DB-Lec 9 & 10

DB-Lec 9 & 10 - Biology 442 Biology Developmental...

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Unformatted text preview: Biology 442 Biology Developmental Biology Developmental Lecture 9 – Cell Fate, Potency and Determination, Mosaic vs Regulative Development and Genomic Equivalence Equivalence Lecture 10 – Pattern Formation Lecture Pattern Cell Fate, Potency and Determination Determination The fate of a cell is the sum of all structures that the cell or fate its descendants will form at a later stage of normal development. With most organisms people studying them have constructed fate maps of the embryo maps Fate map of Xenopus embryo prior to gastrulation How do you construct fate maps of an embryo ? an With the tunicate embryo – styela partita there is a portion of the cytoplasm of the fertilized egg that is yellow (pictured here in red!). This cytoplasm is readily visible and was easily observed to go to cell that would become muscle. To do fate mapping in other embryos new methods had to be developed. had Dye injection into an early blastomere – need a dye which is 1) bright enough to see after dilution through many cell divisions; 2) non-toxic to the cells; 3) can not diffuse from one cell to another. One commonly used method is to couple a fluorescent dye, DiI, to a large metabolically inert carrier molecule which can DiI, not cross cell membranes or go through cell junctions. When these molecules are microinjected into an early blastomere one can follow the fates of all descendents of the injected cell. The ability to trace the fate of descendents of individual blastomeres and cells has made it possible to ask questions about how and when the “potential” of a cell gets restricted to “potential” a particular fate. We refer to this wider range of possible fates that a cell could have as it Potency. Potency If a given cell or blastomere can form all structures or by its self can form a whole embryo we say that it is totipotent. totipotent. (The fertililzed egg is totipotent) If a cell or blastomere can form more structures or cell types than it normally would we say it is pluripotent pluripotent Two types of experiments were used to assess the potency of a cell: 1) Isolation experiments and 2) Heterotopic 1) transplantation experiments. transplantation Cell Determination Occurs as a Part of Embryonic Pattern Formation The determined state is the result of two major types of processes – mosaic development (cytoplasmic specification) mosaic and regulative development (instructive interactions). regulative Mosaic development – localized cytoplasmic determinants: In Mosaic most animals certain components in the egg cytoplasm are distributed unevenly and partitioned into different blastomers. These components are called localized cytoplasmic determinants. determinants They cause blastomeres to be determined or biased toward forming certain cell lineages or body parts. We will discuss these determinants and how they work in Lecture 10. Cytoplasmic specification tends to occur during the earlier stages of embryogenesis and some embryos are more Mosaic in their determination process than others. The other major means of determination is by the process of stepwise instruction and commitment. instruction This involves interaction and exchange of signals among cells. Cells with similar potential we call equivalent cells and cells with different potentials we call non-equivalent. Among equivalent cells – signals exchanged may: 1) Stabilize all cells in the same determined state – community effect. effect 2) Compete to attain a preferred state – Lateral Inhibition. Lateral Example of a community effect An example of Lateral Inhibition: An In the ventral neurogenic region of drosophila embryos, cells are “biased” to form neuroblasts (neuronal precursors) or epidermal cells but all cells are initially equivalent. All of these cells produce a receptor protein – the Notch gene product. The Notch receptor is activated by a ligand which is a transmembrane protein called Delta Delta A cell producing Delta activates its neighbors Notch receptor. The activated receptor initiates a cascade of events that leads to the inactivation of a set of genes that would determine the cell to be a neuroblast causing it to become epidermis instead. Neuroblast gene activity is also necessary for Delta protein synthesis. This means that cells prevented from turning on neuroblast genes (epidermal cells) can not make Delta so that they can not inhibit the Delta producing cells from entering the neuroblast pathway. A Molecular Look at the Molecular Notch/Delta Lateral Inhibition Pathway Inhibition Random event induces cell to make delta. Surrounding cells can’t make delta. Other cells that turn on delta will be further away from the first cell Neuroblast cell Epidermis Get a pattern of neuroblasts surrounded by epidermis Gene product inhibits neuroblast differentiation causing cell to become epidermis. Also cant make delta Embryonic induction is an interaction between non-equivalent Embryonic cells. The result of the interaction is that at least one of the partners changes its determined state. This results in a new cell type in the boundary region between the two interacting partners. We will look at this process in detail in Lecture 11 where we will see how inductive interactions pattern the major body axes. The final body pattern of an organism results from a hierarchy The of determinative events in which each step is dependent on previous steps. previous We will look at some of these cytoplasmic specifications and determinative events and how they lay down the body axes in the next 2 lectures. What is the molecular basis of cell determination? What 3 hypotheses There is evidence for each of these hypotheses The concept of differential gene loss goes back to The Weismann’s theory Weismann’s Somatic cells contain only a fraction of the chromosomal determinants Somatic He based this postulate on the observation of chromosome diminution in all of the somatic cells of the nematode Ascaris during its early cleavage divisions. This loss does not occur in the germ cells. But more recent studies indicate that the same chromatin regions are lost from all somatic cells so this can not be the molecular basis for cell determination !! More recent molecular studies indicate that during Lymphocyte maturation there is irreversible genomic DNA loss genomic The vast number of different antibody proteins that lymphocytes can make is the result of DNA rearrangements in the antibody gene locus as individual lymphocytes are recruited to mature and produce antibodies to particular antigens. Other studies indicated that there might be selective gene amplification in some cell types during development. development. In the later stages of Oogenesis the follicle cells surrounding the Oocyte amplify the cells chorion genes that encode the egg shell protein to make lots of egg shell protein. Frog Oocytes selectively amplify the rRNA genes to make lots of ribosomes during Oogenesis Other evidence argued that these gene/chromosomal loss or amplification events are “isolated” events and could not be “determinative” for most cells. could Regeneration – When a salamander limb is amputated – the epidermal cells form a cap and then the underlying cells dedifferentiate and then respecify to regenerate the limb tissues so muscle can become bone or cartilage etc. Most organisms show the same chromosomes in all somatic cells so chromosome diminution is not widespread is Banding patterns on polytene Banding chromosomes are the same in chromosomes different cells So How do you determine if the genome remains the same or equivalent in later somatic cells ? equivalent In order to determine whether the genomes of later somatic cells were also totipotent Briggs and King (1952) attempted to King transplant the nucleus of a somatic cells into an egg and see if it could produce a whole individual produce Nuclei from mature cells have difficulty readjusting to the rapid mitotic cycles of cleavage stage cells to Many nuclei from somatic cells transferred into enucleated eggs have not completed DNA replication by the time of first nuclear division. 1962 –Gurdon employed a serial 1962 nuclear transfer method to “condition” the adult somatic cell nuclei to rapid divisions again. Nuclei enucleated eggs develop to blastula harvest nuclei and inject into new enucleated eggs This resulted in a better percentage of somatic cell nuclei producing tadpole stage individuals and even 2 fertile adult frogs but efficiencies were still low and if he used nuclei from adult tissue culture cells there were no feeding tadpoles at all. 1997 Wilmut tried nuclear transfer technique with sheep – produce “Dolly” technique Made several major changes: 1) used cultured cells in G0, 2) Fused cultured cell to enucleated egg by electric shock, 3) enucleated egg had to be in meiotic metaphase II Out of 434 sheep oocytes only 1 (Dolly) survived. Interestingly Dolly had short telomeres characteristic of the 6 year old ewe from whom her DNA came. Cloning has been confirmed in cows, cats, mice, and in horses but many of the animals develop debilitating diseases as they mature. The use of animal cloning to produce medically important products medically Milk protein gene promoter After Dolly, Wilmut et al cloned Polly but introduced the gene for human clotting factor IX into the cultured cells before fusing to the enucleated egg. Polly was cloned from fetal sheep fibroblasts. Uses the fertilized egg just before pronuclear fusion as the gene recipient Biology 442 Biology Developmental Biology Developmental Lecture 10 Cytoplasmic Specification Most eggs have certain determinants that are NOT NOT equally distributed in the cytoplasm equally These determinants get preferentially distributed to different blastomers and can restrict the fate of those blastomers. This process is known as cytoplasmic specification and the determinants are know as localized cytoplasmic determinants. determinants In order to demonstrate cytoplasmic localization of developmental determinants it is necessary to demonstrate unequal distribution of a determinant that is important for cell fate specification. important When animal pole is separated from vegetal pole neither half develops Sea Urchin eggs divided along the animal vegetal pole develop into normal larva: each half is Material localized to the vegetal half of the equivalent. egg is necessary for proper development. In the Nematode, C. elegans the P granules are initially scattered through the granules fertilized egg cytoplasm but then they localize and are cytoplasmically segregated to the P1 blastomere which gives rise to the germ cells. gives Localization to the posterior pole starts as the male and female pronuclei fuse Microtubule inhibitors block pronuclei Microtubule fusion but not P granule localization BUT fusion microfilament (F-actin) inhibitors caused microfilament the P granules to clump in the middle of the embryo instead of localizing to the posterior end. These experiments suggested that fertilization activated two cytoskeletal systems; 1) a microtubule based system for 1) moving the pronuclei and 2) a microfilament (actin based) 2) system for transporting P granules and other asymmetries in the fertilized egg. The par proteins (partitioning proteins) are also important for localizing the P granules. These genes were identified in screens for mutants defective in partitioning cytoplasmic materials. Par 2 localizes to cortical cytoplasm nearest sperm pronuclei and Par 3 localizes to the future anterior end of the egg. The Par positioning of these 2 proteins enables Par 1 to localize to the future posterior end of the egg. These proteins align the mitotic spindle, localize transcription factors and are important for localization of P granules. Germ cell development in Drosophila depends on localized cytoplasmic determinants – the Polar Granules. Polar Prior to cellularization the polar granules localize to the posterior pole of the egg. Germ cells “cellularize” early and include the polar granules in their cytoplasm Germ Cells The polar graunles are fibrous, electron dense granules that appear to be composed of aggregates of mRNA and ribosomes. They are not unlike the P granules of Nematodes. Do they play a role in germ cell determination? Experiment 1: UV irradiate posterior pole cytoplasm before nuclei migrate there. pole Result 1: NO Pole Cells! Result NO Experiment 2: Transplant cytoplasm from posterior pole of donor egg. Result 2: Pole Cells! Pole Experiment 3: Transplant Experiment Transplant cytoplasm from anterior pole cytoplasm of donor egg of Result 3: NO Pole Cells! NO Conclusion: These rescue experiments Conclusion: can restore the damage embryos’ pole cells and demonstrate that the component that restores the pole cells is localized to the posterior pole. localized BUT – Is it sufficient ? sufficient Experiment 4: Add the component (pole plasm) at a time or place where it is not normally present and ask if it will result in ectopic pole cells (pole cells in the “new” location or at the “new time”. location Transplant posterior cytoplasm from a Transplant normal wt host to the Anterior of a genetically marked recipient (mwh). genetically Result 4: Get what looks like ectopic pole cells at the anterior end of recipient. pole BUT -- are those ectopic pole cells real germ cells ? Will they produce offspring (mwh) ? cells Problem: Since the ectopic pole cells are at Problem: the anterior end of the embryo they will not be in position to migrate correctly to the developing gonads to mature into germ cells. developing Solution: Transplant the ectopically Solution: expressed pole cells to the posterior pole of another genetically marked host embryo (y w sn) and let them produce flys. sn) Mate those flys which should have both Mate mwh and y w sn gametes to y w sn flys. mwh Result 5: Got some phenotypically Wt flys phenotypically that had to come from the ectopic germ cells that Final conclusion: The polar Final granules are a cytoplasmically localized determinant necessary and sufficient to determine a nucleus for a germ cell fate nucleus How do polar granules get localized to the posterior pole ? pole Several maternal gene products (maternal effects genes) have Several been demonstrated to encode proteins and RNAs that are necessary to assemble the polar granules. The first of these to become localized to the posterior pole is the oskar mRNA. oskar Oskar mRNA is both necessary and sufficient for polar granule formation. It is also necessary for the formation of other posterior abdominal segments. other If Oskar mRNA is injected into the anterior pole of an egg then If Oskar pole cells will also form there and instead of a head and thorax, an abdominal segment will form there as well (2 tails and no head!). and Oskar mRNA is transcribed in nurse cells and transported through cytoplasmic bridges to the anterior end of the Oocyte. through Oskar mRNA is transported to the posterior pole on microtubules It requires the product of the staufen gene to link it to the It staufen kinesin (+ end) motor kinesin At the posterior end the Oskar mRNA is translated into At protein. Oskar protein interacts with the staufen protein and the vasa protein to anchor the oskar mRNA at the posterior end and to form an RNP complex that traps components to form polar granules. polar Another localized cytoplasmic determinant in Drosophila is the Another Bicoid mRNA. Bicoid Bicoid mRNA The Bicoid gene encodes a maternally The synthesized product that acts as a localized anterior determinant that is necessary for head and thorax segment An Po formation. formation. Bicoid Bicoid protein protein In Bicoid minus mutants the In Bicoid embryos have two tails and NO head and throax. head Inject bicoid mRNA into Inject Anterior of a bicoid mutant get wt head to tail pattern wt Wt Bicoid Inject it into posterior of Inject wt egg and get 2 heads and no tail ! and Inject mRNA into mid Inject section of bicoid mutant and get head in middle. and How is the Bicoid mRNA localized to the anterior pole ? Bicoid Localization of bicoid mRNA depends on 3’ UTR Localization AUG UAA AAAAA 5’ cap 5’UTR 5’UTR Coding region 3’UTR Delete 3’ UTR and it does not Delete localize; add it’s 3’UTR to another mRNA and that mRNA will localize. mRNA Bicoid mRNA depends on microtubules and other cytoskeleton components for localization and also needs 3 other gene products, staufen, swallow and exuperantia. staufen swallow exuperantia. Ascidian Embryo – Styela Partita – One of the earliest examples of cytoplasmic localization. examples Following fertilization, the cytoplasm rearranges in part Following dragged by the sperm pronucleus. dragged This rearrangement This results in the formation of localized cytoplasmic determinants which are visible because the cytoplasm is different colors – yellow crescent, grey cytoplasm, clear cytoplasm and dark cytoplasm. cytoplasm. Fate Map of Styela Partita Isolation experiments indicate that any blastomeres that contain the yellow cytoplasm will form muscle. form If you use a needle to “push” some of the yellow cytoplasm into the animal pole blastomers, these blastomers that normally would form ectoderm --- now form muscle! muscle! In microsurgical experiments in which the myoplasm was In removed, the developing embryos not only lacked muscle cells but they also showed no signs of antero-posterior polarity. Instead cells with anterior features only, developed suggesting that the myoplasm is not only a muscle determinant but also an important factor in anterior-posterior patterning. How is the yellow cytoplasm containing these anteriorHow posterior determinants and muscle determinants localized ? The yellow pigment granules of the myoplasm appear from The detergent extraction experiments to be associated with a filamentous lattice which probably contains intermediate filaments which connect to a plasma membrane lamina that underlies the egg plasma membrane and is rich in actin microfilaments. microfilaments. Grey cytoplasm displaced by contraction It is thought that contraction of the It plasma membrane lamina provides the driving force for localization of the myoplasm to the vegetal pole. Inhibited by cytochalasin B (actin). Inhibited Second phase is inhibited by MT Second inhibitors and probably uses the sperm mitotic spindle sperm The myoplasm, posterior determinates contain maternal mRNAs. The One of these mRNA – the Macho-1 mRNA encodes a Zn++ One finger transcription factor. finger Anti-sense oligos to lower the level of Macho-1 mRNA Anti-sense resulted in reduced muscle formation and introduction of Macho-1 mRNA into blastomeres that would not normally make muscle caused them to make muscle. make A mRNA encoding a mRNA T-box transcription factor that activates the muscle specific snail gene is also localized to the yellow cytoplasm yellow In situ hybridization shows location of Macho-1 In mRNA mRNA Cytoplasmic localized determinants initiate the axis formation process in Xenopus (Frog) embryos formation In Drosophila the location of the nurse cells at the anterior In end of the Oocyte imposed an anterior to posterior polarity on the delivery of maternal components that could polarize the cytoplasm. What happens in an egg that does not have this kind of organization ? kind Animal-Vegetal polarity is reflected in the uneven distribution Animal-Vegetal of yolk in Zenopus. of Veg pole has Veg polar granules polar Also mRNAs and proteins localized at both poles but no visible Also markers. markers. Early sign of animal-vegetal polarity is the mitochondrial cloud. Early It is located between the germinal vesicle and the veg pole and It contains mitochondria, ER and ribonucleoprotein particles. contains It is involved in an early path of localization used by several It mRNAs ( Xcat2, Xwnt11 and a non-translatable RNA, Xlsirt) mRNAs These mRNAs are These synthesized in the GV and associate with the mitochondrial cloud but their movement to the veg pole is independent of microfilaments and microtubules. Later their anchorage in the cortical cytoplasm depends on microfilaments. microfilaments. A late pathway of localization is used by 2 mRNAs, Vg1 and VegT. Vg1 is involved in establishing the dorsal mesoderm and dorsal VegT is essential for determining mesoderm and endoderm. mesoderm At the time when Xcat2 and Xlsirt are in the mitochondrial cloud At Vg1 is uniformly distributed in the oocyte. Vg1 Vg1 accumulates around the nucleus and then migrates to Vg1 the vegetal pole. It uses a cone of endoplasmic reticulum that extends from the mitochondrial cloud and runs between the GV and Veg pole. GV Microtubules Microtubules are also involved in the transport. transport. Vg1 mRNA ends up in a thin crescent at the vegetal pole. Vg1 The 3’ UTR is used for transport of the mRNA and if the 3’ UTR The is added to another mRNA is that mRNA will localize to the Veg pole. RNA binding proteins are also RNA involved in anchoring Vg1 RNA to the ER for transport. RNA The prior localization of the The XLsirt RNA is also necessary for Vg1 anchoring at the Veg pole. pole. Microfilaments are also Microfilaments involved in anchoring the RNA. involved ...
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This note was uploaded on 12/09/2010 for the course BIOL 442 taught by Professor Brewster,r during the Spring '08 term at UMBC.

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