Lam, T. Storm, T. Purea, A. MacDonald, K. Crosier and P. Crosier Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, New Zealand The rapid response of the innate immune system to a pathogenic challenge is critical for the ultimate wellbeing of the host. During normal `steady state' haematopoiesis, haematopoietic stem cells (HSCs) undergo a step-wise reduction in lineage potential to generate the full complement of blood lineages, including myeloid leukocytes of the innate immune system that are crucial for host protection. These innate immune cells have a finite life span so a mechanism for rapid replenishment is essential. During the stressed state of infection it has long been known that the host has the potential to regenerate depleted immune cell stocks via `emergency' or `demand-driven' haematopoietic activ to that in mammals with evolutionary conserved genetic programs which drive the formation of analogous blood cell types. In Vertebrates blood cells formation consists in two successive waves. The primitive hematopoiesis produces predominantly erythrocytes and only some primitive macrophages while definitive hematopoiesis provides long term Hematopoietic Stem Cells (HSCs) able to give rise to mature blood lineages. The Notch signaling is an evolutionary conserved regulatory system implicated in regulating cell fate determination in various developmental processes included HSCs self renewal and blood lineages differentiation both in vitro and in vivo. One known inhibitor of Notch activity is the evolutionary conserved adaptor protein Numb. In mice, several lines of evidence suggest that Numb (Nb) and its homologue Numblike (Nbl) play redundant functions in specifying and maintaining neuronal differentiation. The expression of Nb and Nbl has been detected in most of the tissues of mouse embryos including the yolk sac and adult hematopoietic tissues in both mouse and human models. Hence, these findings rise the possibility that Nb and Nbl proteins could play a role also in the embryonic and adult hematopoietic system. It has been proposed that both Nb and Nbl are dispensable for hematopoiesis in adult mice but recent in vitro approaches provided evidences that Numb can modulate the specification of primitive erythrocytes through its interaction with Notch. Numb and Numblike homologues have been identified and cloned also in zebrafish where they are expressed in the whole embryo from the first cleavage stage to organogenesis stages and by 24hpf their expression become restricted to the anterior half of the embryo. Using morpholino antisense oligonucleotide (MO) knock-down of Numb and Numblike we observed embryos in which circulating blood cells are absent or severely reduced starting from the earliest time point that circulation can be detected. In order to assess the presence of differentiated primitive erythrocytes in nb/nbl morphants we analyzed the erythroid hemoglobin content by o-dianisidine staining in 48hpf morphants and we observed that only few differentiated red blood cells were detectable. Furthermore, to gain insight into the erythropoietic defects we injected the nb/nbl morpholino in the transgenic line Tg(gata1: DsRed). Injected embryos at about 28-30hpf showed that, at this stage of development, the overall fluorescence of nb/nbl morphants start to appear strongly reduced and at 48hpf and 72hpf only a little amount of red cells is present within the sinus venosus. Taking together, our results provide the first in vivo evidence of an involvement of Numb and Numblike in erythrocytes determination and differentiation during primitive hematopoiesis. Posters 340 Hematopoiesis and immunology 270 The Hox cofactors Pbx and Meis1 act upstream of gata1 to regulate primitive erythropoiesis L.M. Reaume1, T. Erickson1, A.M. Forrester2, J.N. Berman2, A.J. Waskiewicz1 1 Department of Biological Sciences, University of Alberta, Edmonton, Canada 2Department of Pediatrics and Microbiology/Immunology, IWK Health Centre, Dalhousie University, Halifax, Canada Vertebrate embryos bearing deletions in individual hox genes display a partial loss of blood cells, demonstrating a role for these transcription factors in regulating certain aspects of erythroid lineage specification. However, the overlapping expression patterns and functional redundancy of posterior hox genes have prevented us from defining their role in regulating hematopoietic transcriptional gene networks. The specificity of Hox proteins is achieved through their interaction with other DNA-binding cofactors. These include the Three Amino acid Loop Extension (TALE)class homeodomain transcription factors Pbx (Pre-B-Cell Leukemia Homeobox) and Meis (Myeloid Ecotropic Integration Site). Removal of Pbx generates an anteriorizing phenotype in the zebrafish hindbrain, in which rhombomeres 2-6 take on the identity of rhombomere 1. An identical phenotype results from the loss of Hox1 gene products in Xenopus, indicating the significant role that TALE-class proteins play as Hox cofactors in vivo. In order to model global loss of Hox function in zebrafish primitive hematopoiesis, we have ablated the Hox cofactors Pbx and Meis1. Embryos lacking Pbx and Meis1 fail to produce visible circulating blood cells, and exhibit a severe reduction in the expression of gata1, the earliest marker of erythroid cell fate. Concomitant with a loss of gata1, Pbx and Meis1-depleted embryos initiate, but fail to maintain scl hemangioblast gene expression, and possess increased numbers of pu.1-positive myeloid cells. Furthermore, gata1 overexpression is able to drive scl expression in Pbx and Meis1-depleted embryos. Combined, these results place Pbx and Meis1 upstream of gata1 in the erythropoietic hierarchy. hoxb7a-overexpression rescues gata1 expression in Pbx-depleted, but not Meis1-depleted embryos. We therefore propose a model whereby Hox acts in association with Pbx and Meis1, upstream of gata1, to specify the primitive erythropoietic cell lineage and inhibit myelopoiesis. 341 Posters 271 Hematopoiesis and immunology Using transgenic zebrafish to screen for small molecule inducers of inflammation resolution C. A. Loynes, A. L. Robertson, M. K.B. Whyte, P. W. Ingham, S. A. Renshaw Academic unit of Respiratory Medicine and MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Western Bank, Sheffield, UK Rationale: Diseases of neutrophilic inflammation are common, affect many organ systems, and respond poorly to current therapies. There is a major unmet need to identify new ways to treat such diseases. Neutrophils are usually removed by macrophages having undergone apoptosis, but during inflammatory diseases survival signals delay this apoptosis leading to enhanced inflammation. Methods: We have established a tractable model in transgenic zebrafish expressing GFP in the neutrophil lineage, in which inflammation resolution can be rapidly quantitated in vivo. Sterile physical injury to the tailfin of anaesthetised larvae leads to a reproducible and quantifiable neutrophilic inflammatory response which spontaneously resolves over time. This permits screening of compound libraries to identify compounds which accelerate inflammation resolution. Results: Preliminary experiments have demonstrated the practicality of such screens, and have identified several lead compounds that serve as "proof of principle", demonstrating the utility of this approach in the identifcation of new immunotherapeutics. From the Spectrum collection (MSdiscovery), 960 compounds were tested, of which 12 were shown to have reproducible effects. These include several known anti-inflammatory agents. Of these, a number have been tested and shown to exhibit dose-dependent effects. Active compounds suppressed neutrophilic inflammation to levels below those seen with our positive control, a potent inducer of neutrophil apoptosis, pyocyanin. For example, in control fish the number of neutrophils present at the site of injury at 24 hours post-injury was 30.24+/-1.47. In pyocyanin treated fish it was 19.04+/-1.93, and for one compound it was 12.33+/-1.85 (mean+/- sem, p<0.05 for both treatments vs control, one-way ANOVA with Bonnferroni post-test correction, n=49, 24 and 9 respectively). These compounds are under further investigation for their ability to modulate human neutrophil function and will be assessed for enhancement of resolution of inflammation in mammalian models of neutrophilic pulmonary inflammation. Conclusions: These data show the ability of this model to identify novel therapeutics with dramatic immunomodulatory properties. Some of these compounds may be useful lead compounds for the identification of novel therapeutic entities. Posters 342 Hematopoiesis and immunology 272 Discovery and expression analysis of important markers of T-cell subsets in the zebrafish (Danio rerio) S. Mitra, A. Alnabulsi, C. Secombes and S. Bird Scottish Fish Immunology Research Centre (SFIRC), School of Biological Sciences, University of Aberdeen, UK The zebrafish (Danio rerio) is now recognized as a useful vertebrate model to understand immunity and considerable progress has been made in understanding the molecular basis of normal white blood cell development. Despite this, however, very little is known about T-helper (Th) cell development in fish and whether cell subsets such as Th1, Th2, Th17 and Treg cells exist, as in mammals. Although CD4 and CD8 have recently been characterised in teleosts, the absence of other cell markers, which include T-cell specific transcription factors (Th-POK, STAT6, FoxP3 & T-bet) means we cannot firmly conclude that development and functional properties of T-cell subsets in fish parallel those of mammals. Using these transcription factors that play a critical role during development of T-cell subsets and looking at their distribution in a tissue specific and time specific manner would begin to reveal important details of the immune system of lower vertebrates. Using the zebrafish genome and a synteny approach, we have identified CD4, CD8, Th-POK, STAT6, T-bet, and FoxP3 in zebrafish. Despite high levels of divergence with some of these genes at the sequence level, when compared with other vertebrates, these genes exhibit a striking conservation of gene order, suggesting that selection has maintained gene order among the vertebrates over hundreds of millions of years of evolution. Using quantitative PCR (Q-PCR), Whole In Situ Hybridisation (WISH) and a FoxP3 polyclonal antibody, the expression of T-cell subset markers is investigated in the developing and adult zebrafish. 343 Posters 273 Hematopoiesis and immunology Elucidating the roles of notch ligands, receptors and downstream targets in embryonic haematopoiesis and angiogenesis K. A. McMaahon, J. Rowlinson, M. Gering Institute of Genetics, Nottingham University, UK The Notch pathway has been shown to play an important role in the formation of definitive haematopoietic stem cells (HSCs) in both mouse and zebrafish embryos. In the zebrafish, it is thought that Hedgehog signalling from the notochord induces Vegf signalling in neighbouring somites, which in turn induces notch activity in the dorsal aorta. This then leads to induction of the HSC programme in cells in the ventral wall of the vessel, through up-regulation of genes such as runx1 and c-myb. In support of this, previous work has shown that down regulation of notch signalling in the E3 ubiquitin ligase mutant mindbomb, leads to a loss of runx1 expressing cells in the dorsal aorta. Conversely, overexpression of notch intracellular domain in zebrafish embryos leads to ectopic expression of runx1 in the aortic roof and the vein. Although a general need for Notch has been identified in the formation of zebrafish HSCs, the exact mechanisms by which this pathway regulates the process have yet to be elucidated. We present a detailed dissection of the role of Notch in initiating definitive haematopoiesis. Using morpholino knockdown and over-expression studies, we have identified the notch receptors and ligands responsible for induction of the HSC programme. We also present data suggesting that the putative notch target Gridlock is in fact an upstream regulator of the pathway. In addition, a search for other putative Notch targets in the dorsal aorta has revealed a role for two Notch target genes in regulating angiogenesis. Posters 344 Hematopoiesis and immunology 274 The role of RGS18 in hematopoiesis and megakaryopoiesis S. Louwette1,2, C. Wittewrongel2, C. Vangeet 2,3, J. Arnout1, K. Freson2 1 Proefdierencentrum Katholieke Universiteit Leuven, Belgium; 2Department of Molecular and Cellular Medicine, CMVB, Katholieke Universiteit Leuven, Belgium; 3Department of Woman & Child, section Child and Department of Molecular and Cellular Medicine, CMVB, Katholieke Universiteit Leuven, Belgium Hematopoiesis is a life-long developmental process that involves the differentiation of hematopoietic stem cells. Myelopoiesis and lymphopoiesis are controlled by hematopoietic growth factors, including cytokinesther ROS. HyPer consists of the bacterial H2O2-sensitive transcription factor OxyR fused to a circularly permuted YFP. Cysteine oxidation of the OxyR part induces a conformational change that increases emission excited at 500 nm (YFP500) and decreases emission excited at 420 nm (YFP420). This change is rapidly reversible within the reducing cytoplasmic environment, allowing dynamic monitoring of intracellular H2O2 concentration. We introduced HyPer by mRNA injection into zebrafish embryos to induce global cytoplasmic expression. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting ~3 min after wounding and peaking at ~20 min that extended ~ 100-200 m into the tail fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (DUOX), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role. Posters 346 Hematopoiesis and immunology 276 Zebrafish miR-126 and miR-150 coordinately determine hematopoietic cell fate through c-Myb C. Grabher1, E.M. Payne1, A.B. Johnston1, N. Bolli1, E. Lechman2, J.E. Dick2, J.P. Kanki1, A.T. Look1, 3 1 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA; 2Division of Cell and Molecular Biology, University Health Network, and Department of Molecular Genetics, University of Toronto, Canada; 3Division of Hematology/ Oncology, Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, USA Precise regulatory mechanisms are required to appropriately modulate the cellular levels of transcription factors controlling cell fate decisions during blood cell development. The protooncogene c-myb functions at multiple stages during hematopoiesis, from hematopoietic stemand progenitor cells to fully differentiated cell types, and can affect the definitive specification of lymphoid, myeloid, erythroid and megakaryocyte lineages. Given the spatio-temporal diversity of its developmental roles, the mechanisms controlling the timing and levels of c-Myb activity must be precise and tightly maintained. Here, we demonstrate the direct regulation of c-Myb by miR-126 and the importance of the miR126/c-myb interaction during definitive hematopoiesis in the zebrafish. We show that miR-126 specifically binds to a predicted target site within the c-myb 3'UTR to attenuate translation of its upstream coding region. Knockdown of miR-126 results in increased c-Myb protein levels and promotes erythropoiesis at the expense of thrombopoiesis in vivo, while concomitant knockdown of c-myb alleviates these effects. We further provide evidence that specification of thrombocyte versus erythrocyte cell lineages is adjusted by the combined activity of the miRNAs miR-126 and miR-150. Both microRNAs are sequentially required but not sufficient individually to precisely regulate the cell fate decision between erythroid and megakaryocytic lineages during definitive hematopoiesis. Our observations support the notion that microRNAs not only act to provide precision to developmental programs but also are essential determinants in the control of variable potential functions of a single gene during hematopoiesis. 347 Posters 277 Hematopoiesis and immunology Gene/ Enhancer trap based screen identifies novel transgenic lines with reporter gene expression in haematopoietic and endothelial Cells R. Thambyrajah and M. Gering Institute of Genetics, School of Biology, University of Nottingham, Queen's Medical Centre, United Kingdom Vertebrate haematopoiesis occurs in two waves. The first wave arises from the anterior and posterior lateral plate mesoderm which give rise to primitive myeloid and to primitive erythrocytes respectively. The definitive wave generates haematopoietic stem cells (HSCs), which maintain the blood system throughout adulthood. HSCs are able to self-renew and to give rise to progenitors that differentiate into mature cells of all blood lineages. In zebrafish, the primitive erythrocytes and the definitive wave of haematopoiesis occur in the intermediate cell mass in the trunk of the embryo. As in other vertebrates, HSCs form in close association with the ventral wall of the dorsal aorta (DA). The cells subsequently enter the blood circulation via the vein, settle in the caudal haematopoietic tissue before seeding their final destination, the kidney, the site of adult haematopoiesis in the fish. We have conducted a transposon based gene/enhancer trap screen with the aims a) to identify novel genes involved in vasculogenesis and haematopoiesis and b) to fluorescently label endothelial and haematopoietic cells at different stages of development. Besides identifying a number of transgenic lines with tissue-specific gene expression in various non- haematopoietic and non- endothelial tissues, we have also found two lines that show GFP expression patterns of interest. One transgenic line has expression in primitive erythrocytes while a second line shows reporter gene expression in primitive erythrocytes, in myeloid cells as well as in the ventral wall of the DA. We are currently characterizing the lines further and will present a progress report at the meeting. Posters 348 Hematopoiesis and immunology 278 Characterization of medaka C-MYB mutant BENI FuJI, which displays defective hematopoietic progenitor differentiation; an insight into hematopoietic ontogenesis in medaka A. Moriyama1, K. Maruyama2, A. Kudo1 1 Department of Biological Information, Tokyo Institute of Technology 2Research Center for Radiation Protection, National Institute of Radiological Sciences Vertebrate hematopoiesis is characterized by two evolutionary conserved phase of development; primitive hematopoiesis which is a transient phenomenon in the early embryos that generate primitive erythrocytes and macrophages, and definitive hematopoiesis that takes place in the later stages accompanied with the emergence of hematopoietic stem cell that generate all hematopoietic lineage. Beni fuji (bef), was originally isolated as a medaka mutant that has apparently reduced number of erythrocytes in the peripheral blood. Positional-candidate cloning revealed that the bef mutants have a nonsense mutation in the c-myb gene. Previous studies have shown that c-myb is essential for definitive hematopoiesis and is widely used as a marker gene for the onset of definitive hematopoiesis. To analyze the phenotypes of bef mutants, we performed whole-mount in situ hybridization with gene markers of hematopoietic cells. At st32, the bef mutants showed decreased expression of erythrocyte marker -globin, and myelomonocyte marker l-plastin, and the expression of neutrophil marker, mpo1 was completely lost. These results suggest that the bef mutants have defects not only in erythrocytes but also in other myeloid which indicate that the definitive hematopoiesis is malignant. Surprisingly, in the early stages of st22, we observed a complete loss of l-plastin expression in the anterior region from where the primitive macrophage arises. This result suggests that c-myb may also function in the primitive hematopoiesis, potentially revealing a link between primitive and definitive hematopoiesis. 349 Posters 279 Hematopoiesis and immunology -lapachone treatment causes apoptosis of red blood cells in zebrafish embryos S-P.L. Hwang1,4, Y-T. Wu1, C.Y. Lin1, M-Y. Tsai2, Y-H. Chen4, Y-F. Lu4, C-J. Huang3 1 Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan 221, R.O.C.; 2Graduate Institute of Life Sciences, National Defense Medical Center, National Defense University, Neihu, Taipei, Taiwan 114, R.O.C.; 3Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan 115, R.O.C.; 4Institute of Cellular and Organismic Biology (formerly Institute of Zoology), Academia Sinica, Nankang, Taipei, Taiwan 115, R.O.C. -lapachone, a novel antitumor agent, is currently in phase II clinicaelanoma, how pigment and UVR spectrum interact, and which signaling pathways are crucial for nevi/melanoma transformation and melanoma progression. 351 Posters 281 Diseases models Recapitulating Early Stages of Diabetic Retinopathy in Hyperglycaemic Zebrafish Y. Alvarez, K. Chen, A. Reynolds, N. Waghorne, J. O'Connor and B. Kennedy Conway Institute, UCD, Dublin, Ireland Diabetic retinopathy is the most common complication of type 2 diabetes and the leading cause of blindness in working-age people in developed countries. Some treatments can palliate progression of proliferative diabetic retinopathy (PDR) but a better comprehension of the initial, non-proliferative phase of the disease (NPDR) may reveal interventions that more effectively limit retinal damage. Early stages of diabetic retinopathy are mostly asymptomatic for patients thus, animal models of NPDR are essential. Our objective was to develop a zebrafish model of diabetic retinopathy through the induction of morphological and/or functional changes recapitulating the neuronal damage and vascular microangiopathy observed in the retinas of diabetic patients. The inner retina in adult zebrafish is nourished by an intricate vascular network which shares many features with the human retinal vasculature. Hyperglycaemia was induced in adult zebrafish by treating with 2% glucose on alternative days. 2% mannitol treatment was used as an osmotic control. After 30 days of treatment, retinal morphology and retinal vascular network were analyzed by immuno-histochemistry and transmitted electron microscopy. Ex-vivo electroretinograms (ERG) were conducted to assess electrophysiological function in the treated retinas. Glucose-treated fish display neurodegenerative and vascular hallmarks of early stage diabetic retinopathy. In ~60% of glucose-treated fish, retinal histology is abnormal, displaying a pronounced degeneration of cone photoreceptors. In regards to the retinal capillaries of glucose treated animals, the interendothelial junctions are significantly more open, and the basement membranes are thicker, indicating a breakdown of the retinal blood-barrier function. ERGs confirm defects in cone photoreceptor function in the glucose treated fish. Our data suggest that zebrafish are a useful animal model for research into diabetic retinopathy. Posters 352 Diseases models 282 Using zebrafish embryos to investigate genes implicated in Alzheimer's disease pathology M. Newman, S. Nornes, B. Tucker and M. Lardelli Molecular and Biomedical Science, The University of Adelaide, Australia Alzheimer's disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Zebrafish have orthologues of human PSEN 1 and 2 and APP, therefore they are a useful tool for investigating some of the complex pathways that occur in Alzheimer's disease. We aim to understand the role these genes have in AD pathology by investigating their molecular biology in zebrafish embryos. We have demonstrated that low-level aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. A microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of these phenotypic effects. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. Analysis of zebrafish ccng1 function indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Potentially, the truncated forms of Ccng1 interfere with Ccng2 function in a dominant negative manner. Splicing is observed to change in ageing cells, therefore, aberrant splicing of particular genes may play a role in sporadic forms of AD and other neurodegenerative diseases. 353 Posters 283 Diseases models Zebrafish models for familial Alzheimer's disease P. van Tijn, J.T. Paridaen, C. van Rooijen and D. Zivkovic Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University Medical Centre Utrecht, Utrecht, The Netherlands Autosomal dominant mutations in presenilin 1 (PSEN1) lead to familial Alzheimer's disease (FAD), neuropathologically characterized by amyloid- (A) accumulation. A is formed by PSEN1mediated proteolytic cleavage of the amyloid precursor protein (APP). PSEN1 also cleaves Notch, a protein essential for correct neuronal differentiation during embryogenesis, maintenance of the neural stem cell niche and neurogenesis in adult. Some FAD-PSEN1 mutations lead to impaired Notch signaling whereas others are neutral. We aim to reveal the effects of expression of such different FAD-PSEN1 mutations in the embryonic and adult brain using zebrafish as a model system. To this end, we utilize our recently characterized zebrafish mutant lacking functional Psen1. These Psen1-null mutants are viable and fertile, in contrast to PSEN1-/- mice. Psen1-null mutants show decreased cell proliferation and de novo neurogenesis, which is most pronounced in the cerebellum. In addition, Delta/Notch signaling is impaired. The zebrafish Psen1-null mutant is the first vertebrate model with targeted gene knockout of PSEN1 compatible with life. As a result, we have a unique opportunity to study effects of FAD-PSEN1 with respect to Psen1 lossof-function in the adult brain. By misexpression of human FAD-PSEN1 mRNA in Psen1-null embryos, we aim to dissect the effects of the PSEN1M146V, decreasing Notch signaling in vitro, and PSEN1L286V, neutral towards Notch in vitro, mutations during embryogenesis. In addition, we have generated novel transgenic FAD-PSEN1 lines expressing mCherry-fluorescently tagged human FAD-PSEN1 under the zebrafish psen1 promoter region. In these lines we are investigating effects of FAD-PSEN1 on the neural stem cell compartment during embryogenesis and in adult brain in relation to Notch and APP signaling pathways. Posters 354 Diseases models 284 Deletion of the WD40 domain of LRRK2 in zebrafish provides a model for Parkinson's disease D. Sheng1, D. Qu1, S.S. Ng1, Y.M.A. Lim2, W.H.C. Lee3, S.W. Kin3, E.K. Tan4, T. Lufkin2, S. Jesuthasan5, M. Sinnakaruppan2, J.J. Liu1 1 Human Genetics, 2Developmental Biology and 3Computational and Mathematical Biology, Genome Institute of Singapore, A*STAR, Singapore; 4National Neuroscience Institute and Duke-NUS Graduate Medical School. Singapore; 5Neuroscience Research Partnership, A*STAR, Singapore LRRK2 plays an important role in Parkinson's disease (PD), but its biological functions are largely unknown. Here, we cloned tphenotype, apparent by early somitogenesis, characterized by a massive deficit in neuronal outgrowth and poor fasciculation. We noticed that the gross phenotypes resulting from DISC-1 and Wnt8b loss of function are similar, and showed that partial loss of function of each resulted in a strong phenotype, demonstrating synergy between these genes. Several additional lines of evidence indicate that DISC-1 functions in the Wnt pathway. First, similar changes in forebrain gene expression are observed after loss of DISC-1 or loss of Wnt8b function. Second, loss of DISC-1 function decreased reporter gene expression in a Wnt-responsive reporter zebrafish line, TOPdGFP. Third, a GSK3 inhibitor is able to rescue reporter and endogenous gene expression after DISC-1 loss of function. Fourth, increasing -catenin levels, through an inducible fusion protein, rescues the DISC-1 loss of function phenotype. Finally we were able to rescue the morphant phenotype with human DISC-1 demonstrating the fundamental role of DISC-1 in modulating GSK during zebrafish development. In conclusion, our data identify DISC-1 as an essential positive modulator of the canonical Wnt pathway, and confirm the efficacy of using zebrafish as a tool to explore the function of genes implicated in human mental health disorders. 363 Posters 293 Diseases models Functional characterization of two human disease mutations, PRP31-SP117 and PRP31-AD5, in a zebrafish model for Retinitis pigmentosa J. Yin, C. Winkler Department of Biological Sciences (DBS), National University of Singapore Retinitis pigmentosa (RP) is an inherited eye disease with a prevalence of approximately 1/3500, characterized by a progressive degeneration of photoreceptor cells in the retina, which firstly affects rods and secondarily results in the loss of cones. Different mutations in the general splicing factor PRP31 have been identified as a major cause for the autosomal-dominant form of RP in humans. So far, mostly cell culture models have been used to study the mechanisms underlying RP caused by PRP31 mutations and conflicting results have been obtained from such in vitro approaches. We have used transgenic zebrafish as a powerful animal model for human diseases, to investigate by which mechanisms two human mutations (SP117 and AD5), which cause a frameshift at amino acids 256 and 371 respectively and might result in truncated PRP31 proteins (277 and 469 aa; wildtype 499 aa), lead to photoreceptor cell degeneration. After injection of RNAs encoding both mutant PRP31 variants into zebrafish embryos, we found that the SP117 protein degraded quickly and was no longer detectable at 11hpf, whereas the AD5 variant had a similar stability as injected wildtype PRP31 and showed continuous high levels of expression at 14hpf. For an in vivo assay of protein localization and function, we next generated a rhodopsin-EGFP (rho-GFP) transgenic line, which expresses GFP in the cytoplasm of rods. rho-GFP transgenic embryos were then injected with plasmids encoding wildtype or mutant PRP31 fused in frame to mCherry under the control of the rhodopsin promoter. Embryos injected with wildtype PRP31:mCherry showed transient expression in the nuclei of rods, similar to the situation in AD5:mCherry injected embryos. In contrast, in SP117:mCherry injected embryos, the majority of mCherry signal was detected in the cytoplasm of rods indicating aberrant subcellular localization of the mutant protein. In addition, we found that transient expression of the AD5 mutant resulted in a significant reduction of rhodopsin expressing rod photoreceptors, while expression of SP117 or wildtype PRP31 did not change the number of rhodopsin expressing cells. Hence, our in vivo data suggest that the two analyzed PRP31 mutations act by different mechanisms: Expression of AD5 mutant protein results in rod degeneration possibly in a dominant-negative fashion, whereas mutant SP117 protein is mislocalized to the cytoplasm and unstable and causes rod degeneration by haploinsufficiency due to nonsense-mediated mRNA decay (NMD). Our zebrafis led to changes in neural population or defects in spinal axon projections. In the cases tested, the phenotype could be rescued by overexpression of the native human mRNA but not by some of the disease-related alleles, including novel mutations, thus establishing the usefulness of this approach. For our genomics project we have identified several inherited as well as de novo mutations of synaptic genes that we are validating in zebrafish. Zebrafish thus provide a useful model for validating the pathogenic nature of human brain disease-related alleles. 367 Posters 297 Diseases models G93A-SOD1 transgenic zebrafish as a model of amyotrophic lateral sclerosis S. A. Sakowski, A. S. Busta, J. J. Dowling, E. L. Feldman University of Michigan, Ann Arbor, USA Axonal degeneration precedes symptom onset and motor neuron (MN) cell death associated with amyotrophic lateral sclerosis (ALS; MN disease), making it an appealing target for therapeutic intervention prior to the irreversible loss of MNs. Detailed observations of MN axons and neuromuscular junctions (NMJs) are possible in zebrafish; therefore, they provide an excellent in vivo system to study early events in ALS onset and progression, as well as mechanisms that underlie neuroprotection. Transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in defects in MN outgrowth and axonal branching. To study the mechanisms of neurodegeneration and identify novel treatments for ALS, stable G93A-SOD1 transgenic zebrafish have been generated. These G93A-SOD1 transgenic zebrafish demonstrate a progressive loss of swimming ability. We are currently characterizing this model of ALS by analyzing NMJ integrity, axonal morphology and survival in order to understand the mechanisms of axonal degeneration associated with ALS. Knowledge obtained from the characterization of stable transgenic zebrafish as a model of ALS will greatly advance our ability to treat ALS by identifying points for therapeutic intervention throughout the course of MN degeneration. Supported by the NIH (SAS: NS007222-26), the A. Alfred Taubman Medical Research Institute, and the Program for Neurology Research & Discovery. Posters 368 Diseases models 298 Using zebrafish to investigate presenilin, -secretase, and APP for Alzheimer's disease research L. Wilson, S. Nornes, M. Newman and M. Lardelli Molecular & Biomedical Science, The University of Adelaide, Adelaide, Australia Introduction: An integrated understanding of the exact molecular and cellular basis of Alzheimer's disease (AD) remains elusive. Histological analyses of AD patients have observed lesions containing extracellular deposits of amyloid-beta peptides (A) and intracellular neurofibrillary tangles (NTF). A peptide is a cleavage fragment of the A precursor protein (APP). Aberrant proteolytic processing of APP by -secretase results in an imbalance between A production and clearance that appears to promote neuronal dysfunction and death. Presenilin proteins form the catalytic cores of -secretase complexes. Genetic studies have discovered mutations in APP and PRESENILIN 1 and 2 that contribute to familial autosomal dominant AD. Danio rerio provides an effective vertebrate model for investigating the molecular bases of AD pathology as it possesses orthologues of human PSEN1 and 2, and APP. Results: 1) We have demonstrated that disruption of PSEN1 transcript splicing can have potent dominant negative effects on the function of PSEN1 and the related gene PSEN2. We hypothesise that aberrant splicing of PSEN1 in ageing neural cells may contribute to sporadic AD. 2) We have synthesized mRNA encoding PSEN1 truncations after exon junctions. When injected into embryos, particular truncations have a dominant negative effect. We hypothesise that the aberrant splicing of PSEN (above) produces truncated proteins that act in a dominant negative manner by invading -secretase complexes to prevent the formation of the catalytic site. 3) Currently there is no in vivo assay appropriate for investigating -secretaseactors in a zebrafish model for retinitis pigmentosa H. Dill1, B. Linder1, A. Hirmer1, J. Brocher2, S. G. P. Lee5, S. Mathavan5, A. Gal3, H. Bolz4, C. Winkler2, B. Laggerbauer1 and U. Fischer1 1 Deptartment of Biochemistry, University of Wuerzburg, Germany; 2Department of Biological Sciences, National University of Singapore; 3Department of Human Genetics, University Medical Center Hamburg-Eppendorf, Germany; 4Institut of Human Genetics, University of Cologne, Germany; 5Genome Institute of Singapore, Singapore Retinitis Pigmentosa (RP), an inherited disease leading to blindness, is characterized by progressive rod photoreceptor degeneration with secondary loss of cone photoreceptor cells. The majority of RP-causing mutations affect proteins involved in photoreception. However, mutations in at least three constitutive mRNA splice factors, hPrp3, hPrp8 and hPrp31 have been described that lead to RP. These factors are components of the so-called U4/U6.U5 tri-snRNP particle, an essential part of the pre-mRNA splicing machinery. By screening for RP patients carrying new mutations in splice factors, we identified hPrpf4, another well known tri-snRNP protein as a new RP candidate gene. Biochemical analysis of mutant hPrp4 revealed defective tri-snRNP association. Furthermore we have established a zebrafish knockdown model to gain insight into the mechanism, by which defects in ubiquitously expressed pre-mRNA processing factors cause photoreceptor specific degeneration. In the zebrafish system, mild gene knockdown of zfPrp31 or zfPrp4 leads to severely reduced expression levels of rhodopsin, even though the remaining embryo apparently develops normally. Behavioural tests indicate that zfPrp31 and zfPrp4 morphants exhibit deficits in visual cognition. Thus, we could mimic the main features of RP in zebrafish larvae. In microarray analysis of zfPrp31-morphant retinae, a considerable number of retina-specific genes turned out to be significantly down-regulated, compared to housekeeping genes. Together our data are consistent with the idea that mutations affecting the spliceosomal activity cause RP by influencing splicing of retina specific transcripts. 375 Posters 305 Diseases models Zebrafish, a new model to study rett syndrome G. Gaudenzi1, A. Ghilardi1, A. Guarda2, F. Cotelli1, G. Badaracco2 1 University of Milan, Department of Biology, Italy; 2University of Insubria, Department of Structural and Functional Biology, Italy Rett syndrome (RTT) is a X-linked dominant neurological disorder that affects almost exclusively females, occurring with a frequency of up to 1/10,000 live female births and causing foetal demise in males. After an early period of apparently normal or almost normal development (until 6-18 months of age), this disorder produces a profound mental disability, reduction in speech and purposeful hand movements and reduced brain growth. It is now known that approximately 80% of classic RTT patients have a mutation in the gene encoding methyl-CpG binding protein 2 (MeCP2), a transcriptional repressor involved in chromatin remodeling and the modulation of RNA splicing. Although several murine RTT models were obtained, we propose zebrafish as a powerful vertebrate system to investigate the role of MeCP2 during embryonic development. zMECP2 was previously mapped to linkage group 8 and it is highly similar to mammalian MECP2. Relatively high levels of its expression were found in embryos at 1 to 4 h postfertilization (hpf), after 24 hpf, and in adult brain and eyes. Using a specific antisense morpholino oligonucleotide (MECP2MO) to knockdown gene activity, we observed morphological and behavioral defects resembling phenotypes in RTT. In fact, zMECP2MO injected embryos display spontaneous and touch-provoked motility defects. Moreover, morphants showed a strong reduction of both cephalic nervous system and otic vesicles. Midbrain-hindbrain boundary (MHB) is thin, and both midbrain and hindbrain ventricles appear inflated. A preliminary analysis of morphants with neural marker (otx3, wint1, pax2a) suggests that zMECP2 t one week. Expression of dopaminergic and serotonergic markers is reduced, while markers for other neuronal populations, as well as early brain patterning markers, are unchanged. Thus, this mutant shows a specific loss of some neuronal identities. We are currently exploring whether it can be used as a novel model for selective neurodegeneration to elucidate some of the possible mechanism behind this group of diseases. 383 Posters 313 Analysis of cohesin and condensin genes during zebrafish development M. Moennich, S. Banks, and J. Horsfield University of Otago, Dunedin, New Zealand Diseases models During cell division, the multi-subunit complexes cohesin and condensin have important roles in sister chromatid cohesion and chromosome condensation, respectively. Mutations in the cohesin subunits SMC1A and SMC3, as well as mutations in the cohesin loading factor NIPBL, have been shown to cause the genetic disorder Cornelia de Lange Syndrome (CdLS) in humans. CdLS patients have severe developmental abnormalities, including facial dysmorphism, growth retardation, upper limb anomalies, and mental retardation. It has been shown recently that cohesin does not only function in cell division, but also has key roles in tissue-specific development, such as axon guidance and pruning, skeletal patterning, and hematopoiesis. The mechanism underlying is so far poorly understood, but it has been suggested that cohesin proteins may be involved in transcriptional regulation of developmental genes. To further understand non-canonical roles of cohesin, we analyzed the expression of cohesin subunits in the developing zebrafish embryo and compared it with expression of condensin subunits and the distribution of proliferating cells. Cohesin and condensin subunits are dynamically expressed in a similar, but not identical pattern, indicating a diverse function of the two complexes. The expression pattern of cohesin subunits does not completely overlap with zones of proliferation, supporting the existence of non-proliferative roles. These results suggest that while both complexes are involved in cell division, alternative developmental functions that may underlie CdLS are possible. Posters 384 Diseases models 314 Depletion of zebrafish fukutin family protein activities extends the phenotypic spectrum from dystroglycanopathy to lamininopathy Y-Y. Lin and D. L. Stemple Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom Muscular dystrophies associated with hypoglycosylation of alpha-Dystroglycan are commonly referred to as dystroglycanopathies, in which mutations affect at least six known or putative glycosyltransferase genes, including POMT1, POMT2, POMGnT1, fukutin, fukutin-related protein (fkrp) and LARGE. Allelic mutations in each of these genes can lead to a wide spectrum of clinical severity, ranging from a congenital onset of severe muscular dystrophy with brain malformation to a late onset of milder limb-girdle muscular dystrophy without mental retardation. To date, POMT1, POMT2 and POMGnT1 have been shown to catalyze the O-glycosylation of alpha-Dystroglycan. Over-expressing LARGE circumvents glycosylation defects in cells from some dystroglycanopathy patients, suggesting that LARGE may stimulate an independent sugar modification. The function of Fukutin and FKRP, however, as well as the pathological mechanisms leading to the wide range of clinical severity still remains unclear. To model dystroglycanopathy in zebrafish, we have recovered a novel dystroglycan nonsense allele and inhibited Fukutin family protein function by low-to-high doses of antisense morpholino oligonucleotides (MOs). We show that removal of zebrafish Fukutin or FKRP not only causes reduced glycosylation of alpha-Dystroglycan but also elicits notochord defects associated with loss of Laminin function. Importantly, a complete absence of Dystroglycan leads to loss of Dystrophin localisation, but not loss of Laminin function. The absence of Laminin localisation in fukutin or fkrp MO-injected embryos is mainly a consequence of defective post-translational processing. Taken together, our results strongly suggest that Fukutin family proteins play a pivotal role on the secretion of Laminin, possibly through a novel glycosylation mechanism, and that loss of Laminin function together with hypoglycosylation of alpha-Dystroglycan underlies the wide clinical spectrum of some forms of muscular dystrophies. 385 Posters 315 Diseases models The zebrafish histaminergic system: telencephalic projections, co-transmitters and afferent innervation M. Sundvik and P. Panula Neuroscience center and Institute of Biomedicine/Anatomy, Haartmaninkatu 8, 00014 University of Helsinki, Finland Histamine is a modulatory neurotransmitter involved in e.g. psychiatric disorders and neurodegenerative diseases. Learning, memory, anxiety and sleep are functions regulated by histamine and other neurotransmitters. In zebrafish, lateral telencephalic pallium is proposed to be homologous to the mammalian hippocampus, selectively involved in spatial learning and memory. The medial telecephalic pallium is considered homologous to the mammalian amygdala processing emotions, e.g. anxiety-like behavior. 3D confocal microscopy combined with immunohistochemistry of larval zebrafish brains showed that histaminergic fibers projected from the caudal zone of periventricular hypothalamus to the telencephalon, where some fibers crossed at the commissura anterior to contralateral side to innervate the central part of dorsal telecephalic together with the ipsilateral fibers. Histamine-producing neurons are found exclusively in the hypothalamus which is known to regulate body temperature, hunger, thirst and circadian rhythm. In order to understand the underlying mechanisms of and the role of histamine in these different physiological functions, we identified co-transmitters of the histaminergic neurons in the posterior hypothalamus of zebrafish by immunohistochemistry. In adult zebrafish GABA, galanin and thyrotropin releasing hormone (TRH) were co-localized with histamine in some neurons. TRH-immunoreactive fibers made close contacts with histaminergic neurons. Similar innervation pattern was observed between the histaminergic neurons and the fibers containing the endogenous opioid peptide, met-enkephalin. No met-enkephalin was observed in histaminergic neurons. Taken together, we show that histamine is co-localized with GABA, galanin and TRH in zebrafish which is in accordance with previously published data from rodents. Galanin, GABA, TRH and met-enkephalin are also known to be involved in mediating the physiological functions of hypothalamus, suggesting that together with histamine these neurotransmitter systems can modulate the response of hypothalamus to different stimuli. Previous studies have shown that manipulating the histaminergic system affects learning and memory. Here we present results that are in accordance with those as we show that histamine fibers innervate dorsal telencephalon, an area involved in cognition. Posters 386 Diseases models 316 A zebrafish model of charcot-marie-tooth 2D N. Malissovas1, D. Stainier2 and D. Beis1 1 Developmental Biology, Biomedical Research Foundation, Academy of Athens, Greece 2 Biochemistry aand Biophysics, University of California, San Francisco, USA Cardiac valves derive from endocardial cells and function throughout the life of vertebrates to prevent retrograde blood flow. In a large scale ENU mutagenesis screen, we identified a number of mutants in discrete stages of valve development. In one of this lines, we named metronome, homozygous mutants show retrograde blood flow from 72 hours post fertilization (hpf) onwards, as a result of an impaired cardiac valve development. In parallel, these mutants become progressively immotile by 96 hpf. We found that although filamentous actin is present, the myofibrils of metronome mutants lack the organised fibrillar arrangement seen in wild type siblings. We mapped the mutation with positional cloning and we located it in clone DKEY-276I5 in linkage group 24. We have found that metronome carries a recessive point mutation in the glycyl t-RNA synthetase (gars). Mutations in the human orthologue of gars are responsible for an adult onset distal neuropathy, Charcot Marie Tooth disease (CMT) type 2D and spinal muscular atrophy type V. The mutation in our animal model (T130K) resides next to a previously described human mutation (L129P). Aminoacyl-tRNA synthetases have a broad repertoire of functions beyond translation (they catalyze the first committed step of protein synthesis by linking an aminoacyl to its respective tRNA), including transcriptional and translational regulation as well as cell signalling. We have phenocopied the metronome phenotype by morpholino injections in wild type embryos. gars T130K does not rescue GRS1 mutant yeast and is not associated with cytoplasmic granules in mouse motor neurons cells in vitro, similar to the most severe human mutations (in collaboration with Antony Antonellis and Eric Green, NHGRI, NIH). Using an anti-human (GARS) antibody we have shown that there is a maternally provided protein in the homozygous mutant embryos, which explains why there is no obvious phenotype up to 60 hpf. In parallel, we have indications that T130K interferes with the proper dimerization of GARS, providing an insight in the pathophysiology of CMT. Moreover, we have shown that gars is highly expressed during the developing brain outlining the ventricles, besides a ubiquitous basal expression. We will present our latest data on the mechanism of GARS function in this in vivo model of CMT2D. 387 Posters 317 Diseases models Cardio-facio-cutaneous syndrome alleles are active during zebrafish development and are sensitive to small molecule inhibitors C. Anastasaki1, A. L. Estep2, R. Marais3, K. A. Rauen2 and E. E. Patton1* 1 Institute for Genetics & Molecular Medicine, MRC Human Genetics Unit and The University of Edinburgh, Western General Hospital, UK. 2University of California San Francisco, Department of Pediatrics, UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, USA. 3Cancer Research UK Centre for Cell and Molecular Biology, Signal Transduction Team, The Institute of Cancer Research, London,UK The Ras/MAPK pathway is critical for human development, and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities, and mental retardation. CFC syndrome mutations in BRAF promote both kinaseactivating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinaseimpaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development. BRAF CFC mutations promote an additive effect during development, consistent with both the kinase-active and kinase-impaired BRAF CFC mutations acting as gainof-function mutations during development. Treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug. CFC syndrome has a progressive phenotype, and as many of the phenotypic effects develop postnatally, patients may be helped by systemic therapies after birth. Our work shows the zebrafish system as a tractable tool for medical and research geneticists to explore allele activity and therapeutic potential, and establishes a foundation to propel forward the clinical discussion and scientific strategy for assessing the suitability of using currently available cancer drugs to treat the progressive phenotypes of CFC in children. Posters 388 Diseases models 318 Down regulation of Hccs in medaka recapitulates the phenotype observed in Microphthalmia with linear skin lesions (MLS) syndrome A. Indrieri1,2, I. Conte1, G. Chesi1, P. Bovolenta3, B. Franco1,4 1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 2European School of Molecular Medicine (SEMM), Naples; Italy; 3Instituto Cajal, CSIC, Madrid, Spain; 4Medical Genetics, Department of Pediatrics, Federico II University, Naples, Italy The Microphthalmia with linear skin defects (MLS) syndrome is an X-linked dominant malelethal neuro-developmental disorder associated to mutations in the holocytochrome c-type synthetase (HCCS) transcript. Female patients display unilateral or bilateral microphthalmia and linear skin defects, additional features include CNS malformation and mental retardation. HCCS codifies a mitochondrial protein that catalyzes the attachment of heme to both apocytochrome c and c1 necessary for proper functioning of the mitochondrial respiratory chain. The molecular mechanisms underlying the eye and brain developmental anomalies in this disease are still unknown. Previous studies demonstrated the early lethality of mouse embryonic Hccs knockout stem cells. We thus decided to generate a model for this disease in medaka fish (Oryzia latipes). This model will allow us to overcome the possible embryonic lethality using graded concentrations of either the mutated mRNA or the morpholinos. Specific morpholinos directed against the HCCS transcript have been designed and injected. Our experiments have determined that the morpholinos effectively downregulate the expression of the olhccs gene. Gain of function studies did not result in an aberrant phenotype. Instead injection of both the mutated protein (dominant-negative) and two different morpholinos resulted in a pathological phenotype, which resembles the human condition. As expected, morphants displayed microphthalmia, coloboma, and microcephaly. In addition, unexpectedly, absence of blood pigmentation was also observed. Analysis with specific markers (e.g, Pax6, Six3.2, Otx2, Rhodopsin, Crx, Chx10, Ath5, Syntaxin) showed an abnormal formation of the retinal-pigmented epithelium (RPE) and defects in differentiation of the ventral neural retina. RNA in situ hybridization studies revealed an abnormal domain of expression for connexin43, recently shown to be important for cell proliferation and apoptosis. Tunel and pHH3 assays on morphants, revealed abnormalities in cell proliferation and programmed cell death. Altogether this data suggest a possible role of these mechanisms in the pathogenesis of MLS syndrome and further studies are ongoing to explain the pathogenetic link between Hccs inactivation and the eye abnormalities observed in morphants and affected patients. 389 Posters 319 Diseases models Functional characterization of the SLC7A6OS gene in danio rerio A. Benini, L. Calvarini, A. Bozzato, F. Cignarella, S. Barlati, R. Bresciani, G. Borsani Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy SLC7A6OS is a novel protein coding gene representing a natural antisense transcript (NAT) of SLC7A6 (SoLute Carrier family 7 member A6) in the human genome. RT-PCR studies demonstrated that SLC7A6OS is expressed in all tissues and cell lines we tested. We failed to obtain experimental evidence for a co-regulation of the SLC7A6/SLC7A6OS cis-NAT gene pair in cell models. Transient expression in HeLa cells demonstrated that SLC7A6OS is a soluble protein mainly localized in the cytoplasm. While the SLC7A6OS gene is conserved during evolution, the encoded protein shows no significant sequence identities with other polypeptides of known functions in vertebrates. Although we failed to identify relevant protein domains or functional sites that might provide clues about the biological role of this polypeptide, a low but probably significant level of sequence identity has been detected with Saccharomyces cerevisiae Iwr1. Iwr1 is a factor that associates to RNA polymerase II through the binding to RBP3 (RNA polymerase II subunit 3). This finding suggests that SLC7A6OS may play a role in the regulation of gene expression. More recently, we decided to study the biological role of SLC7A6OS in Danio rerio. We have identified the putative slc7a6os zebrafish ortholog on chromosome 7 adjacent to slc7a6, although there are no evidences that these two genes are NATs in the genome of the teleost. Slc7a6os zebrafish protein is 46% identical to the human counterpart and it also shows a cytoplasmic localization when transiently expressed in HeLa cells. Real time RT-PCR studies demonstrated that slc7a6os is a maternal gene, expressed from the zygote stage to the adult fish. Whole mount in situ hybridization experiments showed that slc7a6os is expressed predominantly in the central nervous system during embryogenesis, and also in the thymus and neuromast of lateral line, during the hatching period. We have investigated the function of the slc7a6os gene by morpholino-induced gene knockdown. The phenotypes observed in injected embryos suggest that slc7a6os is required for early neural development. Posters 390 Diseases models 320 Understanding PINK1 mechanism by gene expression arrays M. Priyadarshini1, J. Tuimala2, Y-C Chen1, P. Panula1 1 Neuroscience Center, Institute of Biomedicine/Anatomy, University of Helsinki, Finland; 2Life science center, CSC - IT Center for Science Ltd, Espoo, Finland One of the challenges in Parkinson's disease (PD) is identifying the biochemical pathways affected by genetic alterations in some key genes causing hereditary PD. Mutations in the PINK1 gene that cause autosomal recessive PD have been described. The Pink1 protein product localises to the mitochondria and cytoplasm. The function of PINK1 is not clearly known but it is a protein kinase, mutations of which enhances sensitivity to ubiquitin proteasome system inhibitors and lowers the threshold to apoptotic cell death. PINK1 mutations may lead to mitochondrial dysfunction and increased sensitivity to cellular stress through a defect in the apoptosis pathway. This study aims at finding novel targets for Pink1. Microarrays on small amounts of RNA can give useful unbiased information of pathways and genes involved. In order to understand the mechanism and different pathways that are altered after PINK1 knockdown with the morpholino oligonucleotides and performed a microarray experiment. A set of four replicates from four different sets of fish were made and hybridised on two-colour gene expression arrays from Agilent. Data was analysed using the R and IPA program programs. We found 237 genes being significantly altered in PINK1 morphants compared to the controls (level of confidence: p>0.01 and log fold change values from -1.6 to +0.9). When the 237 genes were organized into functional categories in biological process pathway by their GO terms, the pathways that were identified as significantly affected (cut off: more than 50% of the genes altered) are: specification of organ axis polarity, cerebellum formation, hindbrain formation, cerebellum development, cerebellum morphogenesis, glutathione biosynthetic process, specification of axis polarity and amoeboidal cell migration. Most of these pathways have links to different forms of PD. The pathways that were significantly altered (p>0.04) based on the commercially available IPA program were cholesterol biosynthesis, oxidative stress, TGF-beta signalling, hypoxia inducible factor signalling pathway, apoptosis and p53 signalling and mitochondrial dysfunction. PINK1 knockdown in zebrafish thus alters several important pathways, which will enable detailed studies on individual genes and novel mechanisms. 391 Posters 321 Diseases models Identification and characterization of the putative co-orthologs of MCOLN1, the gene mutated in mucolipidosis type IV A. Benini1, L. Calvarini1, S. Moleri2, A. Bozzato1, S. Barlati1, M. Beltrame2, G. Borsani1 1 Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy; 2Department of Biomolecular Sciences and Biotechnology, University of Milan, Italy Mucolipidosis type IV (MLIV, MIM 252650) is an autosomal recessive lysosomal storage disorder that causes mental and motor retardation as well visual impairment. MLIV is caused by mutations in the MCOLN1 gene, which codes for mucolipin-1 (TRPML1), a member of the large family of transient receptor potential cation channels (TRP). In mammals, the mucolipin family includes other two members: mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3) encoded by MCOLN2 and MCOLN3 genes, respectively. We have identified in the past the gene responsible for MLIV and subsequently we started to study the pathogenetic mechanism of the disease. More recently, we decided to study the biological role of the MCOLN1 gene in zebrafish. A zebrafish transcriptome- and genome-wide search using the sequences of the MCOLN polypeptides revealed the presence of five different fish genes related to human mucolipins. Being our interest focused on MLIV, we concentrated our efforts on the functional characterization of mcoln1.1 and mcoln1.2, the putative co-orthologs of human MCOLN1. Transient-expression experiments in human HeLa cells demonstrated that fish mcoln1.1 and mcoln1.2 proteins, similarly to human mucolipin-1, localize to late endosomal/lysosomal compartments. Whole mount in situ hybridization experiments show interesting peculiarities in terms of expression pattern of the two zebrafish genes during development. In particular, the expression of mcoln1.1 in the intermediate cell mass (ICM), suggests that this gene, like the human TRPML1 counterpart, may play a role in iron metabolism. Morpholino-mediated knockdown of zebrafish mcoln1.1 causes alterations in vascular development. A more detailed phenotypic characterization of mcoln1.1 morphants is currently underway. Morphological effects due to the ablation of mcoln1.2 function are also being investigated. Posters 392 Diseases models 322 A Fish Model for Duchenne Muscular Dystrophy J. Berger1, S. Berger1, A. Jacoby1, G. Lieschke2, S. Wilton3, P. Currie1 1 Australian Regenerative Medicine Institute, Melbourne, Australia; 2Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; 3Australian Neuromuscular Research Institute, Perth, Australia In humans, mutations within the structural muscle gene Dystrophin (DMD) lead to Duchenne Muscular Dystrophy, one of the most common, lethal disorders. Currently, the mouse null mutant for dmd, the mdx mouse (Hoffman et al., Cell 1987), is the mainly used model for Duchenne Muscular Dystrophy, even though the phenotype of the mdx mouse is extremely mild and does not reflect the human condition. Our laboratory identified the sapje (dmdta222a) zebrafish mutant as a carrier of a null mutation in the orthologous dmd gene (Bassett et al., Development 2003). Further survey of the phenotype revealed that the progression of the muscle atrophy is comparable to that observed in humans. We documented extensive skeletal muscle wasting throughout the larvae lifespan of dmdta222a homozygotes. Necrotic myofibres are replaced by mono-nucleated infiltrates, interstitial connective tissue, as well as neutrophils, indicative for inflammation. In addition, dmdta222a homozygous mutants are characterised by progressively exhausting muscle precursors and broader variation of muscle fibre cross sectional areas. Due to its profound phenotype, the dmdta222a fish closely resembles symptoms of the human disease. We performed a non-complementation screen that led to the identification of novel dmd null alleles. The mutations of the three novel dmd alleles (dmdpc1, dmdpc2, and dmdpc3) encode premature stop codons causing loss of the gene product. Two of these mutants (dmdpc2 and dmdpc3) carry a null mutation in exons (32 and 34, respectively) that are located in the highly repetitive rod domain, which-43 cDNA constructs tagged at both the C-terminus and N-terminus which I will use to determine mutant and WT TDP-43 expression in cell lines, primary motor neurons and zebrafish embryos. Here we demonstrate that mutant TDP-43 causes a specific motor neuron disorder through a toxic gain of function since expression of mutant TDP-43, but not WT, causes selective motor neuron toxicity in primary spinal cord cultures. Further, expression of mutant causes a specific motor phenotype and motor neuron axonal defects in zebrafish embryos. On the other hand, decreased expression using anti-sense morpholino oligonucleotide (AMO) of TDP-43 yielded a similar phenotype in zebrafish; thus a specific loss of function may also be involved in the selective vulnerability of motor neurons to mutant TDP-43. A further characterization of these models will allow us to unravel molecular partners of mutant TDP-43 as well as to allow a better understanding of specific mechanisms of disease involved in mutant TDP-43 caused motor neuron degeneration. We are also generating Tardbp knock-outs in zebrafish as well as mutant TDP-43 transgenic fish lines. These lines will be functionally characterized to determine whether they properly model motor neuron disorders. These lines can then be tested for the screening of a large number of pharmaceutical compounds in transgenic zebrafish expressing mutant TDP-43, thus directly opening avenues for the development of therapies that could delay or prevent disease onset and progression in all ALS patients. 395 Posters 325 Diseases models Zebrafish mitofusin-2 knockdown: a new model for CMT2A neuropathy? G. Bergamin, A. Vettori, E. Moro, G. Polo, G. Vazza, N. Tiso, F. Argenton, M.L. Mostacciuolo Department of Biology, University of Padua, Italy Mitofusin 2 (MFN2) is a large dynamin-like GTPase protein, located in the outer membrane of mitochondria and at the surface of the endoplasmic reticulum. Mfn2 is essential to maintain the organization of mitochondria in cells and it may play a role in the modulation of calcium exchanges between these two cellular compartments. MFN2 gene mutations are associated with Charcot-Marie-Tooth disease type 2A (CMT2A), an hereditary axonal neuropathy characterized by distal muscle weakness and atrophy. To investigate the function of this gene and its role in the pathogenesis of CMT2A, we have identified the zebrafish MFN2 ortholog and used a morpholino antisense oligonucleotide to knockdown mitofusin function. Morphant embryos showed motor impairment. Morphologically they displayed curved tails, disorganized somites, small and underdeveloped eyes and an extensive dilatation of the brain ventricles. Interestingly, this phenotype resembles the central nervous system abnormalities and optic atrophy observed in some CMT2A patients. Shorter and abnormal motor neuron axons were observed on 48 hpf morphant larvae, suggesting that these alterations are likely related to the movement deficits observed in morphants at this stage. Our preliminary results suggest that the abnormalities observed in MFN2 morphants are consistent with the human pathology. Given that a reliable animal model for CMT2A is still lacking, we propose zebrafish as a new and useful tool to dissect the pathogenetic mechanisms underlying CMT2A. Posters 396 Diseases models 326 Nephrocyatin-4 is a ciliary protein required for control ao wnt/b-catenin versus wnt/pcp balance and ciliogenesis in zebrafish embryos 1 C. Burckly 1H. Gaude 2C. Vesque 1R. Salomon 1S. Saunier 2S. Schneider-Maunoury 1 Hereditary nephropathies and kidney development, Inserm U547, Paris, France 2Developmental biology, UMR7622, Paris, France Nephronophthisis, a heterogeneous group of autosomal recessive cystic kidney disease, is caused by mutations in NPHP genes (NPHP1-11), encoding nephrocystins. The nephrocystins share a common subcellular localisation at the primary cilium. Primary cilia play a pivotal role in regulating canonical Wnt/-catenin and non canonical Wnt/PCP (planar cell polarity) pathways. Wnt/PCP is required for polarized converd this, little functional data exists. Through the analysis of multiple alleles we hope to achieve a better understanding of SNPs leading to color variation in zebrafish. We have used electron micrograph analysis to analyze the melanosome morphology of wildtype, albino and golden fish as well as immunofluorescence to show the localization of the products of these genes. As an additional tool we have created a transgenic line expressing a UAS inducible fusion protein of melanoregulin, Melanoregulin was identified in a global gene expression screen for melanophore-related genes and has been shown to specifically localize to melanosomes. Taken together we present a model system capable of greatly expanding our knowledge of pigment variation as well as the function of melanosomes themselves. Posters 400 Diseases models 330 Identification of small molecules that modulate infection of pseudomonas aeruginosa in zebrafish embryos 1 AE. Clatworthy 1J. Lee 1EC. Hett 2M. Mia 1K. Mark 2S. Shaw 1DT. Hung 1 Molecular Biology, Mass. General Hospital, Boston, USA 2Center for Systems Biology, Mass. General Hospital, Boston, USA The increasing prevalence of antibiotic resistance among clinically important human pathogens is a growing threat to human health. There is a clear need to identify and develop novel classes of antibiotics to combat the growing emergence of antibiotic resistant strains. Historically, antibiotics have been identified by their ability to kill (bacteriocidal) or inhibit the growth (bacteriostatic) of bacteria in vitro. This methodology selects for small molecules that target bacterial processes essential for growth, such as cell wall synthesis, DNA replication, RNA transcription, and protein synthesis. While historically this approach has been highly effective, no novel classes of clinically relevant antibiotics have been discovered in over 40 years, with the exception of the recent development of the narrow spectrum drugs daptomycin and linezolid. Importantly, screening bacteria for small molecules that kill or inhibit growth in vitro ignores a wide range of potentially druggable targets such as bacterial virulence factors, bacterial genes essential for viability in the host but non-essential in vitro, and host proteins themselves. Given the current gap in our ability to develop novel antibiotics by traditional in vitro based methods and the growing demand for such drugs, there is a need to reconsider both novel methodologies to identify antibiotic-like molecules as well as alternative ways antibiotic-like molecules could act (novel targets). We are exploring an alternative way of identifying small molecules that have antibiotic-like properties by conducting a chemical screen in a zebrafish embryo infection model. This will allow us to identify compounds that rescue embryos from lethal challenge with the bacterium Pseudomonas aeruginosa, one the one of the most common causes of antibiotic resistant, nosocomial infections in developed countries. We have found that P. aeruginosa infection of zebrafish embryos resembles acute P. aeruginosa infection in rodent hosts. For example, virulence factors required for infection are conserved from rodents to zebrafish (genes involved in type three secretion (pscD) and quorum sensing (lasR and mvfR)) and zebrafish myeloid cells are required for combating infection, similar to rodent models. We have also found that immersion of infected embryos in the anti-Pseudomonal antibiotics ciprofloxacin or imipenem rescues infected embryos from lethal challenge, demonstrating the feasibility of conducting a chemical screen for small molecules that attenuate infection. We are currently screening a chemical library composed of 1120 compounds with known biological activity for compounds that rescue embryos from lethal P. aeruginosa challenge. We plan to use compounds identified in this screen to explore the hypothesis that bacterial virulence factors, bacterial in vivo essential genes, and host gene products are viable targets for antibiotic development. Posters 401 331 Diseases models Chemical-genetic screening for small molecules that alter pigment cell development N. Temperley1, S. Colanesi2, P. Rengtved-Lundegaard1, K. Taylor1, H. Ishizaki1, I. J. Jackson1, J. A Lister3, R. N. Kelsh2, and E. E. Patton1 1 MRC Human Genetics Unit & The University of Edinburgh, Institute for Genetics and Molecular Medicine, UK; 2Biology Programme for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, UK; 3Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, USA Genetic screening in zebrafish has revealed a wealth of mutations in pigment cell development, patterning, and differentiation. In zebrafish, the three pigment cells types are derived from the neural crest: the black/brown melanocytes, the yellow xanthophore, and the silver iridophores. We aim to understand normal pigment cell development and regeneration pathways, with a view to how these pathways may behave during human development and pigmentation, as well as in melanocyte disorders, such as vitiligo, premature hair graying, and melanoma. To complement the available genetic mutants lines, and to identify important molecular tools, we are screening small molecule libraries for compounds that specifically affect the ontogeny and biology of the pigment cells - their development from precursor/stem cells, their proliferation, migration, differentiation, interaction with their environment, and death. In addition to the effects on wild type pigment cells, we are also screening for chemicals that suppress or enhance genetic mutant lines that are deficient for melanocyte and irridophore development and movement, as well as suppressors of small molecules that specifically target melanocytes for cell death. Combined screening results from this screen will be presented, and insight into chemical control of pigment cell pathways will be discussed. Posters 402 Diseases models 332 Zebrafish as a model organism for lowe syndrome M. Hughes, M. Lowe, and I. Barinaga-Rementeria Ramirez Cellular Systems, University of Manchester, Manchester, UK Lowe syndrome is a rare X-linked disease which affects 1 in 100,000 male births, causing disruption to specific tissues, namely those of the brain, eyes and kidney. The genetic cause of the disease has been mapped to mutations within a ubiquitously expressed inositol polyphosphate 5-phosphatase enzyme, termed OCRL1 (oculocerebrorenal syndrome of Lowe). However it is currently unknown how these mutations, which lead to loss of protein expression and activity, cause the disease phenotype. Here we aim to elucidate how the loss of OCRL leads to Lowe syndrome utilising zebrafish as a model organism, studying the effects of OCRL knockdown by morpholino and subsequent rescue on the development of zebrafish embryos. 403 Posters 333 Diseases models Role of the parl genes in Parkinson's Disease etiology using Danio rerio as a model S. Noble and M. Ekker Center for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Canada Familial Parkinson's disease (PD) is associated with mutations in a number of genes, including PINK1 and Parkin. Another candidate gene that may play a role in dopaminergic (DA) neuron death codes for the evolutionarily conserved presenilin-associated rhomboid-like (PARL) protease, located in the inner mitochondrial membrane where it plays a role in regulated intramembrane proteolysis (RIP). Surprisingly, this typically hydrophilic enzymatic reaction takes place in the hydrophobic environment of the lipid bilayer. Thus, it is executed by a unique class of proteases, called intramembrane-cleaving proteases (I-Clips) which include the rhomboid family of proteases. rhomboid-7, the Drosophila orthologue, is genetically upstream and in the same pathway as PINK1 and Parkin. Zebrafish have two parl paralogues, which we arbitrarily designated parl1 and parl2. We found that parl1 is not expressed maternally, but is present from 1 day post-fertilization (dpf) to 7 dpf and is expressed in the adult brain, muscle, li Up to day five the embryos show severe malfunction marked as hyperplastic-dysplastic defects. Double mutants exhibit enhanced cell proliferation and enhanced cell survival after -irradiation compared to siblings. Now, we focus to use the powerful tool of zebrafish lacking PTEN for studying cancer progression. Using state of the art techniques like zf cell culture and transplantation of tumor cells we investigate the role of PTEN during tumorigenesis in vitro as well as in vivo. Posters 406 Cancer 336 Cxcr7 and tumor angiogenesis in zebrafish G. De Sena, S. Buraschi, C. Tobia, and M. Presta Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy Angiogenesis, the growth of new blood vessels from pre-existing ones, plays an important role in tumor growth and metastatic dissemination. CXCR7 (RDC1, CCX-CKR2) has been identified as a chemokine receptor that binds CXCL11/ ITAC and CXCL12/SDF-1 chemokines. Recent studies have shown that CXCR7 is expressed by tumor-associated vessels but not by the normal vasculature, thus suggesting a role for this receptor in tumor angiogenesis. Here, the zebrafish cxcr7 ortholog was cloned and its expression during development was investigated by RTPCR analysis [with significant levels of cxcr7 transcript being detectable from 3 hours postfertilization (hpf) onwards] and by whole mount in situ hybridisation. During late somitogenesis cxcr7 is strongly expressed within somites and in central nervous system precursors. At 24 hpf the expression of cxcr7 in the brain is maintained, associated with a distinct expression in the spinal cord. At 48 hpf cxcr7 is expressed in branchial arches and in posterior lateral line whereas the expression in the brain is limited to diencephalon and midbrain. Knock down of cxcr7 expression by injection of an ATG-targeting morpholino leads to a severe impairment of the systemic circulation in zebrafish embryos. This is paralleled by an interruption at the level of the anterior aortic bifurcation, as shown by loss of expression in this region of the endothelial markers fli1a and VE-cadherin. Also, transgenic tg(kdr:EGFP) embryos injected with cxcr7 morpholino showed a delay in the development of intersomitic vessels. Furthermore, using a zebrafish/tumor xenograft assay developed in our laboratory, we found that cxcr7 downregulation strongly inhibits tumor angiogenesis when zebrafish embryos are grafted with pro-angiogenic mammalian tumor cells. In conclusion, cxcr7 appears to be involved in the development of the vascular system in zebrafish embryo. Also, our data support a role for this chemokine receptor in tumor angiogenesis. 407 Posters 337 Cancer Oncogenic NuP98-HOXA9 suppresses cellular apoptosis and reprograms myeloid hematopoiesis in transgenic zebrafish J.N. Berman1,2,3, A. M. Forrester1,3, E. R. Boyd1,3, C. Grabher4, S. Da'as3, J. T. Dobson1,3, F-B. Kai1,3, A. T. Look4 Departments of 1Microbiology and Immunology and 2Pediatrics, Dalhousie University, 3IWK Health Centre, Halifax, Nova Scotia, Canada, 4Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, U.S.A. The NUP98-HOXA9 fusion oncogene results from a t(7;11)(p15;p15) chromosomal translocation and is associated with inferior prognosis in de novo and treatment-related acute myeloid leukemia (AML), as well as blast crisis in chronic myeloid leukemia (CML). HOXA9, belonging to the highly-conserved HOX gene family of developmental transcription factors is critical for vertebrate hematopoiesis. Elucidating the activity of oncogenic NUP98-HOXA9 may reveal universal mechanisms of leukemogenesis and lead to the design of targeted therapies. The zebrafish is a robust model for studying vertebrate hematopoiesis and leukemogenesis, by virtue of its ex utero development, and conserved genetics and cell biology. We have engineered a transgenic zebrafish harbouring the human NUP98-HOXA9 translocation, and report two novel in vivo phenomena: (1) NUP98-HOXA9 leads to suppression of cellular apoptosis and abnormal cell cyversity of Leiden, The Netherlands PDZ as well as LIM domains are interaction modules, present in a large variety of proteins with diverse biological functions and assorted additional domains. In zebrafish, we identified eleven genes that encode for both a PDZ domain, and one or several LIM domains: five genes of the ALP subfamily (ALP, ALP-like, Elfin, Mystique, and RIL) three of the Enigma subfamily (Enigma, Enigma Homolog, and ZASP), the two LIM kinases (LIMK1 and LIMK2), and the LIM only protein 7 (LMO7). Functionally, all proteins of the PDZ/LIM family share an important trait, they can associate with the actin cytoskeleton. In this study, we analyzed the effects of knock down of PDZ/LIM genes on migration and metastatic behavior of human pancreatic cancer cells. Using a zebrafish xenotransplantation model, we were able to follow metastasis formation and progression in vivo and real time. We implanted human pancreatic cancer cells (PaTu8988-T) into the yolk sac of 2 day post fertilization (dpf) embryos and monitored invasion and metastatic behaviour by confocal laser scanning microscopy. Prior to injections into zebrafish embryos, PaTu8988-T cells were treated with gene-specific small interfering RNAs (siRNAs) for Mystique (PDLIM2, SLIM), RIL (PDLIM4) or LMO7 (PCD1; FBX20). In comparison to control embryos, treatment of pancreatic cancer cells with either one of these siRNAs led to reduced migration and inhibition of the formation of micrometastasis. These results suggest that the three PDZ/LIM genes Mystique, LMO7 and RIL are important for the migratory capacity and metastatic behaviour of this highly-aggressive pancreatic cancer cell line. Posters 410 Cancer 340 Validating novel cancer genes in a zebrafish melanoma model V. Anelli1, C. Santoriello1, D. Rambaldi2, F. Ciccarelli2, M. Mione1 1 IFOM, The Firc Institute of Molecular Biology, 2IEO, Institute of European Oncology, Milan, Italy We have established a model of melanoma progression in zebrafish that is based on the Gal4UAS system, where the crossing between a melanocyte-specific Gal4 line with a UAS:H-RASV12 line results in melanoma development with a high frequency. This binary system allows for target expression/knock down of modulators of oncogene activity in the same cells that undergo transformation, thus allowing precise tumor-specific manipulation of gene expression. We will use this approach to restore the expression of tumor suppressor genes specifically in melanoma cells using the UAS system. Besides known candidates we will test 101 novel candidate cancer genes identified with a bioinformatics approach (Rambaldi et al., 2008). These genes were selected for the low frequency of duplication and the high connectivity in protein networks. As a first step towards the choice of the candidate tumor suppressor to re-express in our melanoma model we carried out quantitative analysis of expression of the 101 genes in several melanoma samples. Data obtained by Real time-PCR analysis from zebrafish melanoma tissue (versus normal skin) demonstrate that the expression of many novel candidate cancer genes is down-regulated (potential tumor-suppressor genes) in zebrafish melanomas whereas only a few are up-regulated (potential oncogenes). We will perform a similar analysis in human melanoma tissue and in melanoma cell lines. The next step will be to restore the expression of the most significantly repressed candidates in our melanoma model. All this work is ongoing and the data obtained will be presented and discussed as well as the strategy used to identify and validate the novel tumor suppressor genes. Rambaldi, D et al, (2008) Low duplicability and network fragility of cancer genes. Trends Genet 24(9):427-30. 411 Posters 341 Cancer Chemical-genetic Approaches in Zebrafish and Yeast Identify Novel Compounds that Sensitize Melanocytes for Cell Death H. Ishizaki1, M. Spitizer2, J. Widenhain2, D. W Melton1, I. J. Jackson1, M. Tyers2, E. E. Patton1 1 MRC Human Genetics Unit & The University of Edinburgh, Institute for Genetics and Molecular Medicine, Crere in this process results in the loss of genetic information and may cause the disease of genetic instability, cancer. Mitotic kinases orchestrate the spindle assembly checkpoint and became attractive targets for anti-cancer drug development. Among the mitotic kinases we became interested in Plk1 and its interaction partner PICH (Plk1-interaction checkpoint helicase). PICH is a putative component of spindle assembly checkpoint as a tension sensor and also involved in DNA decatanation during sister chromatid separation. Despite the considerable interest in PICH, studies exploring the function and importance of PICH during normal cell division are lacking. Therefore we adopted zebrafish embryogenesis model for the easiness of genetic manipulation and abundance of mitotic cells. First, we identified zebrafish ortholog for PICH (zPICH) though bioinformatics. zPICH was readily associated with Plk1 and localized to kinetochores from prometaphase to metaphase, similar to human PICH. zPICH expression was highest at the cellular regions undergoing vigorous proliferation in zebrafish embryos. When the level of zPICH was knocked-down, anaphase bridges were frequently observed, suggesting crucial role of zPICH in proper chromosome segregation. In order to analyze the cell division in detail, we performed time lapse microscopy and found incomplete chromosome separation and cytokinesis for the morphants. These data demonstrate that the zPICH is indeed a human PICH ortholog, which is critical for the sister-chromatid separation. Our results also suggest that the zebrafish model could provide a valuable tool to study the role of mitotic regulators during normal cell cycle and may become a useful vessel to screen drugs targeting them. 417 Posters 347 Cancer Molecular and functional characterization of trim8 genes in zebrafish M. Manzoni1, L. Micale2, G. Paganini1, E. Monti1, G. Borsani1 and G. Merla2 1 Dept. Biomedical Sciences and Biotechnology, University of Brescia, Italy; 2Medical Genetic Laboratory, IRCCS Casa Sollievo della Sofferenza, Poliambulatorio Giovanni Paolo II, San Giovanni Rotondo, Italy TRIM8 is a member of the TRIM/RBCC gene family. TRIM encode proteins characterized by the presence of the tripartite motif, which consists of a RING finger, one or two zinc-binding motifs named B-box and an associated Coiled-coil region (RBCC). In spite of highly conserved structure, which suggests a common basic function, TRIM proteins are involved in a broad range of biological processes and are implicated in several pathological conditions such as Mendelian genetic diseases, cancer development and viral infection. Bionformatic analysis, ChIP and luciferase assays revealed that TRIM8 regulation is modulated by four p53 responsive elements located in the first intron of TRIM8 gene. Importantly, we showed that TRIM8 interacts with and increases the p53 protein stability and it affects p53 transcriptional activation of p21 in mammalian cell lines. TRIM8 is highly expressed in brain and is located within the 10q24.3, a region mostly involved in deletions and rearrangements in brain cancer. Recent investigations revealed that the human TRIM8 gene is down-regulated in a number of glioblastomas and astrocytomas, and studies in mammalian cells suggest it could be involved in a tumor suppression mechanism through the interaction with p53. Our goal is to analyze expression of Danio rerio TRIM8 ortholog(s) and loss-of function phenotype, in order to clarify its developmental and biological role in this animal model. A zebrafish genome-wide search allowed us to detect two orthologs, named trim8.1 and trim8.2, located on chromosome 13 and 12 respectively. Bioinformatic analysis indicates that both trim8.1 and trim8.2 possess the p53 responsive elements and encode a protein with the typical tripartite motives. Zebrafish trim8.1 and trim8.2 expression is detected by RT-PCR starting from early stages of development. In situ hybridizations show a similar expression pattern in the developing central nervous system (forebrain and hindbrain) and eye, in particular in the cerebellum, tectum and retina. We are now generating trim8.1 and trim8.2 knockdown by microinjection of specific morpholino, in order to evaluate the consequences of the silencing of each and both zebrafish trim8 genes. In order to confirm the data obtained in mammalian cells and to shed light on the involvement of TRIM8 in brain tumors onset through p53 interaction, we plan to test the reciprocal effects of trim8.1 and trim8.2 knockdown/over-expression on p53 expression and, vice versa, the effect of p53 silencing/up-regulation on the expression of trim8 genes. Posters 418 Cancer 348 Generation and analysis of zebrafish liver cancer by overexpressing k-rasv12 using mifepristoneinducible cre/lox system A. T. Nguyen1, A. Emelyanov2, C. H. V. Koh1, J. Spitsbergen3, L. Sun1, S. Parinov2 and Z. Gong1 1 Department of Biological Science, National University of Singapore, Singapore; 2Temasek Life Sciences Laboratory, Singapore; 3Department of Microbiology, Oregon State University, USA With histology and gene expression studies of zebrafish tumors closely resembling that of the human, the zebrafish (Danio rerio) has recently emerged as a promising model system to study human cancers. Ras signaling has been at the leading edge of signal transduction and molecular oncology, yet a complete understanding of Ras in human cancer remains elusive. Although KRAS mutations occur in vast majority of human cancers, very little is known about the molecular mechanisms that K-ras drive in tumorigenesis. To study the molecular mechanisms of K-ras oncogenesis, we developed a zebrafish liver cancer model through generation of stable transgenic lines expressing activated K-rasV12 in the liver. Using Activator/Dissociation transposable elements from the maize, we first introduced zebrafish krasV12 oncogene fused with EGFP reporter gene under the liver-specific lfabp promoter and obtained germline transmitted transgenic zebrafish. We observed tumor growth in EGFP-positive livers but also a high mortality in EGFP-positive F1 zebrafish, which hindered the maintenance of these transgenic zebrafish for long-term research. We next used a combination of the mifepristone-inducible LexPR gene expression system and Cre/loxP recombination system to control the expression of oncogenic K-rasV12. Activation of this system successfully induced liver tumor phenotypes, mostly hepatocellular carcinoma, as depicted in our histological examination. By using the inducible transgenic system, we found that the liver tumor could be induced from both fry and adult and we also noticed some GFP-positive tumors in some aged fish, raising the possibility of tumor metastasis. A cDNA microarray approach was then employed to analyze the gene expression profiles of K-rasV12 induced liver tumor formation. Several Ras oncogene family members and genes involved in the MAPK signaling pathway are significantly up-regulated. Other pathways involved in tumorigenesis are also deregulated in our K-rasV12 transgenic model. Consistent with this, our miRNA array data indicated the down-regulation of several miRNAs known to be involved in regulation of expression of kras oncogene. Further analyses of microarray data may help to investigate molecular mechanisms and search for signature pathways leading to K-rasV12 induced liver oncogenesis. We recently also applied the mifepristone-inducible system to transgenic zebrafish expressing K-rasV12 in the pancreas and have also successfully induced tumor formation in this organ and thus demonstrated the general application of the inducible Cre-loxP system in development of transgenic tumor models in zebrafish. 419 Posters 349 Cancer Zebrafish brain represents a reliable niche for xenotransplanted human glioblastoma derived cells E. Rampazzo1, N. Tiso2, F. Pistollato1, G. Del Moro3, A. Della Puppa3, G. te Kronnie1, F. Argenton2 and G. Basso1 1 Hemato-Oncology Laboratory, Department of Pediatrics, University of Padova, Italy; 2 Developmental Biology Laboratory, Department of Biology, University of Padova, Italy; 3 Department of Neurosurgery, University of Padova, Italy Glioblastoma (GBM) is one of the most common primitive malignant tumours, occurring in the central nervous system, which has been shown to contain functionally important subsets of cells with stem-like properties, known as cancer stem cells (CSCs). These subsets of stem cells express primitive markers (i.e. CD133, SOX2, MSI1, BMI1 and Nestin) and are most probably related to the high incidence of relapses in GBM. The presence of CSCs in GMB has been associated with the hypoxic microenvironment typical of brain tumours niches. Thus, a deeper understanding of the parameters characterizing the hypoxic microenvironment, affecting and preserving cancer cells, can help in designing strategies and tools aimed to cure cancer. In recent studies, the zebrafish embryo has been exploited as a novel in vivo model to recreate a transparent and reliable tumour cell niche. We optimized parameters for xenotransplanting primary GBM-derived cells into brain ventricles of zebrafish albino embryos, at 48 hours postfertilization. Transplanted cells were followed for at least 10 days post injection. Cells integrated successfully into the brain and could be tracked, in vivo, by GFP expression or vital staining with a fluorescent dye. By immunocytochemical analyses and in situ hybridisation, tumour grafted cells were checked for the expression of CD133, Nestin and the hypoxic marker Hif-1. As controls, we used ACTB antisense riboprobes and anti-human nuclei antibodies, able to detect all transplanted human cells. Our preliminary observations indicate that at 48 hpf stage, zebrafish CNS microenvironment preserves the GBM immature phenotype. Thus, zebrafish embryos, by mimicking an in vivo hypoxic microenvironment, appear as valuable biosensors for human brain tumours and CSCs behavior studies. Posters 420 Cancer 350 Heritable Zebrafish T Cell Cancer Models from a Mutagenesis Screen J. K. Frazer1,2, N. Meeker1,3, L. Rudner2, D. F. Bradley1,2, A. C. H. Smith1,2, K. Brown4,5, C. Lee4,5, S. L. Perkins6,7, and N. S. Trede1,2 Departments of 1Pediatrics, 2Oncological Sciences, 3Internal Medicine, and 6Pathology, Huntsman Cancer Institute, University of Utah, Salt Lake City, USA; 4Department of Pathology, Brigham and Women's Hospital and 5Harvard Medical School, Boston, USA; 7ARUP Institute, Salt Lake City, USA T cell lymphoblastic lymphoma (T-LBL) and leukemia (T-ALL) are common pediatric malignancies. These diseases have poor prognoses, and their treatments often cause significant morbidities. Unlike many childhood cancers, most T cell neoplasias carry no pathognomonic cytogenetic finding. Consequently, insight into their molecular pathogenesis is incomplete. One key transformation mechanism in T cell malignancy is established, as NOTCH1 activation occurs in about 50% of T-ALL cases. However, many other genetic lesions in these diseases are unknown. To address this deficiency, we pioneered a zebrafish phenotypic screen designed to create new genetic models of T cell neoplasia. We used ENU to randomly mutagenize the germline of adult transgenic zebrafish with T cell-specific GFP expression (lck::EGFP). Then, via fluorescence microscopy, we screened their progeny at juvenile stages for abnormal T cell phenotypes, including the development of GFP+ tumors. Here, we describe the identification and characterization of three different mutants with heritable T cell cancer predisposition. Two mutants exhibit dominant inheritance, while one acts recessively. In all three, disease penetrance is incomplete, demonstrating that other somatically-acquired genetic `hits' are required for malignancies to develop. In each mutant, disease incidence, histologic infiltration, and cellular morphologies resemble human T-LBL and T-ALL. Expression profiles confirm neoplasms are T lineage, and T cell receptor analyses of tumor cells verify their clonality. Malignant cells are transplantable, radiation-sensitive, relapse-prone, and contain leukemia-initiating cells, like their human correlates. Overall, all three mutants recapitulate human T cell malignancy, showing heritable disease predisposition. Current efforts are focused on cloning the mutations conferring inherited cancer risk in each line and the discovery of additional non-inherited genetic lesions contributing to neoplastic transformation. Specifically, comparative genomic analyses have revealed regions with copy number gains or losses in malignant tissues, but not matched control samples, with several recurrent copy number aberrations seen thus far. In sum, we have identified new models of T-LBL and T-ALL. These mutants should provide exciting experimental platforms for study of this important class of human cancers. 421 Posters 351 Cancer Evaluation of Small-molecule PLK1 Inhibitors Using Zebrafish S. Xin, J. Lu, Y. Zhao, N. Wang, S. Li, C. Li, Z. Yang, H. Zhong and S. Lin Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School of Peking University, Shenzhen, China PLK1 (Polo like kinase 1) is one of the key regulators which control mitotic entry, spindle assembly, chromosome segregation, and cytokinesis in cell cycle. Since PLK1 expression is abnormally up regulated in several tumors, it has been regarded as a good target for cancer therapy. A number of PLK1 small molecule inhibitors have been developed as chemical biology tools and potential anti-cancer drug candidates. However, the specificity of these inhibitors is not thoroughly examined. Here, we use whole zebrafish embryo assay coupled with genetic analysis to address this issue. Bioinformatics analysis revealed zebrafish genome has all of the PLK family members which share very high homology with their human counterparts. In particular, PLK1 has a nearly identical 3-D structure between zebrafish and human. To determine if zebrafish can be used to evaluate specificity, we selected two published PLK1 inhibitors, LFM-A13 and ON01910, and one homemade PLK1 inhibitor, PLK1-Yang. When added at 2-cell stage, all of these inhibitors prevented embryos from dividing and caused cells to fuse into one large cell. When added at shield stage, a later stage when zygotic mRNA transcription program is initiated, embryos survived for 3 days but showed different phenotypes for each compound. Embryos treated with LFM-A13 were relatively normal. Embryos treated with ON01910 didn't properly develop trunk and tail while the head structure was relatively normal. Embryos treated with PLK1-Yang had shorter body axis and deformed head structure, which were similar to the phenotypes of zebrafish that are genetically deficient of plk1. These data suggest that the three PLK1 inhibitors had different off-target activities and PLK1-Yang was most specific to PLK1 among them. Our studies also suggest that zebrafish can be used as an efficient in vivo model to rapidly evaluate specificity of small molecule chemical inhibitors designed to other target proteins. Posters 422 Cancer 352 Formation of liver tumor by inducible expression of c-Myc in a tet-on transgenic system in zebrafish Z. Li1, H. Zhan1, Z. Zeng1, J. Spitsbergen2, and Z. Gong1 1 Department of Biological Science, National University of Singapore; 2Department of Microbiology, Oregon State University, USA C-myc (MYC) is frequently observed to be overexpressed in human cancers. Its role in tumorigenesis has also been shown in transgenic models of rodents. Although c-myc overexpression has been found in intestinal adenomas and hepatic adenomas in an APC-deficient zebrafish, its function in transgenic zebrafish studies has only been proved in T-cell leukemia. Despite that these studies have indicated that c-myc is involved in tumorigenesis, the exact mechanism of c-myc involving in the tumor initiation and progression and its target genes remains undetermined. In order to investigate the function of c-myc as well as its underlying biological processes involved in liver cancer formation, we have established a transgenic zebrafish model by using the Tet-on system to conditionally express mouse c-Myc in the liver under the control of the zebrafish lfabp (liver fatty acid binding protein) promoter. After 14 days of treatment with the inducer Doxycyclin starting from 21 dpf, 100% (n= 74) of transgenic fish showed apparently enlarged abdomen, whereas none (n=23) of the non-transgenic siblings showed such phenotype. Histological analysis showed that overexpression of mouse c-Myc in zebrafish liver caused hyperplasia of liver at early stage and followed by the development of hepatocellular adenoma when c-Myc was overexpressed for a longer time. Brief withdrawal of Doxycycline greatly reduced the c-Myc expression and alleviated the enlarged liver phenotype. Compared to the activation of c-Myc from 21 dpf, less time is needed to obtain liver overgrowth when c-Myc was activated from early developmental stages. In addition, we also observed in some of the treated embryos that liver cells were detached and migrated to other tissues including blood. Our observations indicated the potential of our c-Myc transgenic zebrafish as a model in the study of liver cancer progression and reversal. In the ongoing experiments, we are going to activate transgenic c-Myc at different ages to investigate its role in liver cancer formation during different stages of development and growth. Microarray studies will be carried out for understanding of the c-myc pathway from transcriptomic analyses and we expect that these experiments will provide new insights into mechanism of c-myc involving in the liver cancinogenesis. 423 Posters 353 Cancer Zebrafish is a powerful system in studying Aurora-A function in mitosis and development H-Y. Jeon and H. Lee Department of Biological Sciences, College of Natural Sciences, Seoul National University, Korea Aurora-A is a serine/threonin kianse, which regulates many intricate processes during mitosis. It is localized at centrosomes and microtubules during mitosis, implicating roles in the control of centrosome and spindle function. Since the discovery of Aurora-A overexpression in cancer cells and induction of cell death with its inhibition, Aurora-A specific inhibitors drew much attention as promising anti-cancer drugs. Despite the emerging interest for clinical applications, our understanding how Aurora-A functions in normal cells and organisms is poor. Here, we performed loss-of-function experiments in zebrafish embryos, as the model stages normal cell cycle and development. In order to block the function of zebrafish Aurora-A (zAurA) we utilized a translation blocking morpholino or a small molecule inhibitor, MLN8054. After the morpholino injection or the inhibitor treatment, zebrafish developed short trunk with severe growth retardation and cell death. The developmental defects might be attributable to the abnormal mitotic progression as the cells in the morphants manifested monopolar spindles and increased expression of mitotic markers suggestive of mitotic arrest. The embryos treated with MLN8054 also exhibited abnormal spindle formation including monopolar and multipolar spindles, similar to the morphants. In order to analyze the fate of individual cells after blocking zAurA, we generated Histone H2B-GFP transgenic zebrafish and carried out time-lapse microscopy. In the live-imaging, the morphants displayed mitotic delay and/or mitotic slippage in most cells. Intermittently, we also observed chromosome bridges and cell death. Taken together, our data demonstrate that zAurA is essential for the bipolar spindle formation and that its loss primarily results in mitotic arrest. Our study reveals that zebrafish is a powerful model in studying mitosis. Furthermore, zebrafish embryogenesis is an efficient in vivo system to test the efficacy of mitotic inhibitors, let alone assess the functions of mitotic kinases. Posters 424 Regeneration 354 Kidney Damage and Regeneration in Zebrafish Larvae C. Cianciolo Cosentino and N. Hukriede Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, USA The vertebrate kidney has an innate ability for recovery and regeneration following acute injury. During the recovery phase renal epithelial tubular cells are known to proliferate actively and differentiate to reconstitute tubular epithelium. The regeneration process is thought to have many parallels with the growth and maturation that occur during kidney organogenesis. However, the source of proliferating cells that repopulate the injured nephron, and the relative contribution of intra-renal versus extra-renal cells is poorly understood. A better understanding of the mechanisms of nephron repair may provide important clues for the development of new therapies for the treatment of acute kidney injury. Fish possess a remarkable regenerative ability and are able to respond to renal injury by nephron neogenesis. The zebrafish pronephric kidney thus provides a simplified and suitable model for the study of kidney regeneration. To explore the link between kidney repair and embryonic development we induced gentamicin mediated proximal tubular damage in the larval zebrafish kidney. To model damage in real-time, we utilized transgenic lines for lhx1a and pax2a, two genes known to play key regulatory roles during kidney embryonic development. We assayed for damage in terminally differentiated pronephric kidneys that do not express either reporter. The goal was to determine if we could visualize reactivation of either reporter and if so whether cells expressing GFP were found in the existing tubules or in a distant population of cells. In both Tg(lhx1a:eGFP) and Tg(pax2a:eGFP) lines we saw reactivation. Our current data suggests that the GFP positive cells are localized outside the kidney and could possibly be ascribed to a local population of progenitor cells. In the future, by determining the origin of the reactivated cells and being able to manipulate their numbers, we hope to develop methodologies that could eventually be used as an alternative therapy for acute kidney injury. 425 Posters 355 Canonical Wnt Signalling in the Development of the Zebrafish Optic Tectum M. Varga, R. Young and S. W. Wilson Cell and Developemntal Biology, University College London, UK Regeneration The optic tectum (OT) is one of the primary processing centers of visual information in vertebrates. During the embryonic development of teleost fish, it develops from the simple neuroepithelium of the mesencephalic alar plate into a complex, multilayered structure, containing at least eleven different cell types. The continuous post-embryonic growth of the OT shows that its germinative zone retains proliferating cells during the whole life of the fish, providing a good model to study neural stem cell maintenance and post-embryonic neurogenesis. We provide evidence for the existence of a Wnt-responsive, proliferative cell population in the dorsal OT. These cells are epithelial in their character and abut the mesencephalic roof-plate, which is the likely source of Wnt signals. With a combination of loss-of-function and gain-of function experiments, we show that Wnt signalling is essential for cell-cycle progression in these cells. We hypothesize that this population of cells contains the neural stem cells of the OT and the proximity of the roof-plate defines the niche where these stem cells can persist. Posters 426 Regeneration 356 A small molecule screen for motor neuron regeneration in zebrafish G. Becker1, T. Becker1, A. Norris1, M. M. Reimer1, P. Rengtved-Lundegaard2, E. Patton2 1 Centre for Neuroregeneration, University of Edinburgh, Summerhall, Edinburgh, UK; 2Medical Research Council Human Genetics Unit, University of Edinburgh, UK Recently, we have demonstrated that adult zebrafish are capable of motor neuron regeneration (Reimer et al., 2008, J Neurosci 28:8510-8516). However, humans cannot do this, for example in motor neurone diseases, and it remains a mystery as to why not. Small molecules could be useful in stem cell therapy of motor neuron diseases and there is a need for novel small molecules that drive motor neuron differentiation from stem cells. In order to find such compounds, we use transgenic embryonic zebrafish containing motor neurones that express grn/repair would have interesting therapeutic implications. To simulate the ischemic injury of myocardial infarction caused by coronary artery occlusion, we set up a treatment to cause acute hypoxic stress in adult zebrafish. In order to elucidate possible mechanisms of injury repair, we focused our attention on the search for cardiac progenitors. Adult zebrafish heart expresses genes involved in cardiac development and, at low levels, a gene homologous to a mammalian stemness marker (kita). We observed the up-regulation of kita 18 hours after zebrafish exposure to hypoxia and we are currently analyzing possible differential expression of other key development genes that might be involved in the repair of localized ischemic damages and might suggest a mechanism of differentiation of cardiac progenitors present in the adult heart. 429 Posters 359 Regeneration Expression profiling of neural precursor cell markers in the adult zebrafish optic tectum H. Tanaka1,2, Y. Ito1, H. Okamoto1,2 and T. Ohshima1 1 Department of Life Science and Medical Bio-Science, Waseda University, 2-2Wakamatsu-cho, Shinjuku-ku, Tokyo, Japan. 2Laboratory for Developmental Gene Regulation, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1Hirosawa, Wako, Saitama, Japan In adult teleost brain, proliferating cells are observed in broad area while these cells are located in the restricted regions in mammalian brain. In the optic tectum of adult zebrafish brain, most of proliferating cells are distributed in the caudal margin of periventricular gray zone (PGZ). However, whether these cells have properties of neural stem or precursor cell is still unknown. To confirm this point, we examined expression patterns of known neural stem/ precursor cell markers along with bromodeoxiuridine (BrdU) labeling in the optic tectum PGZ of adult zebrafish. We found that the PGZ of optic tectum is largely divided into three distinct regions, one mitotic region and two non-mitotic regions which we designate as superficial and deep layer of PGZ. These regions are distinguished by the differential expression patterns of neural stem/precursor or neuronal cell marker genes, such as pcna, gfap, sox2, fabp7a, cntfr, msi1, elavl3 and neurod. We also analyzed cell linage of proliferating cells from 24 hours to 2 months after BrdU incorporation. We found that few BrdU-positive cells were still observed in the mitotic region in 2 months after BrdU incorporation. We also found that most of the cells exited cell cycle and showed strong elavl3 expression between 2 weeks and 1 month after BrdU incorporation. Then, these cells differentiated into the cells in the superficial layer of PGZ. Interestingly, some populations of BrdU-positive cells which ceased cell proliferation were elavl3-negative and maintained expression of sox2, fabp7a, cntfr and msi1. In contrast to elavl3positive cells, these cells incorporated into deep layer. These findings suggest that most of the progenitor cells differentiated into cells consisting mature PGZ. However, some populations may not differentiate and maintain neural stem/progenitor-like properties in the adult optic tectum. Posters 430 Regeneration 360 Expression of Telomerase and Telomere Length are Unaffected by Either Age or Limb Regeneration in danio rerio T. C. Lund, T. J. Glass, J. Tolar, B. R. Blazar University of Minnesota, Division of Pediatric Hematology/Oncology/Blood and Marrow Transplant, Minneapolis, USA The zebrafish is a rapidly growing model for studying many aspects of biology. Recently, ztert, the teleost homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintain telomerase activity for much of its adult life. We have looked systematically at several individual organs of the zebrafish with regard to both telomere length and telomerase activity through stages of its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years old by Southern blot anae biological mechanism by which transcription factors regulate development it is important to identify their targets. We have previously approached this problem using chromatin immunoprecipitation followed by analysis of transcription factorassociated DNA on genomic microarrays (ChIP-chip). Whilst such studies have yielded valuable data they are limited by the coverage of the available microarrays. A superior alternative now available to us is chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq data have numerous advantages over microarray data including increased coverage of the genome and no loss of data between genome releases. Here we will present our ChIP-seq methodology, preliminary data for No tail and Eomesodermin genomic binding and a comparison with our ChIP-chip data. 441 Posters 371 Genomics Establishment of medaka full-length cDNA resources -An activity of NBRP MedakaK. Naruse1, Y. Yoshimura1, X. Shen1, T. Okubo2, T. Shin-I3, Y. Minakuchi3, H. Kagoshima3, K. Ohishi3, A. Toyoda3, T. Aizu3, T. Watanabe3, Y. Yamazaki3, A. Fujiyama3 and Y. Kohara3 1 National Institute for Basic Biology, 2Graduate School of Agricultural and Life Sciences, The University of Tokyo and 3National Institute of Genetics, Japan Although medaka genome sequences are now publicly available through Ensembl, USCS genome browser and UT genome browser, the annotation of medaka genome using species specific data such as medaka cDNA and protein is very limited. To overcome this situation, we are now conducting the large scale full-length cDNA sequencing project. We have constructed seven full-length cDNA libraries (early embryogenesis, organogenesis, fry stage, male liver, brain, testis and ovary) with V-capping method (Kato et. al., DNA Res, 12:53-62, 2006). We picked up 24960 clones from each library and sequenced both end of the clones. After mass alignment of all data, we identified over 18000 unique clones with the difference of 3'end. We are now sequencing whole sequences of each representative clone. About 30 % of clones from cDNA libraries of the organogenesis stage and brain are unique in each library. Number of unique sequences in liver cDNA library is only 12.5 %. We observed the variation of transcription start site in the genes, whose transcripts are relatively abundant. All data and clones are searchable and open for public through NBRP Medaka website ( Posters 442 Genomics 372 FISHTRAP: An Insertional Mutagenesis Screen in Zebrafish using the Ac/Ds transposon system K. Sampath, H. N. B. Quach, R. Hua, A. Ramanathan, K. Balasubramanian, A. Emelyanov, S. Parinov Temasek Life Sciences Laboratory, 1 Research Link, NUS, Singapore We are using the Ac/Ds transposon to carry out a transposon insertion screen in zebrafish. The transposon system we are using is multifunctional, and contains a gene-trap reporter, an enhancer-trap reporter, and a deleter cassette. In the year 2007-2008, we performed a pilot screen using maize Ac/Ds transposon system, which was recently reported to yield in a high rate of germ-line transmission in zebrafish (Emelyanov, 2006). We tested the efficacy of several Ds insertion cassettes, and identified one which gave a germ-line insertion frequency of ~24%. We find that founders, on average, harbor 3-4 inserts. Expression of the gene trap fluorescent reporter in progeny of injected founders was found in multiple cell types during embryogenesis, including the central nervous system, notochord, circulatory system pronephric ducts, liver, and intestine. F1 progeny of the Ds insertion lines were confirmed by PCR, and flanking sequences from 74 insertion lines obtained by Thermal Asymmetric InterLace (TAIL) PCR. We have performed phenotypic characterization of F2 fish, and have identified insertion mutants with defects in the digestive organs, CNS, heart etc... Our pilot gene trap insertion screen shows that the maize Ac/ Ds can be used successfully to trap genes in zebrafish. We have now embarked upon a large-scale functional genomics screen as a collaborative effort involving multiple zebrafish laboratories in Singapore. The Ds insertions from the FISHTRAP screen will be made available to the larger research community in due course, and has the potential make a significant impact in the field of zebrafish functional genomics. 443 Posters 373 Genomics Havana and Vega: Providing Manual Annotation for the Zebrafish Community G. K. Laird, S. Donaldson, J. P. Almeida-King, D. M. Lloyd, H. K. Sehra, K. Howe, B. Reimholz, S. Trevanion, J. Torrance, J. G. Gilbert, J. L. Harrow, T. Hubbard Wellcome Trust Sanger Institute, Cambridge, United Kingdom The zebrafish genome is being sequenced and analysed in its entirety at the Wellcome Trust Sanger Institute. The manual annotation is provided by the Human and Vertebrate Analysis and Annotation (HAVANA) group and is released at regular intervals via the Vertebrate Genome Annotation (Vega) database ( The Vega database is a central repository for high quality, frequently updated, manual annotation of vertebrate finished genomic sequence. Our annotation is completed in close collaboration with the Zebrafish Information Network (ZFIN) (, which has enabled us to provide an accurate, dynamic and distinct resource for the zebrafish community as a whole. Our manual annotation is based on the reference genomic sequence that has been derived from clones meticulously finished in-house to an accuracy of over 99.99%. This method of annotation produces a wealth of reliable information that may not be readily or accurately identified by current automated efforts. Such as, for example, non-coding genes, pseudogenes, complex gene structures, clusters and re-arrangements. All structures are supported by evidence provided by the latest species-specific and cross-species cDNA, EST and/or protein homologies (UniProt) and facilitate the identification of novel splice variants, and poly-A features. New zebrafish transcriptome sequence from the Illumina (Solexa) Genome Analyzer has recently provided a valuable new source of data. Extremely deep coverage from a range of developmental stages and adult tissues has proven an excellent resource for manual annotation. Gene models have been extended and previously fragmented genes joined. In addition, further alternative splices have been identified and previously un-described genes highlighted. Full clone annotation of our current finished clone path for chromosomes 2, 4, 5, 8, 9, 10, 13, 18, 19, 20, 22 & 23 is available in Vega. In addition we have annotation for the majority of ZFIN cDNAs that map to our current assembly. These two annotation strategies are on-going and we aim to finish full annotation of the rest of the genome within the next few years. Posters 444 Genomics 374 Elucidation and comparative analysis o fan MHC haplotype in the CHORI-1073double-haplois zebrafish HK. Sehra, JP. Almeida-King, GK. Laird, DM. Lloyd, S. Donaldson, K. Howe, B. Reimholz, J. Torrance, W. Chow, JGR. Gilbert, M. Larbaoui, E. Griffiths, RD. Storey, KA. Auger, G. Kerry, S. Trevanion, D. Stemple, JE. Collins, JLA. Harrow, T. Hubbard. Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK The major histocompatibility complex (MHC) comprises a group of genes involved with the adaptive and innate immune systems. Whilst it is known to be present amongst vertebrates, it remains poorly characterised outside of mammals. Its role in disease susceptibility and resistance makes the region a focal point in comparative genomics, hence there is a need for further data from a wider variety of species. The MHC core regions in the zebrafish reference genome currently consist of several haplotypes; this is not conducive for describing a region known to display major variation. We have therefore selected a tile path of BACs from the double haploid (DH) fish allowing the replacement of these mosaic regions with a single haplotype. The prior sequencing and annotation of the human, pig and dog MHCs has shown that in these cases the MHC exists as a tightly linked cluster, encoding over 200 immune related proteins contained within three sub-regions. The chicken MHC, in contrast, contains just 19 genes. In zebrafish, whilst we have identified a core set of orthologous genes grouped together on chromosome 19, many other members are found scattered across the genome. Similar disparate organisations have been described in other teleosts: in medaka an orthologous core region of class I genes is found on chromosome 11 whereas the class II genes are spread over several chromosomes, and segregation analysis in stickleback has shown class I and class II loci assort independently of each other. This suggests that the different teleost lineages evolved independently following the initial whole genome duplication, via large-scale genomic rearrangements. As the zebrafish is an excellent model for developmental studies, we anticipate that the elucidation of its MHC combined with genomic comparisons with other lineages will improve the understanding of function of these immunological genes. 445 Posters 375 Genomics Genetic analysis of clonal characteristics of medaka nuclear transplants generated from the somatic cell nuclei transfer to non-enucleated diploidized eggs 1 T. Adachi, 1E. Bubenshchikova, 2M. Shinya, 3M. Kinoshita, 1E. Sawatari, 1H. Hashimoto, 1 Y. Wakamatsu 1 Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan; 2Model Fish Genomics Resource, Natl. Inst. of Genetics, Mishima, Japan 3Graduate school of agriculture, Kyoto University, Kyoto, Japan Nuclear transplantation is a key technique for cloning animals using somatic cells. We previously reported a novel method for generating diploid and fertile medaka fish through the transfer of adult somatic cell nuclei to diploidized eggs. Since we used non-enucleated eggs as recipients, we simply accepted that the recipient nuclei would contribute, at least in part, to the formation of nuclear transplants. Interestingly, the resultant transplants have exhibited only the genotypic and phenotypic traits of donor clones to date. To further clarify the nuclear origin of these donor clone-like transplants, we sought to determine whether the transplants had traces of the recipient DNA in their genome. Populations of Japanese medaka consist of two groups, a northern population and a southern population. HNI-I is one of the representation of northern population and d-rR strain belongs to southern population. Since these two strains are genetically distinct from each other and the numerous DNA polymorphisms known to exist between them have all been well characterized, these polymorphisms could be utilized to effectively identify the origin of the DNA in the transplants. We successfully prepared three adult diploid nuclear transplants using the medaka HNI-I strain as the donor and the d-rR strain as the recipient. We therefore assayed the genotypes of the transplants using 96 polymorphic PCR markers and genomic DNA isolated from the anal fins of all three transplants and from the internal organs (brain, eye, liver, kidney, spleen, muscle) of one of the transplants. The results showed that, at all of the chromosomal locations examined - four sites on each of 24 chromosomes - the polymorphisms obtained showed the transplant genotypes to be identical to HNI-I, indicating that the nuclei of the three transplants originated from the donor. We therefore conclude that the putative medaka clones produced by our new method are true clones of the donor. Posters 446 Genomics 376 Genome-wide functional screen for the enhancers in zebrafish I. Kondrychyn, M. Garcia-Lecea, V. Korzh Developmental Biology, IMCB, Singapore, Singapore In vertebrate genome the enhancers can be scattered over large distances in non-coding sequences. In respect of coding regions they can be located upstream, downstream and in intronic regions. Such scattered distribution makes difficult the identification of enhancers by functional assays. The enhancer trap technique is the effective approach for monitoring gene activity despite on the difficulties of direct identificaebrafish, maintain their insulator capacity when tested in this model. 451 Posters 381 Genomics Reverse Genetics in Australia: Fishing for novel zebrafish mutants S. Berger1, J. Berger1, G.J Lieschke2, J.K. Heath and P.D. Currie1 1 Australian Regenerative Medicine Institute, Monash University, Clayton, Australia; 2Cancer and Haematology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; 3 Colon Molecular and Cell Biology Department, Ludwig Institute for Cancer Research, Parkville, Australia The use of zebrafish as a vertebrate genetic model organism relies mostly on investigating gene function in a forward genetic manner, where identified phenotypes lead to the discovery of novel genes. At the moment, fewer methods are available for reverse genetics, where a gene of interest is knocked out to study its function. There are currently two approaches available for reverse genetics: TILLING and deep sequencing. TILLING (Target Induced Local Lesions In Genome) has initially been established for plants and invertebrates and was shown to be applicable to zebrafish also (Wienholds et al., 2002). Deep sequencing allows the analysis of large groups of DNA samples, thereby detecting mutants with high throughput and low costs. Requirement for either of those methods is the generation of randomly mutagenised fish that can either be kept alive during the screening process or can be used to set up a frozen sperm / DNA library that allows access to mutants far beyond the time limit of a zebrafish lifespan. We have treated wild type males with N-ethyl-N-nitrosourea (ENU) and have subsequently outcrossed the mutagenised males to wild type females. The resulting mutants were used to perform sperm freezing. Simultaneously, tissue for DNA extraction was collected. Amplicons of known genes across the entire genome were sequenced to calculate the mutagenesis rate. The analysis confirmed a high saturation of mutations wit a mutagenesis rate of 1 base-pair exchange every 122 kb. Furthermore, as proof of principle, we resurrected one identified mutant via In Vitro Fertilization from a frozen sperm sample that was stored for 2 years. The remaining mutant F1 females were crossed into a Tg(acta1:GFP)zf13 background. The resulting F2 families were screened for muscle mutants. This F2 screen resulted in the identification of two fin mutants, two heart mutants, two novel dystrophic and one muscle mutant. We will now continue to perform state of the art ENU mutagenesis and will collect a total number of 5000 frozen sperm samples with accompanying DNA samples that will allow researchers from the entire community to screen for mutants of their interest. After identification of such, the frozen sperm sample will be used for the resurrection of the mutant through In Vitro Fertilization and the researcher will receive the fish for further analysis. Posters 452 Genomics 382 The Zebrafish Genome: an Update of Mapping and Sequencing Progress S. Sims and L. Matthews Sequencing, Wellcome Trust Sanger Institute, Cambridge, UK The Sanger Institute is in the final months of its nine year project to sequence the Zebrafish genome, funded entirely by the Wellcome Trust. At 1.36Gb in size, this is the largest singlecentre sequencing project to be taken to HTGS Phase 3 in the world. The strategy used began with libraries of BAC clones, fingerprinted, mapped and sequenced to "gold standard" (at least 99.99% accuracy in euchromatic sequence), with the assembly being aided and informed by the addition of Whole Genome Shotgun (WGS) data. In 2008, a new Heat Shock map was generated by analysis of markers from 48 homozygous fish; this map now forms the basis for final clone selection and gap closure. Many regions of the genome appear to have high levels of haplotype diversity; BAC and fosmid libraries generated from a single doubled haploid fish (CH-73/1073) have been used to resolve such regions and extend clone coverage. Fluorescence in situ hybridization (FISH) is being used to help anchor contigs, resolve order and estimate gap sizes along chromosomes. We discuss sequencing progress and present examples of some of the unique challenges faced in finishing this genome, and report results from trials using the new technology platforms. Tools which aid the coordination and analysis of the genome are described. The latest assembly can be viewed at which includes links to pre-Ensembl where the new assembly zv8 can be found. The sequencing project moves into maintenance phase in January 2010. 453 Posters 383 Genomics The Zebrafish Mutation Resource: Towards a TILLING knockout in every protein coding gene R. Kettleborough; E. Busch-Nentwich; C. Herd; F. Fenyes; C. Torroja; F. van Eeden; D. Stemple Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK Target selected mutagenesis, or TILLING (for targeting induced local lesions in genomes), is a proven method that allows mutations to be identified in virtually any gene. We have previously shown that capillary-sequencing can be used to effectively identify mutations in a high throughput manner. A library of Zebrafish non-sense alleles is being created by the Zebrafish Mutation Resource, each with an associated morphological and molecular phenotype. Phenotypic data will be published via ZFIN (using an adapted version of the Phenote database). Information about the mutations identified by the project can be found on our website: http://www.sanger. The advent of next generation sequencing technologies has significantly changed the amount of mutations that can be identified and has opened up the possibility of identifying a nonsense allele in every protein coding gene within a reasonable time frame and budget. We have designed a strategy that will allow us to amplify and sequence thousands of exonic fragments on a single lane of the Illumina Solexa sequencing platform. A primer design program has been written that selects 100bp exonic fragments that are most likely to give a non-sense allele by ENU mutagenesis. These fragments (up to 4000 exons per experiment) are amplified across DNA pools from 24 mutagenised zebrafish, and each pool is run on one lane of the Solexa platform. Automated data analysis then searches the sequence for all potential non-sense alleles in the amplified fragments, and putative non-sense alleles are confirmed by capillary sequencing. Using this method the Zebrafish Mutation Resource aims to produce >1000 knockouts over the next 2 years, creating a rich resource for the zebrafish research community. Posters 454 Genomics 384 Integration of heat-shock, mgh and t51 genetic maps into zebrafish genome assembly (zv8) C. Torroja1, J. Torrace2, B. Reimholz2, K. Howe2, D. Stemple1 1 Team31. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK; 2 Team71. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK Due to the choice of genetic map (T51) on which previous Zebrafish assemblies have been built, there have been many discrepancies between genetic mapping positions and the position of the markers represented in these assemblies. Analysis of the correlation between Zv7 and the three genetic maps (HS, MGH and T51) highlights this problem. Despite the fact that the T51 map was used to anchor physical map fingerprint contigs (FPCs) in Zv7, there is poor correlation between the assembly and the map. Indeed, for Zv7 the average correlation between the three maps and the assembly is only around 0.7. This is an important issue for researches trying to map their mutations as well as for the genome sequencing project, making the task of identifying real gaps between FPCs and resolving them very difficult. To improve Zv8 quality we have reorganized FPC order and orientation by combining the existing genetic maps: Heat Shock (HS), MGH meiotic maps and the T51 radiation hybrid map. We have reorganized the FPCs, prioritising the meiotic maps HS and MGH for long-range order and chromosome assignment, then using the T51 radiation hybrid map mostly to resolve local order and orientation. In this process we consider an FPC as an indivisible unit. The FPC provides a link between the markers mapped on the FPC and therefore makes an association between the three different maps. We have used this association to integrate map position data derived from the three different maps by sequence alignment between genetic markers and the sequence of each FPC. By using all three maps we increase the coverage and resolution to the maximum possible given available information. We see a striking improvement in the correlation between the Zv8 assembly and each of the genetic maps, which is now at an average of 0.96 for each chromosome. In addition, we find that Chromosome 4 has a significantly increased size now closer to the size predicted by flow cytometry. Assignment of sequence to Chromosome 4, however, needs to be interpreted with caution. The long arm of Chromosome 4 contains very repetitive sequence, which could lead to mis-localisation of FPCs. 455 Posters 385 The Zebrafish Genome Sequencing Project: Web resources K. Howe, W. Chow, B. Reimholz, J. Torrance and T. Hubbard Informatics, Wellcome Trust Sanger Institute, Cambridge, UK Genomics The Sanger Institute has recently released a new integrated zebrafish genome assembly, Zv8. This genome assembly shows major improvement compared to its predecessors. It contains a higher precentage of high-quality finished clones and these clones have been ordered and oriented by careful use of the genetic maps HS and MGH and the T51 radiation hybrid map. In this process, we identified further artificial duplications arising from the use of several haplotypes and those have subsequently been removed. Gaps between the finished clones have been filled with contigs from a whole genome shotgun (WGS) assembly with a greater fold coverage than was used for Zv7. The Zv8 assembly has been fully annotated using the Ensembl gene build pipeline and is accessible in Ensembl ( Ensembl now also offers a track for community annotation. Please enter your annotation at Annotation/ Finished clone sequence only with manual annotation is provided in the Vega browser (vega. With the sequencing project nearing its completion, we will eventually create a finished clone only assembly and provide integrated manual and automated annotation for it displayed in Ensembl. We will present the new data to the community and demonstrate the use of the available web resources during the poster sessions and in a one-day workshop (details to be advertised). We will also help with individual problems regarding the use of these resources. Please come and see us! Posters 456 Bioinformatics and system biology 386 Meta-analysis of rapamycin modulated expression profiles in the zebrafish and human O. Konu1, C. Sucularli1, K. D. Kaya1, A. R. Ozturk1, H. Ozdag2 1 Bilkent University, Department of Molecular Biology and Genetics, Ankara, Turkey; 2Institute of Biotechnology, Ankara University, Turkey Recent high-throughput gene-expression profiling and transcriptional control studies help deciphering the functional components in the genomes. In this context, large numbers of gene expression profiles under different physiological and pathological conditions are being generated for multiple vertebrate species ranging from zebrafish to humans. Meta-analysis of such multivariate datasets provides unique opportunities for comparative analysis of geneexpression modules. Accordingly, we performed a microarray experiment to decipher the effects of rapamycin on ZF4 transcriptome; and then meta-analyzed this dataset using a series of public zebrafish and human Affymetrix microarray data. We developed a meta-analysis tool in which selected human and zebrafish Affymetrix datasets were made accessible through a web-interface for statistical testing and visual representation using R modules ( Our findings indicated that maternal expression of up- and down-regulated genes bath. A day after the treatment, GFP expression was observed in some organs such as gonads, a liver and fins. Secondly, we employed a thin metal probe whose temperature can be controlled and monitored. By inserting this probe into the body cavity, noninvasive heat treatment was possible. This method successfully allowed us to induce gene expression in a tiny area of ovary. 461 Posters 391 Emerging technologies Viral cell transduction in the adult zebrafish brain I. Rothenaigner1, R. Jagasia1, A. Lepier2, P. Chapouton1, B. Bahn1, L. Bally-Cuif1 1 Helmholtz Center Munich- German Research Center for Environmental Health, Department Zebrafish Neurogenetics; 2Institute of Physiology, University of Munich The adult zebrafish brain shows in contrast to mammals an enormous potential to produce new cells. Proliferating zones, visualized so far with the proliferation markers PCNA and MCM5 or by incorporation of the thymidine analog BrdU, can be found in several brain regions along the entire anterior-posterior axis. These germinal zones, mostly located close to the brain ventricle, contain self-renewing progenitor cells that show features of adult mammalian neural stem cells. BrdU tracing experiments showed that cell division can be followed by the formation of postmitotic neurons. The aim of our study was now to develop a tool to analyze the long-term fate of neural progenitor cells by permanently labelling them. For this purpose we established virus-mediated transduction of these cells in the adult live zebrafish brain. We chose retrovirus-based systems, which have the advantage that after infection the transgene is stably integrated into the genome of the target cells. We injected MLV- and HIVbased GFP-encoding retroviruses into the brain ventricles of live adult zebrafish. Preliminary results show infection of cells along the ventricular zones with two types of retroviruses and promoters. Further experiments will classify the set of successful technical combinations and reveal the long term fate of infected cells using immunohistological stainings of neuronal and glial markers. This method will permit the long-term genetic tracing of progenitor cells in the live zebrafish adult brain and will help to further identify the molecular mechanisms controlling stem cell maintenance and differentiation. Posters 462 Emerging technologies 392 Development of living color transgenic medaka for biomonitoring aquatic contamination H.B.G. Ng and Zhiyuan Gong NUG Graduate School and Department of Biological Sciences, National University of Singapore With the advent of GFP reporter gene, it is feasible to apply the living color transgenic fish in monitoring water contamination. Previously, our laboratory has generated a GFP transgenic medaka line under the estrogen-inducible promoter from vitellogenin gene and demonstrated that the Tg(mvtg1:gfp) transgenic line faithfully responded to estrogen and other estrogenic compounds by displaying green fluorescence color and thus provided a convenient biomonitoring tool amendable for online surveillance of estrogenic compounds (Zeng et al., Eng. Sci. Tech. 39:9001). In the current presentation, we report the generation of second biomonitoring transgenic medaka line using a stress-inducible promoter, hsp70, with the designation of Tg(hsp70:gfp). The transgenic line was generated with the aid of maize Ac/Ds transposon system. Out of 12 founders screened, we obtained germline transmission of the transgene from 10 of them, indicating a high efficiency of the Ac/Ds system to aid transgene integration. All of the transgenic lines we obtained show strong ubiquitous GFP induction within 4 hours of heat shock treatment at 37oC for two hours in 2 dpf or older embryos. The expression of GFP is consistent with that of endogenous hsp70 mRNA after heat shock. In addition, the transgenic line also responded to several heavy metals including mercury, arsenic and cadmium. Thus the newly developed transgenic line may be used for monitoring stressed caused by heavy metals and potentially other chemicals to be protein and impaired negative feedback may account for the observed increase in hypothalamic corticotropinreleasing-hormone (CRH) and proopiomelanocortin (POMC) mRNA as well as plasma cortisol in basal, non-stressed state. Behavioral testing reveals significant increase in freezing behavior of the homozygote fish in isolated arena following repeated exposures to similar conditions. This behavior is inhibited by acute (Diazepam) and chronic (Fluoxetine) anxiolytic and antidepressants treatments. Accordingly, changes in serotonin transporter mRNA levels before and following SSRI treatment, point toward a potential role for brainstem serotonergic system in these behaviors. Finally, mutant fish that are allowed visual interaction with other fish do not display the freezing behavior, suggesting social buffering effect. The result presented in this study emphasizes the uniformity in symptoms and machinery of stress-induced anxiety disorders across vertebrates and validates the use of zebrafish GRs357 mutant for exploring the neural basis of certain aspect of human disorder. 471 Posters 401 Emerging technologies an online neuroanatomical resource T. A. Hawkins1, K. Turner1, M. Folgueira1, J. D.W. Clarke2, S. W. Wilson1 1 Department of Cell and Developmental Biology, Anatomy Building, UCL, London, UK; 2MRC Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Hospital Campus, London, UK A complete description of the connections of all the neurons of the zebrafish nervous system, ie. the zebrafish connectome, is some years from being a reality. For this epic task to be completed, some key technologies remain to be developed. However, the developing zebrafish is likely to be a candidate for the first `complete' first-draft vertebrate connectome because of its relative simplicity and accessibility. We are taking what might be considered the first tentative steps towards this enticing goal through the development of This is a web-based neuroanatomical resource we aim to be a repository for all neuroanatomical knowledge about the zebrafish. The data contained within should provide a spring-board for the next steps toward the more distant goal of the connectome. Currently, the resource consists of annotated image data derived from in-house high-resolution confocal imaging of a selection of transgenic lines (originally from many sources) which have specific expression patterns in one or more neuroanatomical structures. Embryonic or larval specimens are fixed and usually counter-stained, incorporating one of several brain-specific neuroanatomical antibodies for orientation, the specimens are then mounted and imaged at high resolution using scanning confocal microscopes. The stacks are processed using volocity software (Improvision) and projections are loaded into our database where they are annotated. These annotated data are then web-browseable by neuroanatomical structure. Detailed descriptions or `tutorials' about specific brain structures have also been written, these use a selection of the image data to describe in detail the structure in question. To ensure interoperability we are maintaining parity with the ZFIN anatomical ontology in our lists of neuroanatomical structures for annotating and tutorial production. The project is at an early stage: we have used data from about 30 transgenic lines and have produced a handful of fully-fledged tutorials to the level of detail we want to be standard for all structures. We are currently trying to secure funding both for the further development of the neuroinformatic side of the project (particularly to be able to present data in 3D space), to gather together more brain-specific transgenic imaging to populate the database, to write more tutorials and to extend detail to the level of individual neuronal morphologies, perhaps by using brainbow-type technologies. We are interested in obtaining data from transgenic lines, or in obtaining the lines themselves, where expression clearly demarcates specific brain struafish and found that it is expressed at the apical surface of the retina. Furthermore, using time-lapse approaches, we observe that during zebrafish retinal development slit1b morphants show delayed apical retraction. In these morphants, RGCs extend axons at similar stages as in wildtype but do so while still retaining an unretracted apical process. Interestingly, a previous report has demonstrated that Slit can induce downregulation of the cell surface adhesion molecule, N-cadherin, through Robo receptor activation. Studies are under way to test if a similar mechanism applies to apical retraction during RGCs differentiation. To do this, we use a combination of immunocytochemistry and in vivo live-imaging approaches. Our study will shed light on the molecular events underlying apical retraction in RGCs and might uncover general mechanisms during neuronal differentiation in other model systems. Posters 474 Live imaging 404 Live Imaging of Developing Connectivity in the Spinal Circuit of the Zebrafish Embryo E. Warp, C. Wyart, F. Del Bene, H. Baier, E. Y. Isacoff Molecular and Cell Biology, UC Berkeley, USA Before sensory systems provide input to the nervous system, networks such as the spinal cord, retina and hippocampus display spontaneous, rhythmic bursts of action potentials. Global patterns of this activity have been shown to be locomotor-like in vertebrate models such as the rat and chick, with synchronization in ipsilateral regions of the spinal cord and alternation between the left and right side of the cord. We are interested in how these highly-coordinated activity patterns arise in development. The genetic accessibility and transparency of zebrafish allow us to apply optogenetic techniques to monitor the activity of confined populations of spinal cord neurons in an intact animal and through developmental time. Using the UAS:GAL4 system, we are targeting the genetically-encoded calcium indicator GCaMP to motoneurons to observe their activity patterns during different stages of development. At 20 hours post-fertilization (hpf), three hours after the onset of spontaneous activity in the spinal cord, calcium transients in ipsilateral motoneurons are tightly synchronized and alternation is present between the left and right side. Two hours earlier (18hpf), however, spontaneous events in individual motoneurons are independent of each other, and neither synchronization nor alternation is observed. To further understand how the network progresses from sporadic to coordinated activity, we are using time-lapse calcium imaging to follow the development of correlations between individual neurons during this developmental period. An analysis of when and where correlated and anti-correlated groups appear will provide insight into the steps taken during the maturation of the underlying circuit. 475 Posters 405 In-Vivo quantification of cellular processes by high-resolution microscopy D. Schul, Manfred Schartl, Gregory Harms, Toni Wagner Physiological Chemistry I, Biocentre, University of Wuerzburg, Germany Live imaging Cells have to carry out numerous functions that range from replication and energy conversion to molecule transport. In-vivo there are distinct cell types with different morphological properties and consequently variable functions. Currently there is no tool to identify, track and measure physical parameters of cells and nuclei, during vertebrate development allowing to correlate them to cell function. Basal parameters like volume and shape of organelles should be linkable with cellular processes. We want to develop a computer-based method that automatically recognizes and analyzes various cell identities in-vivo by their cytoplasmic/nucleic volume ratio and shape descriptions in living zebrafish embryos. Therefore, we want to generate a double-labeled fishline, to examine and analyze distinct cell types and automate their correlation with the imaging softwares Image J and Volocity. This method could be a basic tool for the identification and quantification of cellular processes like migration or cell division in-vivo. To reach these aims, we already constructed a multicistronic vector that contains minitol2integration sites to generate transgenic fishlines. With this construct we are able to label the membranes and nuclei of the fish embryos with different fluorescent proteins. Up to now, we injected this construct into embryos and were able to preliminarily analyze these embryos by confocal microscopy. We are able to identify single cells of living embryos in these confocal stacks, concluding that the live-imaging with this multicistronic construct works as expected. Consequently, it should be possible to expand this method by using a third fluorescent protein to visualize cellular processes and organelles. Posters 476 Live imaging 406 In toto imaging of somitogenesis and muscle formation in FlipTrap zebrafish embryos F. Ruf-Zamojski1, S. Megason2, L. Trinh1 and S. E. Fraser1 1 Biology, California Institute of Technology, Pasadena, CA, USA 2Systems Biology, Harvard Medical School, Boston, MA, USA Somitogenesis and myogenesis are highly conserved mechanisms in vertebrate species that give rise to skeletal formations. It is therefore important to observe these processes with high resolution to understand how muscles form, are maintained and degenerate throughout life. To that end, we combined two powerful methods in living zebrafish embryos to study muscle formation and degeneration with high-resolution and non-invasively: 1) in toto single-cell imaging using "embryo arrays", individual cell labeling and confocal microscopy, and 2) a FlipTrap genetic screen to isolate lines with citrine fusion protein expression in muscles and to create cre-lox conditional mutants. This novel screening approach has so far identified 10 lines with functional fluorescent protein fusions to endogenous muscle proteins and is used to assess in vivo the specific function of the trapped genes, before and after disrupting their function with cre-lox recombination. We show that the trapped proteins recapitulate endogeneous protein expression using immunostaining, and we compare the distribution of the specific mRNAs by combining imaging of the trapped protein with in situ hybridization. Time-lapse microscopy of the fluorescent protein allows us to watch the process of muscle formation and the distribution of the trapped proteins with unprecedented resolution in living zebrafish embryos. Together, these two approaches enable us to gain more insights into the roles played by specific proteins in muscle formation and maintenance. 477 Posters 407 Live imaging Identification and Functional Analysis of the Gene Regulatory Network that Regulate a Primary Sensory Neuron Lineage in the Developing Zebrafish Spinal Cord using In toto Imaging and FlipTrap Screen R. Noche and Sean Megason Department of Systems Biology, Harvard Medical School, Boston, USA The zebrafish spinal cord is a powerful model system to watch developmental circuits function in vivo. Early in development, the spinal cord emerges from a sheet of neuroepithelial cells that converge toward the midline to form a neural tube. In zebrafish and other anamniotes such as the frog, the boundary between the neuroectodermal sheet and non-neural ectoderm, the neural plate border (NPB), has been shown to give rise to both neural crest (NC) and RohonBeard (RB) neurons, which are early-born mechanosensory neurons essential for the embryonic and larval escape response. Loss of transcription factors that are expressed in the NPB, such as Prdm1a and Olig3, were shown previously to reduce the number of NC and RB cells. Thus, an "equivalence group" was proposed to give rise to both cell types. However, the complete cell lineage of RB neurons and the gene regulatory network (GRN) that induce, specify, and determine this lineage are unknown. We are using in toto imaging technology to elucidate the complete cell lineage of RB neurons. First, we are doing in vivo time-lapse confocal and twophoton imaging of zebrafish embryos wherein all the cell nuclei and membranes are tagged with different fluorescent proteins. Then, we are analyzing our imaging data sets to generate the RB lineage tree using GoFigure2, an open source image analysis and segmentation software suite being developed in our lab. Recently, a candidate gene approach had been used to characterize proposed components of the RB lineage GRN. To define novel components of the RB lineage GRN, we are screening for genes that are expressed in the RB lineage using the FlipTrap, a novel conditional gene trap mutagenesis approach also developed in our lab. FlipTraps generate a tissue-specific and functional endogenous fusion with yellow fluorescent protein. In the presence of Cre recombinase, truncated and mutant versions of the FlipTrap fusion are tagged with red fluorescent protein. Together, these studies will enable us to generate a mechanistic synthesis of how a biological program is executed in a developing embryo. Posters 478 Cell movement 408 The Par/aPKC Complex Controls The Vectorial Migration Of Medaka Macrophages In Vivo 1 C. L. Crespo, 1R. Molteni, 1B. Clissi, 2 P.Keller, 3J. Wittbrodt and 1R. Pardi 1 Leukocyte Biology Unit, S. Raffaele University School of Medicine, Milan, Italy; 2Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany; 3Heid elberg Institute for Zoology-Univ. of Heidelberg,Germany The establishment and maintenance of cell polarity is a requirement for leukocyte migratory response to inflammatory cues. A conserved polarity complex consisting of Par3, Par6 and atypical PKC (aPKC) in conjunction with small GTPases of the Rho Family temporally and spatially controls polarization in several cell types. We used a Medaka (Oryzias latipes) transgenic line as a model system to explore the functional role of this signaling complex in vivo during wound-triggered macrophage polarization and directed migration. By using a transient transgenesis approach, we overexpressed specifically in macrophages the orl_Par3 dominant-negative deletion mutant known to interfere with Par3/aPKC binding. Compared with control cells, orl_Par3 mutantexpressing macrophages display a prolonged "sensing time" with non-polarized status and, as an outcome, a delayed response to the injury. Closer inspection of cell morphologies reveals increased number of unpolarized pseudopodes and elongated phenotypes in mutant-expressing cells. Accordingly, blocking PKC signaling with a myr-PKC pseudosubstrate inhibitor impairs macrophage directional motility when compared with untreated cells. RhoA signaling is contributing as well to stabilize macrophage asymmetry as abnormal cell elongation and concomitant reduction in cell speed occurs in the presence of a Rho Kinase pharmacological inhibitor. Collectively, our data support the hypothesis that the Par/aPKC complex and its effector GTPases are involved in regulating the directional migration of myeloid cells responding to inflammatory cues in vivo. 479 Posters 409 Cell movement Potential role of mesoderm and extracellular matrix in organising polarization and morphogenesis of zebrafish neuroepithelium C. Araya and J. Clarke MRC Centre for Developmental Neurobiology, KCL, London, United Kingdom The development of the zebrafish neuroepithelium is a highly coordinated morphogenetic process in both time and space. This involves the convergence of neuroepithelial cells towards the dorsal midline and the generation of the apico-basal cell polarity. One of the key tissues that might actively contribute to co-ordination of this process is the adjacent mesoderm. Analyses by time-lapse microscopy have revealed that during the initial stages of neurulation, neural plate and mesoderm move in a tightly co-ordinated way. In addition we are analysing neural morphogenesis in a series of mesoderm mutant embryos. For example, in embryos with defective mesoderm convergence due to the reduction of Has2 function, we find that neural plate convergence is severely affected. Furthermore embryos that completely lack head mesendoderm due to loss of Nodal signalling, develop a neural primordium with chaotic apico-basal organisation. Timelapse microscopy demonstrates that the normal highly co-ordinated movements of neurulation are severely disrupted in these mesoderm-less embryos. Although, Nodal mutant embryos show defective neural tube morphology, our experiments suggest that lack of mesoderm rather that Nodal signalling itself is required for neural tube development. In addition, we demonstrate that apico-basal organisation and neural tube morphogenesis can be rescued in mesodermless mutant embryos by replacing mesoderm. Although, we do not know the molecular nature of such tissue-interaction, components of the extracellular matrix (ECM), which are expressed between these two embryonic tissues may to have an important role in this process. We are currently testing whether ECM and mesoderm together can regulate the co-ordination of neural plate cell movements and organise their polarity. Posters 480 Cell movement 410 Regulation of planar cell polarity signalling by the prenylation pathway M. Tada1, M. Kai1, N. Buchan1, and C-P. Heisenberg2 1 Department of Cell and Developmental Biology, University College London, UK; 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany During vertebrate gastrulation, the body axis is established by a variety of co-ordinated and directed movements of cells. One of these movements is convergence and extension (CE), which is regulated by a non-canonical Wnt/planar cell polarity (PCP) pathway. From our forward genetic screen, we have identified 3-hydroxy-3-methyglutaryl-Coenzyme A reductase 1b (hmgcr1b) gene as a dominant enhancer of the silberblick (slb)/wnt11 CE phenotype. hmgcr1b mutant embryos exhibit only very mild CE phenotype during gastrulation while showing a thicker yolk extension at pharyngula stages. Notably, abrogation of hmgcr1b also enhances the CE defects of other core PCP mutants/morphants. The prenylation pathway is one of branches downstream of HMGCR, and has been implicated for lipid modification at the C-terminus of proteins. To test the possibility that the prenylation pathway regulates activities of the PCP pathway, we abrogated farnesyl transferase (FT) or geranylgeranyl transferase (GGT) function using morpholinos on PCP mutant/morphant backgrounds. Consistent with the notion that FT preferentially performs lipid modification on to proteins with the CAAX motif including the core PCP protein Prickle (Pk), abrogation of FT, but not GGT, enhances the pk1a or pk1b morphant CE phenotype, suggesting the specificity for targets of the prenylation enzymes. 481 Posters 411 4D time lapse analysis of zebrafish eye development H. Otsuna, K. Kwan, C-B. Chien University of Utah, Dept. Neurobiology & Anatomy, Salt Lake City, USA Cell movement To understand the development of tissues, biologists have commonly used sectioning or SEM to visualize their morphology. However, these methods require fixation, preventing analysis of different time points during the development of the same animal, and thus obscuring the dynamic aspects of morphogenesis. To circumvent this problem during our study of zebrafish eye morphogenesis, we carry out live 4D imaging of the developing eye primordium from 12 to 24 hpf, collecting complete z-stacks every 3.5 min after labeling chromatin and cell membranes with histone-mCherry and EGFP-CAAX, respectively. Here we describe several analysis methods that we have developed for these 4D datasets. We analyzed lens and retina morphology over time using manual segmentation in Amira. We developed custom software for manual 4D cell tracking. The LongTrack application allows us to track individual cells either forwards or backwards through time, and which can export XYZ coordinates or volume images of the tracked cells. Tracks of individual cells can then be combined with the eye segmentation as polygon meshes, allowing us to visualize changing eye morphology and cell movements within this developing tissue. We have also begun to analyze the relative contributions of cell division and cell migration to eye morphogenesis. We measure cell numbers from the histone-mCkine SDF1, which is present along the path of migration, and by its two receptors CXCR4 and CXCR7. cxcr4b is expressed in the leading cells but not in the trailing cells of the primordium, and cxcr7 is expressed in the reciprocal pattern (highly in the trailing cells and not at all in the leading cells). Our results suggest that CXCR4 is required for the migration of the primordium and that CXCR7 provides the directionality. Recent work by others have shown that the WNT/FGF system also plays a role in driving migration ( Nechiporuk and Raible, 2008, Lecaudey et al, 2008, Aman and Piotrowski, 2009). We have identified yet other components of the network that regulates migration of the primordium and we will report their interactions with the two systems, CXCR4/CXCR7 and WNT/FGF. Posters 484 Cell movement 414 Timely differentiation of rod photoreceptors depends on a feedback regulatory loop between NeuroD and Six6 R. Marco-Ferreres, I. Conte, L. Beccari, E. Cisneros, J. M. Ruiz, N. Tabanera and P. Bovolenta Instituto Cajal, CSIC and CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Timely generation of distinct neural cell types in appropriate numbers is fundamental for the generation of a functional retina. In vertebrates Six6, a transcription factor of the Six/sine oculis family, is initially expressed in multipotent retina progenitors and then becomes restricted to differentiated retinal ganglion and amacrine cells as well as to the proliferative ciliary marginal zone. How Six6 expression in the retina is controlled and what are its precise functions is still unclear. To begin to address this issue we have used bioinformatic searches and transgenic approaches in medaka fish (Oryzias latipes), identifying a highly conserved regulatory enhancer in the Six6 locus responsible for its expression in differentiating and adult retina. Search for transcription factor binding sites, luciferase and ChIP assays together with gain-of-function studies indicated that NeuroD, a bHLH transcription factor, directly binds an "E-box" sequence present in this enhancer, regulating Six6 expression in the retina. NeuroD-induced Six6 overexpression in medaka fish embryos promotes unorganized retinal progenitor proliferation, in agreement with previous Six6 gain-of-function studies. Notably, as the retina differentiates, NeuroD-mediated Six6 over-activation impairs photoreceptor differentiation, with no apparent changes in other retinal cell types. Interestingly gain- and loss-of function of Six6 induces changes in NeuroD expression levels and alterations in the expression and localization of the photoreceptor differentiaion marker, rhodopsin. Together these results indicate that NeuroD is a direct regulator of Six6 expression in the eye development and suggest that the appropriate generation of photoreceptor precursors and their subsequent differentiation requires a NeuroDSix6 feedback regulatory loop. Supported by: BFU2007-6177 and CAM, P-SAL-0190-2006 485 Posters 415 Cell movement olSox2-mediated activation of olSix3.2 promotes telencephalic versus eye field specification L. Beccari, I. Conte, E. Cisneros, N. Tabaneras and P. Bovolenta Instituto Cajal, CSIC and CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Vertebrate forebrain derivatives are specified at early stages of gastrulation in the anterior neural plate by the overlapping expression of several transcription factors including Six3, a member of the Six/sine oculis family of homeobox containing transcription factors. The medaka fish (Oryzias latipes) genome contains two Six3 paralogs: olSix3.1 and olSix3.2. The combined expression pattern of these two genes recapitulates that of the mammalian or avian Six3, with a preponderant expression of olSix3.1 in the eye and of olSix3.2 in the telencephalic and thalamic regions, suggesting a possible regulatory and functional sub-specialization of the two genes as consequence of their independent evolution after genome duplication. To begin to test this possibility and decipher the logic of Six gene regulation, we have identifrticularly pronounced in the liver at 3 dpf. In addition to a metabolic role of Lxr the ubiquitous and temporal expression pattern suggests a developmental role. Indeed, the knock down of Lxr expression by morpholino antisense oligonucleotids induces developmental defects in zebrafish embryos such as impairment of general lipid distribution, reduced yolk utilization and cartilage defects. 501 Posters 431 Gene regulation Abnormal neurogenesis, angiogenesis and haematopoiesis in zebrafish lacking group 4 paralog of Hox genes A. Anusha Amali, T. S. Fiona, C. Winkler and M. Featherstone School of Biological Sciences, Nanyang Technological University, Singapore The vertebrate central nervous system contains many classes of neurons with specialized properties. In the developing hindbrain, the functional loss of individual and combinations of Hox genes has revealed their role in specifying rhombomere identity and neuronal development. However, the interaction of Hox genes with transcriptional program to specify distinct neuronal lineages within the rhombomeres remains to be elucidated. Haematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form haematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of haematopoietic and vascular progenitor cells. The first haematopoietic and endothelial progenitors are derived from a common embryonic precursor, termed the haemangioblast. The genetic cascades that regulate the differentiation of the haemangioblast to haematopoietic and endothelial cells are largely unknown. In general, much of embryonic development is coordinately regulated by temporal and spatial expression of transcription factors, such as the Hox gene family. Here, through the morpholino knock down of group 4 paralog of Hox genes, we have identified an early defect in neurogenesis, angiogenesis and haematopoiesis. We demonstrate that single and combined knockdown of 4th group of Hox genes results in a loss of olig2, nkx 2.2 and islet-1 expressing progenitors that gives rise to somatic motoneurons and branchio motoneurons. Alcian blue staining showed that the pharyngeal cartilages in the morphants were malformed or abolished. The differentiation of endothelial cells is tightly connected with the formation of blood vessels during development. Vasculature was examined in Tg Fli:EGFP embryos, where the fli1 promoter drives expression of enhanced green fluorescent. Loss of hoxc4a and d4a function results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, failure in intersomitic vessel (ISV) development and sprouting, and lack of blood circulation. Remarkably, morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers such as flk-4, flk1 and fli-1 and the vein marker flt4. Also blood pooling was observed in the cardiac region, eye and in yolk. Taken together, these results demonstrate for first time that paralog group 4 genes play a significant role in haematopoiesis and angiogenesis. Posters 502 Gene regulation 432 Transcriptional Regulation of the Stem Cell/Progenitor Gene c-myb M. Gering and J-C. Hsu Institute of Genetics, School of Biology, University of Nottingham, Queen's Medical Centre, UK The transcription factor c-Myb plays an essential role in stem/progenitor cell proliferation and differentiation in the blood, the brain and the intestine. It is also known to be expressed in a number of other tissues, including the neural retina and the olfactory placode. Little is known about the transcriptional regulation of c-myb. This is why we are investigating the transcriptional regulation of c-myb in zebrafish embryos. In zebrafish embryos, c-myb expression is detected in the retina, the intestine, the olfactory placode, the branchial arches, the brain and or DNA binding. For our analysis of hand2 as an activator/repressor, we created super-activator (VP16)- and super-repressor (Engrailed - EnR)-hand2 constructs. To examine whether Hand2 action requires DNA binding or HLH dimerization, we created basic (R100D)- and HLH (F110P)-hand2 mutant constructs. Capped mRNA generated from each of these constructs was injected into wild type and hans6 zebrafish embryos at the 1-2 cell stage. Embryos were fixed at 5 days post fertilization and stained with alcian blue to analyze cartilage development. While our analysis is ongoing, we have thus far found that injection of all of our constructs into wild type embryos leads to mispatterning of the ceratohyal, while hand2R100D also results in loss of the ceratobranchials. When injected into hans6 mutants, we find that hand2R100D rescues all ventral cartilages, while hand2F110P only rescues the anterioventral cartilages. Injection of VP16-hand2 also only rescues the anterio-ventral cartilages, while injection of EnR-hand2 rescues both the anterio- and posterio-ventral cartilages. Based on this preliminary data, it appears that Hand2 functions differently in the development of the anterior and posterior pharyngeal skeleton and that some of these functions occur independently of DNA binding. 509 Posters 439 Gene regulation Ribosomal protein L11 (RPL11) deficiency in zebrafish leads to a selective upregulation of P53modulatory nucleolar proteins A. Chakraborty, T. Uechi, H. Torihara and N. Kenmochi Frontier Science Research Center, University of Miyazaki, Japan Ribosome biogenesis is a tightly controlled multi-step process that begins in the nucleolus and ends with the formation of mature ribosome in the cytoplasm. Several nucleolar proteins play critical role in pre-RNA processing and pre-ribosomal assembly within the nucleolus. Recent findings indicate that impairment of ribosome biogenesis induced by the decrease of an RP or mutations in nucleolar proteins activate the p53 pathway presumably through a nucleolar stress or ribosomal stress. Studies in cell lines have identified RPL11 as a major inhibitor of the p53MDM2 regulatory loop. However, our previous data demonstrate that knockdown of RPL11 activates the p53 pathway in zebrafish, which is contrary to the cell line-based results. In an effort to investigate how an RP deficiency activates the p53 response, we examined the expression of several nucleolar protein genes in L11-deficient embryos (morphants). A total of nine nucleolar proteins (wdr36, wdr55, bap28, rrs1, bxdc1, ncl, gnl3, pes, and bop1), already identified for their role in ribosomal assembly, were analyzed in this study. Quantitative and semi-quantitative RT-PCR revealed a selective upregulation of gnl3 and bop1 transcripts in the morphants whereas the expression of other nucleolar protein genes was similar to that in wild-type embryos. In situ hybridization showed enrichment of gnl3 and bop1 in the head region of the morphants that displayed morphological abnormalities and an increased p53 localization. Furthermore, a timecourse analysis at various stages post fertilization indicated that overexpression of gnl3 and bop1 coincided with the activation of p53 response in the morphants. Interestingly, aberrant expression of Nucleostemin (the human homolog of gnl3), and Bop1 activates p53 in human and mouse cells respectively. These results suggest that an activated p53 response in L11-deficient embryos could be due to an overexpression of nucleolar proteins involved in the p53 pathway. Posters 510 Gene regulation 440 prdm genes in zebrafish craniofacial development L. Kwok1,2, D. E. Clouthier2 and K. B. Artinger2 1 Cell Biology, Stem Cells and Development Graduate Program, 2Department of Craniofacial Biology, University of Colorado Denver - Anschutz Medical Campus, Aurora, USA The zebrafish prdm gene family is comprised of a set of 17 genes that all contain an N-terminal PR/SET domain to mediate protein interactions, and a variable number of DNA-binding zinc finger domains. prdm1a, a member of the prdm gene family,01, 206 M. Tsang 203 S. Tsuruoka-Kinoshita 54 L.C. Tu 150 B. Tucker 353 J. Tuimala 391 K. Turner 472 L.T. Turner 77 E. Turola 489 M. Tyers 412 C.R.Tyler 228 U Authors Index S. Uji 199 J.A. Ulloa 318 A.U. Uong 77 A. Urasaki 317 J. Urban 252 B. Urbanyi 148 R.A. Uribe 268 V E. Vaccari 257 D. Vallone 49 F. vanBebber 377 T. van Boxtel 357 B.J.C. van den Bosch 374 Y.U. van der velden 373 L. van der Aa 519 G. van der Goot 182 P. van Duijn 177, 406 F. van Eeden 454 C. Vangeet 345 V. van Heyningen 514 M. van Lohuizen 373 J. van Noort 62 T. Van Raay 483 C. van Rooijen 354 P. van Tijn 354, 436 L.Varadi 148 M. Varga 143, 426 G. Vatine 49 G. Vazza 396 J. Veerkamp 299 J.A. Vega 250, 284 534 P. Vella 313 M.I. Vera 263 H. Verkade 57 P. Vernier 383 C. Vesque 179, 397 A. Vettori 396 E. Villa 489 D.H. Vlecken 409 D. Volpin 171 V. Von Berg 192 E. Voronezhskaya 153 M. Voz 173, 210 W H. Wada 54 N. Waghorne 314, 352 S. Walmsley 329 C.N. Walpita 191 G.W. Wang 224 M. Wang 365 N. Wang 422 W-D. Wang 320 X.G. Wang 164 F.C. Wardle 441 E. Warp 293, 475 P. Washbourne 269, 279 A.J. Waskiewicz 232 S. Watabe 194, 506 M. Watanabe 196 B. Weger 59 J. Wegner 292 G. Weidinger 180, 435 Authors C. Weiler 504 F.U. Weiss 409 B. Wendik 156, 302 M. Westerfield 292 T.T. Whitfield 105, 218, 358 M.K.B. Whyte 342 A. Wichmann 470 J. Widenhain 412 B. Wilde 415 D. G. Wilkinson 459 B. Willems 211 T. Wilm 483 C. Wilson 143 L. Wilson 369 S.W. Wilson 426 S. Wilton 393 R. Wingert 195 C. Winkler 211, 364, 502 J. Winkler 298 V. Wittamer 30, 97 M. Witte 90 J. Wittbrodt 479 C. Wittevrongel 345 N. Wittkopp 504 S.A. Wolfe 88 T. Wolfsberg 69 G.K. Wong 474 B.K. Wu 166 C.C. Wu 149 Y-T. Wu 350 C. Wyart 467, 475 Authors Index X S. Xin 422 P. Xu 154 Y. Xu 157, 187 Z A. Zakrzewska 72 A. Zaucker 68, 148 Z. Zeng 423 H. Zhan 423 B. Zhang 22 P.J. Zhang 157 Y. Zhang 157 Z. Zhang 365 X. Zhao 253 Y. Zhao 422 X. Zheng 154 H. Zhong 422 W. Zhou 278 Y. Zhou 95 A. Zink 413 L. Ziv 471 D. Zivkovic 239 D. Zizioli 161 L.I. Zon 95 L.Z. Zon 26 N. Zoppi 378 T. Zygmunt 297 Y M. Yahmi 519 M. Yamaguchi 188, 235 T. Yamamoto 214 J. Yan 206 H-J. Yang 206 L. Yang 517 P-H. Yang 206 S. Yang 154 Z. Yang 422 C. Yanicostas 233, 434 Y.J. Yeh 151 D. Yelon 324 L. Yieh 268 C. Yin 483 T.Y. Yokogawa 224 Y. Yoshimura 235 A. Yoshizawa 189 J. Yost 46 R. Young 426 S. Young 270 R.S. Yu 47 535 Authors First published june 2009 Arti Grafiche Ramberti, Rimini, Italy Organizing Committee Marina Mione - Milan, Italy Karuna Sampath - Singapore, Singapore Francesco Argenton - Padua, Italy Massimo Santoro - Turin, Italy Organizing Secretariat Adria Congrex srl and Meetitaly Via Sassonia, 30 - 47900 Rimini - Italy Congress Venue Palazzo dei Congressi Piazza John Kennedy, 1 00144 Rome - Italy Tecniplast know-how surges into aquatics, with passion. Presenting Aquatic Solutions, the dedicated Structure and dedicated team of highly qualified specialists offering reliable, innovative, all-round solutions. Masterly technological know-how, the hallmark of all Tecniplast products for scientific research, is now manifest in the in-house research, development and manufacturing of solutions for aquatic holding facilities. Aquatic Solutions is the Tecniplast Division entirely devoted to the study of the very special requirements of this segment. It comprises a team of qualified specialists working out of well-serviced laboratories with the latest equipment and machinery. They have developed an array of innovative solutions specially designed to be compatible with automation systems (cleaning, storage and handling). Tecniplast aquatic products are custom-designed to meet the needs of all those using fish and amphibians in their scientific research, while providing users with unequalled local presence and support. Find out now all the solutions that fit for you on the website ... View Full Document

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