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Mechanisms Molecular of Germline Stem Cell Regulation Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Marco D. Wong, Zhigang Jin, and Ting Xie Stowers Institute for Medical Research, Kansas City, Missouri 64110; email: tgx@stowers-institute.org Annu. Rev. Genet. 2005. 39:173 95 First published online as a Review in Advance on August 30, 2005 The Annual Review of Genetics is online at http://genet.annualreviews.org doi: 10.1146/ annurev.genet.39.073003.105855 Copyright c 2005 by Annual Reviews. All rights reserved 0066-4197/05/12150173$20.00 Key Words germline stem cells, self-renewal, differentiation, niche, asymmetric cell division Abstract Germline stem cells (GSCs), which can self-renew and generate differentiated progeny, are unique stem cells in that they are solely dedicated to reproduction and transmit genetic information from generation to generation. Through the use of genetic techniques in Drosophila, Caenorhabditis elegans, and mouse, exciting progress has been made in understanding molecular mechanisms underlying interactions between stem cells and niches. The knowledge gained from studying GSCs has provided an intellectual framework for de ning niches and molecular regulatory mechanisms for other adult stem cells. In this review, we summarize recent progress and discuss conserved mechanisms underlying GSC self-renewal and differentiation by comparing three GSC systems. Because GSCs and other adult stem cells share stemness, we hope this review will help de ne fundamental principles of stem cell regulation and provide further guidance for future studies of other adult stem cells. 173 Contents INTRODUCTION . . . . . . . . . . . . . . . . . REGULATORY MECHANISMS OF GERMLINE STEM CELL SELF-RENEWAL AND DIFFERENTIATION IN THE DROSOPHILA OVARY . . . . . . . . . . General Features of the Drosophila Ovarian GSCs and their Niche . . . . . . . . . . . . . . . . . . . . . . . . . BMP, Piwi, and Yb Function in the Niche to Regulate the Maintenance and Division of GSCs . . . . . . . . . . . . . . . . . . . . . . . . Intrinsic Factors Play Essential Roles in GSC Maintenance and Differentiation . . . . . . . . . . . . . . . . . . . Extrinsic Signals Regulate the Function of Intrinsic Factors to Control GSC Self-Renewal and Differentiation . . . . . . . . . . . . . . . . . . . REGULATORY MECHANISMS OF GERMLINE STEM CELL SELF-RENEWAL AND DIFFERENTIATION IN THE DROSOPHILA TESTIS . . . . . . . . . . General Features of the Drosophila Testicular GSCs and their Niche . . . . . . . . . . . . . . . . . . . . . . . . . Niche Signals, BMP and Unpaired, Control Testicular GSC Self-Renewal . . . . . . . . . . . . . . . . . . Different Classes of Intracellular Factors Regulate Testicular GSCs . . . . . . . . . . . . . . . . . . . . . . . . . REGULATORY MECHANISMS OF GERM CELL FATE 174 SPECIFICATION IN C. ELEGANS . . . . . . . . . . . . . . . . . . . . General Features of the C. elegans GSCs and their Niche . . . . . . . . . A Notch-Like Signal from the DTC is Both Necessary and Suf cient for Controlling GSC Self-Renewal and Proliferation . . . . . . . . . . . . . . . . . . Intrinsic Factors Regulating RNA Stability and Translation Play Essential Roles in GSC Maintenance and Differentiation . . . . . . . . . . . . . . . . Interplay Between Notch-Like Signaling and Intrinsic Factors Is Critical for Controlling GSC Self-Renewal and Differentiation . . . . . . . . . . . . . . . . REGULATORY MECHANISMS OF GERMLINE STEM CELL SELF-RENEWAL AND DIFFERENTIATION IN MAMMALS . . . . . . . . . . . . . . . . . . . . . General Features of the Mouse GSCs and their Putative Niche . . . . . . . . . . . . . . . . . . . . . . . . . Signals from Sertoli Cells Control the Maintenance and Differentiation of GSCs . . . . . . . Different Classes of Intrinsic Factors Control GSC Maintenance and Differentiation in the Mouse Testis . . . . . . . . . . . . . . . . . . . . . . . . . SUMMARY AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . 182 182 175 175 183 Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. 175 183 178 184 179 184 180 184 180 185 180 182 186 187 INTRODUCTION With their remarkable ability to self-renew and undergo differentiation, stem cells are crucial to development and tissue homeostasis (68, 88). Interest in stem cell research has bur174 Wong geoned since the successful culture of human embryonic stem cells (hESCs), which are able to generate various differentiated cell types (81, 92). In addition to ESCs, stem cells in a variety of adult tissues are also able to generate Jin Xie Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. one or several differentiated cell types throughout an individual s lifetime. Germline stem cells (GSC) are dedicated to producing gametes for transmission of genetic information from generation to generation and, therefore, are true immortal stem cells. The sterility resulting from GSC loss can be easily recognized, and it facilitates the identi cation of extrinsic signals and intrinsic factors in genetic model systems such as Drosophila, C. elegans, and mouse. Furthermore, GSCs are easily identi ed in the anatomically simple Drosophila ovary and testis and have enabled the rst elucidation of relationships between stem cells and their microenvironment or niche (48, 95, 105). The investigation of the GSC niche and the regulatory mechanisms of stem cell self-renewal in Drosophila has provided guiding principles for study of adult stem cells in other systems, because relationships between stem cells and their niches are conserved. Cultured mouse GSCs and stem cell transplantation make it feasible to elucidate molecular regulatory mechanisms of mammalian GSCs (44, 53). This review summarizes our current understanding of GSC regulation, highlights conserved molecular mechanisms, and predicts future challenges. REGULATORY MECHANISMS OF GERMLINE STEM CELL SELF-RENEWAL AND DIFFERENTIATION IN THE DROSOPHILA OVARY General Features of the Drosophila Ovarian GSCs and their Niche The identi cation of stem cells poses unique challenges particularly in mammalian systems because stem cells are rare and indistinguishable from early differentiated progeny (68, 88). The Drosophila ovarian GSC system circumvents this problem by virtue of its simple anatomy, unique molecular markers, and a linear arrangement between stem cells and their differentiated progeny (30, 60, 102). At the tip of the germarium, the anterior end of the ovarioles, 2 to 3 GSCs can be identi ed by their anteriorly located spectrosome (spherical fusome) and anchorage to cap cells through DE-cadherin-mediated cell adhesion (Figure 1; Table 1) (87). The fusome is a germ cell-speci c organelle rich in membrane skeletal proteins such as Spectrins and Hu-li tai-shao (Hts) (21) (62). The cap cells form a niche that regulates the behavior of GSCs, perhaps with some contributions from terminal lament (TF) and inner germarial sheath (IGS) cells close to GSCs (105). These GSCs undergo asymmetric division: The daughter that remains in the niche retains GSC identity, while the other daughter cell moves away from the niche to differentiate into a cystoblast. The cystoblast also has a spectrosome and undergoes four rounds of synchronous division with incomplete cytokinesis to form a 16-cell cyst containing a branched fusome and ring canals connecting individual cystocytes (21). The well-de ned morphology and the linear fashion in which GSCs and their progeny progress throughout the germarium, along with available genetic tools in Drosophila, have facilitated the investigation of GSC maintenance and differentiation. For example, by using a heat-inducible FLP ( ippase, a DNA recombinase)-FRT (FLP recognition target)mediated recombination technique to produce a marked mutant GSC clone that can be compared with the control GSC clone sideby-side in the same germarium, we can determine the role of a particular gene in stem cell self-renewal, differentiation, and division (104). With a well-characterized niche and readily identi able GSCs, the Drosophila ovary represents an excellent model system to investigate stem cell biology in vivo at the molecular and cellular level (30, 60, 102, 103). Stem cell: a unique undifferentiated cell that has the ability to self-renew and generate differentiated cell types GSC: germline stem cell Intrinsic factors: factors that act within the cell to control its behavior Niche: the molecular milieu or microenvironment formed by support cells that express regulatory molecules that promote stem cell self-renewal and block differentiation Self-renewal: the ability of a stem cell to regenerate itself IGS: inner germarial sheath Asymmetric cell division: cell division generating two daughter cells that have different cell fates Extrinsic factors: factors that function outside the cell for controlling its behavior BMP, Piwi, and Yb Function in the Niche to Regulate the Maintenance and Division of GSCs The GSC asymmetric cell division can be partially attributed to the extrinsic factors emanating from the niche. Regulatory molecules, www.annualreviews.org Germline Stem Cell Regulation 175 Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Figure 1 Major signaling pathways and intrinsic factors for GSC self-renewal and differentiation in the Drosophila ovary. The BMPs (Dpp and Gbb), Yb, Piwi, and Hh are expressed in the TF/cap cells and control GSC self-renewal. In the GSC, Pum/Nos and Vasa are required for controlling self-renewal, and Piwi regulates GSC division. GSCs are anchored to their niche by DE-cadherin-mediated cell adhesions. Gap junctions formed by Zpg are important for GSC maintenance and CB differentiation. Bam is required for cystoblast differentiation but is repressed in GSCs by Mad/Med complexes. However, this repression of Bam is overcome in CB, partially through Smurf. BMP: bone morphogenetic protein bone morphogenetic proteins (BMPs) and Piwi, are produced from cap cells to modulate ovarian GSC maintenance and division via intracellular signaling pathways (Figure 1; Table 1). Two BMPs, Dpp and Gbb, are expressed primarily in cap cells (86, 104) and serve as short-range signals to activate BMP signaling in GSCs through type I (Tkv and Sax) and type II (Punt) BMP receptors to nuclear complexes, Mad [a founding member of SMA and MAD (SMAD) family pro176 Wong teins; a BMP-speci c SMAD], and Medea (SMAD4, also known as Co-SMAD) to control their self-renewal and division (42, 104). Mutations in dpp, gbb, or downstream components lead to GSC loss by premature differentiation and slower division rates (86, 104, 105). Overexpression of dpp, but not gbb, completely blocks cystoblast differentiation, resulting in proliferation of GSC-like tumors throughout the germarium (86, 104). These studies indicate that BMP signaling is necessary and Jin Xie Table 1 Extrinsic signals and intrinsic factors that are required for regulating GSC function Functions Species D. melanogaster Pathways and genes BMP: dpp, gbb (niche) tkv, put, mad, Med (GSC) smurf (CB) JAK/STAT: upd (niche) hop, stat92E (GSC) Piwi/Yb: piwi (niche) Yb (niche) EGF: stet (germline cells) Egfr, raf (somatic cyst cells) Ovary GSC self-renewal/division (43, 86, 104) CB differentiation (10, 11, 13) Unknown GSC self-renewal (16, 17) GSC self-renewal (51, 52) CB differentiation (80) Unknown Testis GSC self-renewal (45, 85) Unknown GSC self-renewal (5, 48, 95) GSC self-renewal (16) Unknown Gonialblast differentiation (80) Gonialblast/spermatogonia differentiation (49, 93) Unknown Unknown Gonialblast differentiation (45, 79) Spermatogonial proliferation (33, 65) Unknown Potential niche anchorage of GSC GSC spindle orientation (107) GSC survival and differentiation (29, 91) GSC spindle orientation (107) Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Translational regulation: pum/nos (GSC) vasa (germline cells) bam/bgcn (CB) GSC self-renewal (2, 12, 25, 31, 61, 98) GSC maintenance/survival (89) CB differentiation (28, 59, 64, 65, 69) CB differentiation (4, 14, 58) Niche anchorage of GSC (87) GSC maintenance/survival and differentiation (29, 91) Unknown sxl, orb (CB) Cell adhesion: E-cadherin/arm (niche and GSC) Gap junction: zpg (germline cells) Cell cycle regulators: APC1, APC2, cnn (GSC) cycB (PGC and GSC) C. elegans Notch: lag-2 (niche) glp-1, lag-1, lag-3 (GSC) Translational regulation: fbf-1/fbf-2 (GSC) gld-1, gld-2, gld-3, nos-3 (differentiated germline cells) GDNF: GDNF (Sertoli cells) Ret, GFR 1 (GSC) BMP: BMP4, BMP7, BMP8a, BMP8b (germline cells) SCF/c-Kit: SCF (Sertoli cells) c-Kit (differentiated spermatogonia) Transcriptional regulation: Plzf (spermatogonia) Translational regulation: nos-2 (germline cells) PGC/GSC proliferation and GSC maintenance (99) GSC maintenance (99) GSC and germline cell proliferation (1, 3, 20, 23, 37, 56, 75) GSC self-renewal and proliferation (19, 57, 100, 109) Promoting meiosis (24, 35, 38, 40, 41, 55, 63, 97) M. musculus GSC self-renewal (44, 53, 66) Germline cell viability (76, 110) Unknown Spermatogonia differentiation (22, 71, 108) GSC maintenance (9, 15) GSC maintenance (94) GSC, germline stem cell; PGC, primordial germ cell; CB, cystoblast. www.annualreviews.org Germline Stem Cell Regulation 177 suf cient for GSC self-renewal. Since Gbb and Dpp likely use common signal transducers, it remains to be determined why dpp, but not gbb, is suf cient to block germline cell differentiation when overexpressed (34, 46). Although GSCs normally undergo asymmetric cell division, they are also capable of dividing symmetrically to generate two GSCs when both of the daughters remain in the niche (105). Surprisingly, partially differentiated cells such as 2-, 4-, and 8-cell cysts revert back to GSCs when the niche signal Dpp is provided (43). It remains to be determined how Dpp signaling can completely turn off the active differentiation program in cystocytes. Understanding this phenomenon would provide more insight into how GSC self-renewal and differentiation is regulated, and it may allow the regeneration of stem cells from differentiated cells in future regenerative medicine, if such cell fate reversal also exists in mammalian stem cells. Like BMPs, Piwi and Yb are expressed in niche cells and are also involved in GSC maintenance (Figure 1; Table 1). Loss-offunction mutations in piwi and Yb cause rapid loss of GSCs, while Yb and piwi overexpression increases GSC-like or cystoblast-like germ cells (16, 17, 51, 52, 61). Yb is a novel intracellular protein that regulates piwi and hh (hedgehog) expression (51, 52), whereas Piwi is the founding member of the piwi family genes containing conserved PAZ and Piwi domains that bind to RNAs (16). Hh signaling appears to play a redundant role with Piwi to control GSC maintenance (52). Although the requirement of Piwi in the niche for GSC maintenance is well established, how this is accomplished remains unclear. As a cystoblast moves away from the cap cells, it becomes surrounded by cellular processes of IGS cells, raising a possibility that IGS cells regulate cystoblast differentiation. Indeed, a study on stet function has revealed the link between IGS cells and germ cell differentiation (80). In stet mutant ovaries, the cellular processes of IGS cells are severely 178 Wong Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. reduced, and spectrosome-containing single germ cells accumulate, suggesting that stet is required for cystoblast differentiation. stet, expressed in germ cells, encodes a membrane protease similar to Rhomboid that can cleave and release EGF ligands (96). Supporting a role of EGF signaling in IGS cells, the activated MAP kinase accumulates in wild-type IGS cells but is reduced in stet mutant IGS cells. However, it remains to be determined which EGF ligand is activated by Stet in germ cells and how IGS cells control germ cell differentiation. Intrinsic Factors Play Essential Roles in GSC Maintenance and Differentiation Two classes of intrinsic factors govern GSC self-renewal or differentiation: self-renewing factors and differentiation-promoting factors (Figure 1; Table 1). Pumilio (Pum) and Nanos (Nos) are de ned as intrinsic selfrenewing factors because mutations in these genes cause premature GSC loss (25, 31, 61, 98). They are RNA binding proteins that form protein complexes that repress translation of mRNAs in Drosophila embryos (2). vasa (vas), encoding a Drosophila homologue of eukaryotic initiation factor 4A, is also likely required for ovarian GSC selfrenewal, because vas mutant germaria contain few degenerate or growth-arrested germ cells (89). These studies indicate that translational regulation plays a critical role in GSC selfrenewal. Furthermore, E-cadherin-mediated cell adhesion and Cyclin B also participate in controlling GSC self-renewal (Figure 1; Table 1). E-cadherin and its interacting partner Armadillo (Arm, -catenin), expressed in GSCs and cap cells, form adherens junctions, which anchor GSCs to cap cells during niche formation and help recruit GSCs to the niche (87). The main function of adherens junctions is to keep GSCs in close proximity to niche cells to receive maximal BMP signaling for self-renewal. Cadherin-mediated cell Jin Xie adhesion represents a conserved mechanism for anchoring stem cells in the niche in a variety of systems (26). Cyclin B is speci cally required in germ cells for promoting division of PGCs and GSCs and possibly for controlling GSC maintenance (99). Presumably, Cyclin B promotes PGC and GSC division through regulating cell-cycle progression by activating CDK1. However, it remains unclear whether it controls GSC maintenance indirectly by regulating cell-cycle progression or directly by interacting with the GSC maintenance machinery. Taken together, different classes of intrinsic factors play distinct roles in controlling GSC self-renewal and proliferation. Several differentiation-promoting factors are involved in cystoblast differentiation in the ovary; these include Bam (Bag of marbles), Bgcn (Benign gonial cell neoplasm), Orb, and Sxl (Sex lethal) (4, 14, 58, 59, 64, 69). Among these, Bam, a novel protein, and Bgcn, related to DExH-box RNA binding proteins, are essential differentiation factors: Mutations in bam and bgcn germline cells abolish cystoblast differentiation, leading to a cystoblastlike germ cell tumor phenotype (28, 65, 69); overexpresssion of bam triggers GSC differentiation and consequently germ cell depletion in the ovary (70). Genetic interactions between bam and bgcn suggest that their gene products may form protein complexes that regulate the mRNA stability or translation. Mutations in orb and Sxl, encoding proteins involved in the regulation of mRNA polyadenylation and translation, respectively, cause an accumulation of GSC-like or cystoblast-like cells mixed with early differentiated cysts, implying that these genes play unessential roles in cystoblast differentiation (4, 14, 58). In addition, a gap junction connexin, Zpg (Zero population growth), is present in cytoplasmic membranes of GSCs and their differentiated progeny in the ovary and testis and is required to maintain GSCs and to promote germ cell differentiation (29, 91). Loss of zpg function results in partial GSC loss due to cell death and accu- Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. mulation of a few undifferentiated single cells. Because Zpg is a gap junction component, it likely helps transport small molecules from supporting somatic cells to control the survival of GSCs and the differentiation of their progeny. zpg may function in parallel with bam to regulate germ cell differentiation (29). Pum and Nos control GSC self-renewal through repressing a Bam-independent differentiation pathway (12, 31, 90). bam transcription is not upregulated in pum and nos mutant GSCs, although bam mutant germ cells that are also mutant for pum can differentiate. In summary, different classes of differentiation factors work synergistically to drive cystoblast differentiation by negatively regulating functions of self-renewing factors. Extrinsic Signals Regulate the Function of Intrinsic Factors to Control GSC Self-Renewal and Differentiation Recent studies on Drosophila ovarian GSCs have revealed that extrinsic signals impinge on intrinsic factors to control their functions. In GSCs, bam is actively repressed through a transcriptional silencer in the bam promoter (13). BMP signals, Dpp and Gbb, from niche cells activate BMP signaling, leading to formation of SMAD complexes, which directly bind to the bam silencer to repress bam expression in GSCs (11, 86). Since BMPs function as short-range signals, cystoblasts with insuf cient BMP signaling fail to repress bam expression and consequently upregulate bam to promote differentiation. Similarly, via an unidenti ed mechanism, the niche Piwi is required to repress bam in GSCs for their selfrenewal (12, 90). As both the BMP- and Piwimediated signaling pathways are essential for controlling GSC self-renewal, Piwi may converge with BMP signaling by regulating the stability, production, processing, and/or activation of BMP molecules in the niche or BMP signal transduction in GSCs to repress bam expression (12, 90). The observation that a mutation in smurf, encoding a ubiquitin E3 ligase www.annualreviews.org Germline Stem Cell Regulation 179 Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. that regulates degradation of phosphorylated Mad, can rescue a piwi GSC loss phenotype indicates that Piwi-mediated signaling and BMP signaling must intersect at the level of, or above, smurf (12). Therefore, extrinsic and intrinsic factors work in a coordinated manner to promote GSC fate over cystoblast fate, when instructive signals are received from the niche. In cystoblasts, Bam initiates a differentiation program and promotes cyst formation. It is proposed that Bam interacts with Bgcn and represses Pum/Nos complexes to promote differentiation (Figure 1) (12, 90). Although Dpp and Gbb only activate BMP signaling in GSCs due to their short-range action, there is also a backup system for actively repressing BMP signaling in the cystoblast. Namely, upregulated Bam in the cystoblast can serve as a negative regulator of BMP signaling, and Smurf, which functions in CBs and descendents, negatively regulates BMP signaling activities by targeting phosphorylated Mad for degradation (10). Therefore, precise control of BMP signaling in GSCs and cystoblasts is crucial for these cells to achieve a critical balance between self-renewal and differentiation. ferentiated gonialblast that moves away from the niche. The gonialblast, a counterpart of the cystoblast, also undergoes four rounds of synchronous division with incomplete cytokinesis to form a 16-cell cluster with a branched fusome (27, 47). Each gonialblast or its descendent is surrounded by two somatic cyst cells (SCCs) that control its continuous differentiation (32, 36). These cysts cells are functionally similar to IGS cells in the ovary and are important for germ cell differentiation (49, 80, 93). Niche Signals, BMP and Unpaired, Control Testicular GSC Self-Renewal In the Drosophila testis, Janus Kinase-Signal Transduction and Activator of Transcription (JAK-STAT) and BMP signaling are indispensable for GSC self-renewal (45, 48, 85, 95) (Figure 2; Table 1). Upd produced by the hub acts as a short-range signal to activate JAK (Hopscotch, Hop) and STAT (STAT92E) downstream components in GSCs. Removal of either hop or stat92E causes GSC differentiation by disrupting self-renewal, whereas ectopic expression of Upd increases the number of GSC-like or gonialblast-like cells in the testis, indicating that JAK-STAT signaling is both suf cient and necessary for GSC renewal. Therefore, JAK-STAT signaling plays an instructive role in GSC selfrenewal in the testis similar to that of the Dpp/BMP signaling pathway in the ovary. Remarkably, differentiated germ cells revert back into GSCs when stat function is restored in a temperature-sensitive stat mutant (5). As in the ovary (43), this study suggests that niche signals maintain GSCs not only by preventing differentiation but also by reprogramming differentiated cells back into stem cells when all GSCs are lost from the niche. It is important to identify downstream target genes of JAK-STAT signaling in GSCs to understand how it controls GSC self-renewal. gbb appears to have a stronger effect on GSC maintenance than dpp in the testis, REGULATORY MECHANISMS OF GERMLINE STEM CELL SELF-RENEWAL AND DIFFERENTIATION IN THE DROSOPHILA TESTIS General Features of the Drosophila Testicular GSCs and their Niche As in the Drosophila ovary, GSCs and their niche are also well de ned in the Drosophila testis by virtue of its simple anatomy and availability of molecular markers (48, 95, 106, 107). Seven to nine spectrosome-containing GSCs are located at the apical tip of the testis and directly contact the hub cells that function as a GSC niche (106). Similar to ovarian GSCs, they divide asymmetrically and give rise to one GSC that remains in the niche and one dif180 Wong Jin Xie Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Figure 2 Major signaling pathways and intrinsic factors for testicular GSC self-renewal and differentiation in the Drosophila testis. The GSCs are likely anchored to the niche via DE-cadherin-mediated adhesions and are enveloped by SSCs. Gbb/Dpp and Upd, expressed in hub cells (HC), activate BMP and JAK-STAT signaling for GSC self-renewal, respectively. Bam is repressed in GSCs and gonialblasts by Gbb/Dpp signaling and is expressed in spermatogonial cells. In the GSC, APC1, APC2, and Cnn are required for correct spindle orientation. Gap junctions formed by Zpg are important for GSC maintenance. EGFR signaling in somatic cyst cells (SCC) is activated by an unknown ligand and is important for gonialblast differentiation and spermatogonial cell (SG) proliferation. which is consistent with their relative expression levels in the hub and SCCs (45, 85). Removal of BMP downstream components (punt, tkv, mad, and Med) causes severe GSC loss similar to the disruption of JAK-STAT signaling in the testis, indicating that both BMP and JAK-STAT signaling play essential roles in controlling male GSCs either by regulating each other or working in parallel. On the other hand, dpp overexpression only partially suppresses differentiation of gonialblasts, whereas gbb overexpression has no obvious effect. Therefore, BMP signaling plays a permissive role for GSC self-renewal in males. Although Piwi-mediated signaling is also required for GSC maintenance in the testis, it remains unclear which cells require its func- tion for GSC self-renewal and whether it interacts genetically with JAK-STAT and BMP pathways. piwi is expressed in early germ cells, hub cells, and somatic cysts of the adult testis, and mutations in piwi lead to premature loss of GSCs (61). As discussed earlier, Piwi-mediated signaling may interact with the BMP pathway potentially through Smurf and thereby work cooperatively to sustain testicular GSCs (12). The IGS equivalent cells, SCCs, are also involved in regulating gonialblast differentiation (49, 80, 93). An unidenti ed SCC signal received by the gonialblast requires the EGFR and Raf-mediated MAP kinase signaling cascade in SCCs (49, 93) (Figure 2; Table 1). Genetic mosaic www.annualreviews.org Germline Stem Cell Regulation SCC: somatic cyst cell JAK-STAT: Janus Kinase-Signal Transduction and Activator of Transcription 181 Symmetric cell division: cell division generating two identical daughter cells Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. analysis reveals the requirement of Egfr and raf functions in SCCs for regulating gonialblast differentiation/proliferation. The GSC population in the Egfr or raf mutant testis remains active longer than wild type. Finally, stet also functions in the germ cells to control gonialblast differentiation since stet mutant testes have more gonialblast-like cells than wild type (80). Because EGFR signaling is involved in regulating functions of IGS cells and SCCs, it will be interesting to see whether the same EGF ligand in the germ cells is responsible for activating EGFR signaling in both IGS cells and SCCs and what IGS/SSC signal(s) controls cystoblast or gonialblast differentiation. Different Classes of Intracellular Factors Regulate Testicular GSCs As for the ovarian GSCs, E-cadherin and catenin also accumulate between GSCs and hub cells (107), and are likely involved in anchoring GSCs (Figure 2). The accumulated E-cadherin at the GSC and hub interface may serve as a platform for binding APC2, a Drosophila Adenomatous Polyposis Coli (APC) homolog, to the GSC cortex to orient the mitotic spindles perpendicular to the niche (107). APC2 and two integral centrosome components, centrosomin (cnn) and APC1, are required for the orientation of the stem cell spindle. Loss-of-function mutations in apc1, apc2, and cnn result in mispositioned centrosomes and misoriented spindles, giving rise to symmetric cell division and the accumulation of more GSCs around the hub (107). This study demonstrates that intrinsic control of spindle orientation is crucial for maintaining a stable number of stem cells in the niche in addition to extrinsic niche signals. It remains to be seen whether this intrinsic mechanism of orienting the stem cell spindle is a universal mechanism for ensuring that only one of the two stem cell daughters maintains stem cell identity in other stem cell systems. Although bam and bgcn are essential for cystoblast differentiation, they are dispens182 Wong able for gonialblast differentiation because bam or bgcn mutant gonialblasts form germ cell cysts (33). However, bam alone is suf cient to cause male GSCs to differentiate because bam overexpression leads to GSC depletion (45, 79). In order to maintain GSC self-renewal in the testis, bam needs to be repressed. Indeed, it is actively repressed in testicular GSCs by BMP signaling initiated by Dpp and Gbb similar to the Drosophila ovary (Figure 2; Table 1). However, bam and bgcn play essential roles in restricting four rounds of cyst division as bam and bgcn mutant cysts continue to divide after the fourth division (33). In addition, communication between gonialblasts and their surrounding SCCs via Zpg-mediated gap junctions is also important for gonialblast differentiation and early germ cell survival (91). Although Pum and Nos in the Drosophila ovary, Pumilio homologs in C. elegans, and Nos in mice have been demonstrated to be required for controlling GSC self-renewal (19, 31, 94, 98), its role in GSC self-renewal in the Drosophila testis awaits determination. Furthermore, little is known about how niche signals control GSC self-renewal except for the involvement of BMP signaling in repressing bam expression in GSCs. The genetic and molecular relationships between extrinsic signals and intrinsic factors in the testis will require thoroughgoing investigation in the near future. REGULATORY MECHANISMS OF GERM CELL FATE SPECIFICATION IN C. ELEGANS General Features of the C. elegans GSCs and their Niche The gonad of the C. elegans hermaphrodite consists of two symmetrical, U-shaped tubes that are connected to a common uterus. GSCs are located in the mitotic region (MR) that directly contacts the distal tip cell (DTC), although speci c individual GSCs have not yet been precisely de ned. GSCs and their Jin Xie Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. early progeny continue to proliferate in the MR, and the germ cells moving away from the DTC terminate their mitotic activities, enter meiosis, and eventually develop into mature eggs or sperm (20). The fact that only the germ cells that contact the DTC maintain GSC identity suggests that the DTC functions as a GSC niche. Consistent with this idea, the somatic DTC is shown to be required for maintaining germline mitosis by laser ablation experiments (50). Strikingly, the C. elegans gonad displays an arrangement of stem cells and differentiated cell types resembling those in the Drosophila ovary and testis. Powerful genetics and the simple gonadal anatomy have made C. elegans GSCs a productive system to study the regulation of stem cell selfrenewal, proliferation, and differentiation. A Notch-Like Signal from the DTC is Both Necessary and Suf cient for Controlling GSC Self-Renewal and Proliferation Genetic studies have identi ed a Notch signaling cascade essential for maintaining GSCs in C. elegans (Figure 3; Table 1). The DTC expresses a Delta-like Notch ligand, LAG2, whereas the mitotic germ cells express a Notch-type receptor, GLP-1 (20, 37). LAG-2 binding triggers proteolytic cleavage of GLP1 to generate a truncated intracellular domain for transport to the nucleus, where it forms protein complexes with other transcription factors, LAG-1 and LAG-3, to control target gene expression. LAG-1 is a CSL [CBF1, Su(H), Lag-1]-type transcriptional regulator, and LAG-3 is a glutamine-rich protein that possibly tethers LAG-1 and the cleaved GLP-1 intracellular domain (23, 75). Loss of GLP-1 and LAG-2 function causes GSC loss and consequently premature entry into meiosis (1, 37, 56). In contrast, constitutive GLP-1 activity prevents entry into meiosis and causes germ cell overproliferation (3). Together with the DTC ablation experiments, these studies show that the DTC functions as a GSC niche and GLP-1/Notch signaling activated Figure 3 Signaling pathways regulating the mitosis and meiosis switch in C. elegans germline. The LAG-2/Delta ligand expressed from the DTC binds to the GLP-1/Notch receptor on the germ cell to activate GLP-1/Notch signaling, which may act on FBF-1 and -2 to ensure mitosis through inhibiting two regulatory branches that promote meiosis; GLD-3/GLD-2 and GLD-1/NOS. GLD-3/GLD-2 may activate meiosis-promoting mRNAs like gld-1 mRNA and GLD-1/NOS promotes meiosis by repressing mRNAs critical for mitosis. GLD-1 facilitates meiosis by blocking translation of glp-1 mRNA. by LAG-2 from the niche directly controls GSC self-renewal and proliferation. The direct targets of the GLP-1/Notch signaling pathway remain to be determined. Furthermore, it is unclear whether the C. elegans niche maintains GSCs through a population mechanism or a stereotypic asymmetric division mechanism as in Drosophila, although current data favor the former. DTC: distal tip cell Intrinsic Factors Regulating RNA Stability and Translation Play Essential Roles in GSC Maintenance and Differentiation As in Drosophila, two classes of intrinsic factors control GSC maintenance or differentiation (Figure 3; Table 1): self-renewing factors www.annualreviews.org Germline Stem Cell Regulation 183 and differentiation-promoting factors. Two nearly identical Pumilio-like RNA binding proteins, FBF-1 and FBF-2, control GSC selfrenewal and proliferation. In fbf-1 fbf-2 double mutants, germline proliferation is initially normal, but GSCs are prematurely depleted owing to differentiation and entry into meiosis (19, 109). Pumilio-like proteins usually regulate protein translation by binding to the 3 untranslated region (UTR) of a target mRNA to inhibit its translation (101). FBF1 functions redundantly with FBF2 to promote mitosis but has an opposite effect on netuning the size of the mitotic region, re ecting a regulatory circuit for maintaining a GSC population (57). The size of the mitotic region is precisely controlled as FBF-1 and FBF-2 regulate each other s expression, and this reciprocal repression is probably direct through FBF binding sites located in fbf-1 and fbf-2 3 UTRs. Several differentiation genes have been identi ed for controlling the entry into meiosis: gld-1, gld-2, gld-3, and nos-3. gld-1 functions redundantly with gld-2 to promote meiotic entry and/or inhibit germ cell proliferation (41) (Figure 3; Table 1). Unlike other nos genes in Drosophila and mice, nos3, one of three C. elegans nos genes, promotes differentiation by enhancing GLD-1 accumulation (35). gld-2 gld-1 and gld-2 nos-3 double mutants have a tumorous germline phenotype due to a defect in meiotic entry, whereas gld-1, gld-2, and nos-3 single mutants exhibit normal meiotic entry (35, 41). The GLD1 and GLD-2 pathways are both thought to regulate expression of target genes at a posttranscriptional level on the basis of their molecular identities. GLD-1 is a STAR/KH domain translational repressor (38, 40, 78), while GLD-2 is a catalytic subunit of a poly(A) RNA polymerase (55, 97). GLD3, a BicaudalC homolog, genetically interacts with GLD2 to promote meiosis by activating mRNAs critical for meiosis (24). Moreover, GLD-3 and GLD-2 form a heterodimeric enzyme that polyadenylates and may activate meiosispromoting mRNAs such as gld-1 mRNA. 184 Wong As expected, two classes of intrinsic factors regulate each other. For example, FBF2 regulates the size of the mitotic region by means of repressing the translation of meiosispromoting gld-1 and gld-3 mRNAs (57). Similar to Drosophila, intrinsic factors that regulate mRNA stability and/or translation play key roles in controlling GSC maintenance and germ cell differentiation in C. elegans. Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Interplay Between Notch-Like Signaling and Intrinsic Factors Is Critical for Controlling GSC Self-Renewal and Differentiation In Drosophila, niche signals repress expression of differentiation-promoting genes and thereby maintain stem cell self-renewal. In C. elegans, GLP-1/Notch signaling also inhibits the activities of meiosis-promoting genes, gld1, gld-2, and nos-3, although the mechanism currently is unclear (35, 41). Notch signaling may activate FBF translational repressors that repress the functions of differentiationpromoting genes (Figure 3) (24, 100, 109). In order for germ cells to differentiate, GLP-1/Notch signaling has to be shut off by differentiation genes. Indeed, GLD-1 facilitates meiosis through down-regulating GLP1/Notch signaling in the distal region by binding to the 3 UTR of glp-1 mRNA to block translation (63). Therefore, GLP-1/Notch signaling and intrinsic factors must precisely balance positive and negative regulatory actions to determine whether germ cells remain in the mitotic cycle or enter the meiotic cycle. REGULATORY MECHANISMS OF GERMLINE STEM CELL SELF-RENEWAL AND DIFFERENTIATION IN MAMMALS General Features of the Mouse GSCs and their Putative Niche Stem cell transplantation, simple anatomy, and genetics make the mouse testis a powerful Jin Xie model to study complex regulatory networks of mammalian GSCs and their niche. The spermatogonial stem cells (referred to as GSCs here for consistency) are a subset of single cells (Asingle or As ), which are located along the basement membrane of seminiferous tubules in the mouse testis (7). GSCs selfrenew or produce a differentiated As daughter that divides to form a pair of interconnected spermatogonial cells called Apair . The Apair spermatogonial cells can divide synchronously to form a chain of interconnected spermatogonial cells that subsequently become differentiating spermatogonia, spermatocytes, spermatids, and sperm cells, which is reminiscent of cyst formation in the Drosophila ovary and testis. GSCs and early differentiated spermatogonia are morphologically alike and thus indistinguishable. GSCs are very rare (1 in 5000) cells in the adult mouse testis based on transplantation studies (7). Fluorescenceactivated cell sorting (FACS) in conjunction with the transplantation assay for GSCs has identi ed the antigenic pro le of GSCs as v integrin /dim 6 -integrin+ Thy-1lo/+ C-Kit (54). Sertoli cells, the somatic cells in the seminiferous tubules that physically interact with GSCs, likely constitute a functional GSC niche by providing growth factors that promote GSC self-renewal and/or proliferation. Several studies support the idea that these cells regulate the maintenance of the stem cell pool. First, transplantation of GSCs into infertile male mice has shown that Sertoli cells can indeed support GSC maintenance and spermatogenesis (8). Second, Sertoli cells produce GDNF (glial cell line-derived neutrophic factor) that controls GSC maintenance in a dosage-dependent manner (66). Furthermore, transplantation of Sertoli cells into the mouse testes that are defective in Sertoli cell function demonstrates that Sertoli cells are indeed essential for maintaining spermatogenesis (84), but it is still not clear whether and how Sertoli cells alone can constitute the GSC niche. A series of transplantation experiments in mice and rats suggest that the GSC niche in Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. the mammalian testis is very dynamic during development (72, 73, 77, 82, 83). The most urgent issue in studying GSCs in the mouse testis is to de ne the physical structure the of niche and its associated signals. Although most mammalian females were believed to lose the capacity for germ cell self-renewal during fetal life, a recent study argues that juvenile and adult mouse ovaries possess mitotically active germ cells that continuously replenish the follicle pool (39). Despite compelling experimental evidence, it remains to be seen whether these observations can be duplicated in other mammals. If those GSCs exist in the peripheral epithelial layer of the ovary, a new avenue will be opened to study GSCs in mammals and further investigate how GSCs are regulated differently in both sexes. GDNF: glial cell line-derived neurotrophic factor Signals from Sertoli Cells Control the Maintenance and Differentiation of GSCs One extrinsic factor involved in GSC selfrenewal and proliferation is GDNF, which is released from Sertoli cells (66) (Figure 4; Table 1). GDNF binds two heterologous receptors, Ret and GFR- 1 which are expressed in spermatogonial cells. gdnf +/ mice lose their GSCs prematurely in testes, indicating that GDNF is essential for GSC selfrenewal. Overexpression of GDNF causes accumulation of GSC-like cells. Two recent studies show that male mouse GSCs can be cultured and expanded in vitro in the presence of GDNF for more than 6 months and can reconstitute long-term spermatogenesis and restore fertility when transplanted to sterile recipient mice (44, 53). These ndings support the hypothesis that Sertoli cells must contribute to the function of the GSC niche. In addition to GDNF signaling, BMP signaling also has a role in GSC maintenance (76, 110). Multiple BMPs, BMP4, BMP7, and BMP8 are expressed in male germ cells, while BMP4 is also expressed in Sertoli cells (76, 110), which is in www.annualreviews.org Germline Stem Cell Regulation 185 Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Figure 4 Extrinsic and intrinsic factors for GSC self-renewal/differentiation in the mouse testis. The putative niche cell, Sertoli cells, which exhibit functional polarity, express GDNF, BMPs, and SCF to promote self-renewal and differentiation by binding to their receptors GDF 1/Ret, BMPs and C-Kit, respectively. The myoid cell may assist the niche function. Intrinsic factors for self-renewal include Plzf and Nos-2. contrast to restricted expression of BMPs only in somatic niche cells in both the Drosophila ovary and testis. Intriguingly, targeted disruption of these genes has revealed that they all play important but redundant roles in maintaining the viability of germ cells, including GSCs (76, 110). It remains unclear whether they are required for GSC self-renewal as well. Therefore, from C. elegans, Drosophila to mice, extrinsic signals from their niches play instructive roles in controlling GSC self-renewal. Differentiation of GSC progeny in mice also depends on extrinsic signals. The stem cell factor (SCF) produced by Sertoli cells activates c-Kit, a tyrosine kinase receptor for SCF, to promote the differentiation of GSC progeny (22, 71, 108) (Figure 4; Table 1). Loss-of-function mutations in c-kit cause an arrest in an early step of spermatogonia differentiation, suggesting that the SCF/c-Kit 186 Wong pathway is required for germ cell differentiation and survival (22, 71, 108). The identi cation of immediate target genes controlled by SCF/c-Kit signaling will be crucial for understanding how germ cell differentiation is regulated in mice. Different Classes of Intrinsic Factors Control GSC Maintenance and Differentiation in the Mouse Testis Two different intrinsic self-renewing factors have been identi ed in mice, nanos2 and Plzf (promyelocytic leukemia zinc- nger) (9, 15, 94). nanos2 is expressed predominantly in male germ cells, and a nanos2 mutation results in complete GSC loss (94). In contrast, nanos3 is expressed in primordial germ cells (PGCs) of both sexes, and a nanos3 mutation leads to complete loss of germ cells in both sexes. As discussed earlier, one nanos gene Jin Xie Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. maintains PGCs as well as GSCs in the Drosophila ovary, which is in contrast to two genes sharing these roles in mice. It would be interesting to see whether pumilio-like genes are also required for maintaining PGCs and GSCs in mice since its Drosophila counterpart functions in the same protein complex with Nanos to repress translation and maintain GSCs. plzf-null mice have lost their ability to maintain GSCs in the testis, and its function is required only in GSCs (9, 15). Plzf, a member of the POK (POZ and Kruppel) family of transcriptional repressors, is expressed in early undifferentiated spermatogonial cells. It can potentially recruit members of the mammalian Polycomb family, such as BMI1, to link epigenetic modi cations to transcriptional control. Since BMI1 has recently been shown to be required for self-renewal and proliferation of hematopoietic and neural stem cells (67, 74), it would be worthwhile to see whether BMI1 is also required to maintain GSCs in the testis. It is equally important to know how Plzf and Nanos2 interact with the known GDNF signaling pathway to control GSC self-renewal in the testis and to understand how the transition from a GSC to a differentiating As is regulated intrinsically. Figure 5 A conceptual relationship between GSCs and their niche. The niche cell produces short-range self-renewing signals, and self-renewing intrinsic factors such as Pum/Nos promote self-renewal by repressing expression of differentiation-promoting factors. In differentiating cells, however, this process is reversed to initiate differentiation. SUMMARY AND FUTURE DIRECTIONS A comparative analysis of GSC systems in Drosophila, C. elegans, and mice has elucidated some fundamental principles and strategies for GSC self-renewal and differentiation (Figure 5). First, GSCs are situated in specialized regulatory niches to ensure selfrenewal. Cap cells function as a GSC niche in the Drosophila ovary, hub cells form a GSC niche in the Drosophila testis, and the DTC is a GSC niche in C. elegans, as demonstrated by cell biological and genetic studies (18, 106). In the mouse testis, GSC transplantation experiments have revealed the existence of a GSC niche but its structure has not yet been de ned (6). Second, GSC niches in Drosophila and C. elegans exhibit structural asymmetry to ensure that the GSC daughters remaining inside the niche self-renew, and the others outside the niche generate differentiated cells. Third, short-range niche signals prevent GSCs from differentiation and thereby maintain stem cell self-renewal. Niche signals, BMPs in the Drosophila testis and ovary, Upd in the Drosophila testis, and LAG-2 in C. elegans gonad all function over a short distance to maintain GSCs. However, it is unknown whether GDNF is also a short-range signal that acts speci cally on GSCs in the mouse testis. Fourth, intrinsic factors regulating mRNA stability and/or translation are conserved for their ability to regulate GSC self-renewal and differentiation. In Drosophila and C. elegans, the majority of intrinsic factors are related to regulation of mRNA stability and/or translation. Notably, Pum and Nos are conserved translational repressors that are involved in GSC self-renewal from Drosophila, C. elegans to mice. Finally, the interplay between extrinsic signals and intrinsic factors is critical for GSC self-renewal. In the Drosophila ovary and testis, BMP signaling directly represses expression of a differentiation-promoting www.annualreviews.org Germline Stem Cell Regulation 187 factor, bam, in order to maintain GSCs, whereas, in C. elegans, GLP-1/Notch signaling is implicated in repressing functions of differentiation-promoting genes, such as gld genes. It will be interesting to see whether these conserved GSC regulatory mechanisms are also utilized by other adult stem cells. Despite the commonalities of GSCs in different systems, obvious differences exist between them. First, a different combination of extrinsic signals is needed for different GSCs. For example, only one GLP-1/Notch signaling pathway is required for GSC selfrenewal in C. elegans, Piwi and BMP signaling are needed for controlling GSC selfrenewal in the Drosophila ovary, while Piwi, BMP and JAK-STAT signaling contribute to GSC maintenance in the Drosophila testis, and GDNF and, likely, BMPs work together to control GSC maintenance in the mouse testis. Because these different combinations of signaling pathways are suf cient to prevent GSC differentiation, these differences may re ect distinct developmental histories of different GSCs. Second, the same extrinsic signals exhibit some differences in their ability to regulate GSCs. One example is how Dpp plays slightly different roles in the Drosophila ovary and testis. Dpp plays an instructive role in controlling GSC self-renewal in the Drosophila ovary, but it plays a minor role in GSCs of the Drosophila testis. Third, some intrinsic factors are needed in only one GSC system but not in others to direct GSC self-renewal and differentiation. GSCs in Drosophila, C. elegans, and mice form differently during early development; their different developmental histories may confer GSCs distinct properties, which thus require the use of different intrinsic factors to control their self-renewal and differentiation. For example, bam has not been identi ed and shown to be required for germ cell differentiation in C. elegans, while many intrinsic factors identi ed in C. elegans, such as gld genes, have not been shown to be required for GSC differentiation in Drosophila. Together, these differences in the mechanisms regulating GSC behavior have revealed that different combinations of extrinsic signals and intrinsic factors can achieve the same purposes of controlling self-renewal and differentiation. The knowledge gained from studies on GSCs and their niches in Drosophila and C. elegans has provided an intellectual framework for de ning stem cells and their niches in mammalian systems. Moreover, the signaling pathways identi ed for controlling GSC self-renewal have also been shown to regulate different adult stem cell types in mammals. Although we have learned so much from studying GSCs in different systems, many questions remain. What constitutes a GSC niche in the mouse testis? How is GSC niche formation regulated in different species? How does signaling initiated by niche cells interact with intrinsic factors to control GSC selfrenewal and differentiation? Whether and how do GSCs regulate their niche function? Is GSC aging due to intrinsic aging or niche aging? The answers to these questions will greatly advance our understanding of GSC regulation and will also provide insight into how adult stem cells are regulated in general. Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. SUMMARY POINTS 1. The regulatory microenvironment or niche directly controls GSC asymmetric division and self-renewal. 2. GSC niches exhibit structural asymmetry to ensure that only one of the two GSC daughters remains in the niche for self-renewal. 188 Wong Jin Xie 3. Niche signals function at short range to act directly on GSCs to prevent them from differentiation and thereby control self-renewal. 4. Different combinations of niche signals are needed for GSC self-renewal in different systems. 5. The same niche signal may function differently in different GSC systems. 6. Differentiation of GSC daughters is controlled by extrinsic signals from their surrounding somatic cells. 7. Several classes of intrinsic factors are involved in controlling GSC self-renewal and differentiation. Pumilio and Nanos families of proteins are conserved intrinsic factors for GSC self-renewal. Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. 8. Intimate interplay between extrinsic factors and intrinsic factors is critical for GSC self-renewal. ACKNOWLEDGMENTS We would like to thank Daniel Kirilly for making all the gures and the other Xie laboratory members for stimulating scienti c discussions. Owing to space constraints, we apologize to colleagues whose work was not mentioned or discussed in detail. The authors are supported by Stowers Institute for Medical Research and N.I.H. (1R01 GM64428-01). LITERATURE CITED 1. 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Development 131:1365 75 46. Khalsa O, Yoon JW, Torres-Schumann S, Wharton KA. 1998. TGF-beta/BMP superfamily members, Gbb-60A and Dpp, cooperate to provide pattern information and establish cell identity in the Drosophila wing. Development 125:2723 34 47. Kiger AA, Fuller MT. 2001. Male germ-line stem cells. In Stem Cell Biology, ed. DR Marshak, RL Gardner, D Gottlieb, pp. 149 88. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press 48. Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT. 2001. Stem cell selfrenewal speci ed by JAK-STAT activation in response to a support cell cue. Science 294:2542 45 49. Kiger AA, White-Cooper H, Fuller MT. 2000. Somatic support cells restrict germline stem cell self-renewal and promote differentiation. Nature 407:750 54 www.annualreviews.org Germline Stem Cell Regulation This paper reports that Nos and Pum may function together to repress PGC and GSC differentiation in a Dpp-independent manner. Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. This paper provides experimental evidence challenging the dogma that GSCs do not persist in postnatal mammalian ovary. This study shows that differentiated germ cell cysts can revert back into GSCs in early female gonads and in the adult ovary. The GSCs regenerated from differentiated germ cells function normally. This study shows that the hub cells function as a GSC niche in the Drosophila testis by demonstrating that they produce Upd that activates JAK-STAT signaling to control GSC self-renewal. 191 This study uses laser ablation to show that the DTC is required for maintaining germ cells in C. elegans. Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. The rst paper showing that GSCs in the mouse testis can be cultured and expanded in vitro.These cultured GSCs provide a powerful system for studying their regulation in vitro. The rst paper showing that Pumilio is required for maintaining GSCs in the Drosophila ovary. This paper identi ed the rst signal from Sertoli cells, GDNF, that is necessary and suf cient for controlling GSC self-renewal and proliferation. 50. Kimble JE, White JG. 1981. On the control of germ cell development in Caenorhabditis elegans. Dev. Biol. 81:208 19 51. King FJ, Lin H. 1999. Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis. Development 126:1833 44 52. King FJ, Szakmary A, Cox DN, Lin H. 2001. Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary. Mol. Cell. 7:497 508 53. Kubota H, Avarbock M, Brinster R. 2004. Growth factors essential for selfrenewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. 101:16489 94 54. Kubota H, Avarbock MR, Brinster RL. 2003. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc. Natl. Acad. Sci. USA 100:6487 92 55. Kwak JE, Wang L, Ballantyne S, Kimble J, Wickens M. 2004. Mammalian GLD-2 homologs are poly(A) polymerases. Proc. Natl. Acad. Sci. USA 101:4407 12 56. Lambie EJ, Kimble J. 1991. Two homologous regulatory genes, lin-12 and glp-1, have overlapping functions. Development 112:231 40 57. Lamont LCS, Bernstein D, Wickens M, Kimble J. 2004. FBF-1 and FBF-2 regulate the size of the mitotic region in the C. elegans germline. Dev Cell. 7:697 707 58. Lantz V, Chang JS, Horabin JI, Bopp D, Schedl P. 1994. The Drosophila orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity. Genes Dev. 8:598 613 59. Lavoie CA, Ohlstein B, McKearin DM. 1999. Localization and function of Bam protein require the benign gonial cell neoplasm gene product. Dev. Biol. 212:405 13 60. Lin H. 2002. The stem-cell niche theory: lessons from ies. Nat. Rev. Genet. 3:931 40 61. Lin H, Spradling AC. 1997. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development 124:2463 76 62. Lin H, Yue L, Spradling AC. 1994. The Drosophila fusome, a germline-speci c organelle, contains membrane skeletal proteins and functions in cyst formation. Development 120:947 56 63. Marin V, Evans TC. 2003. Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1. Development 130:2623 32 64. McKearin D, Ohlstein B. 1995. A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells. Development 121:2937 47 65. McKearin DM, Spradling AC. 1990. bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis. Genes Dev. 4:2242 51 66. Meng X, Lindahl M, Hyvonen ME, Parvinen M, deRooij DG, et al. 2000. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287:1489 93 67. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. 2003. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425:962 67 68. Morrison SJ, Shah NM, Anderson DJ. 1997. Regulatory mechanisms in stem cell biology. Cell 88:287 98 69. Ohlstein B, Lavoie CA, Vef O, Gateff E, McKearin DM. 2000. The Drosophila cystoblast differentiation factor, benign gonial cell neoplasm, is related to DExH-box proteins and interacts genetically with bag-of-marbles. Genetics 155:1809 19 Wong 192 Jin Xie 70. Ohlstein B, McKearin D. 1997. Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells. Development 124:3651 62 71. Ohta H, Yomogida K, Dohmae K, Nishimune Y. 2000. Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF. Development 127:2125 31 72. Orwig KE, Ryu BY, Avarbock MR, Brinster RL. 2002. Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes. Proc. Natl. Acad. Sci. USA 99:11706 11 73. Orwig KE, Shinohara T, Avarbock MR, Brinster RL. 2002. Functional analysis of stem cells in the adult rat testis. Biol. Reprod. 66:944 49 74. Park IK, Qian D, Kiel M, Becker MW, Pihalja M, et al. 2003. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423:302 5 75. Petcherski AG, Kimble J. 2000. LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway. Nature 405:364 68 76. Puglisi R, Montanari M, Chiarella P, Stefanini M, Boitani C. 2004. Regulatory role of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation in the immature mouse. Eur. J. Endocrinol. 151:511 20 77. Ryu BY, Orwig KE, Avarbock MR, Brinster RL. 2003. Stem cell and niche development in the postnatal rat testis. Dev. Biol. 263:253 63 78. Saccomanno L, Loushin C, Jan E, Punkay E, Artzt K, Goodwin EB. 1999. The STAR protein QKI-6 is a translational repressor. Proc. Natl. Acad. Sci. USA 96:12605 10 79. Schulz C, Kiger AA, Tazuke SI, Yamashita YM, Pantalena-Filho LC, et al. 2004. A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage. Genetics 167:707 23 80. Schulz C, Wood CG, Jones DL, Tazuke SI, Fuller MT. 2002. Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells. Development 129:4523 34 81. Shamblott MJ, Axelman J, Wang S, Bugg EM, Little eld JW, et al. 1998. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA 95:13726 31 82. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. 2001. Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proc. Natl. Acad. Sci. USA 98:6186 91 83. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. 2002. Germ line stem cell competition in postnatal mouse testes. Biol. Reprod. 66:1491 97 84. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. 2003. Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation. Biol. Reprod. 68:1064 71 85. Shivdasani AA, Ingham PW. 2003. Regulation of stem cell maintenance and transit amplifying cell proliferation by tgf-beta signaling in Drosophila spermatogenesis. Curr. Biol. 13:2065 72 86. Song X, Wong MD, Kawase E, Xi R, Ding BC, et al. 2004. Bmp signals from niche cells directly repress transcription of a differentiation-promoting gene, bag of marbles, in germline stem cells in the Drosophila ovary. Development 131:1353 64 87. Song X, Zhu CH, Doan C, Xie T. 2002. Germline stem cells anchored by adherens junctions in the Drosophila ovary niches. Science 296:1855 57 88. Spradling A, Drummond-Barbosa D, Kai T. 2001. Stem cells nd their niche. Nature 414:98 104 www.annualreviews.org Germline Stem Cell Regulation Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. This paper shows that niche signals maintain GSCs by directly repressing expression of differentiationpromoting genes. It also shows that gbb, a Drosophila homolog of BMP5-8, is also essential for maintaining GSCs and repressing bam expression. The rst paper showing that Ecadherin-mediated cell adhesion is essential for anchoring and recruiting GSCs to their niche. Cadherins are also involved in anchoring adult stem cells in mammalian systems. 193 Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. 94. This paper shows that two mouse nanos genes are involved in regulation of PGC and GSC functions in mice. nano2 is required for GSC maintenance in the testis, while nanos3 is required for PGC maintenance in both sexes. 95. This study shows that the hub cells function as a GSC niche in the Drosophila testis by providing Upd that activates JAK-STAT signaling in GSCs for self-renewal. 98. This study shows that nanos is required for preventing the differentiation of PGCs and GSCs in the Drosophila female gonads. 104. This study demonstrates that Dpp/BMP2-4 is essential for controlling GSC self-renewal and division. The rst study using mutant clonal analysis to study gene function in the control of GSC self-renewal. 194 89. 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Conserved role of nanos proteins in germ cell development. Science 301:1239 41 95. Tulina N, Matunis E. 2001. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK- STAT signaling. Science 294:2546 49 96. Urban S, Lee JR, Freeman M. 2001. Drosophila rhomboid-1 de nes a family of putative intramembrane serine proteases. Cell 107:173 82 97. Wang L, Eckmann CR, Kadyk LC, Wickens M, Kimble J. 2002. A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419:312 16 98. Wang Z, Lin H. 2004. Nanos maintains germline stem cell self-renewal by preventing differentiation. Science 303:2016 19 99. Wang Z, Lin H. 2005. The division of Drosophila germline stem cells and their precursors requires a speci c cyclin. Curr. Biol. 15:328 33 100. Wickens M, Bernstein D, Crittenden S, Luitjens C, Kimble J. 2001. PUF proteins and 3 UTR regulation in the Caenorhabditis elegans germ line. Cold Spring Harb Symp. Quant. Biol. 66:337 43 101. Wickens M, Bernstein DS, Kimble J, Parker R. 2002. A PUF family portrait: 3 UTR regulation as a way of life. Trends Genet. 18:150 57 102. Xie T, Kawase E, Kirilly D, Wong MD. 2005. Intimate relationships with their neighbors: Tales of stem cells in Drosophila reproductive systems. Dev. Dyn. 232:775 90 103. Xie T, Spradling A. 2001. The Drosophila ovary: an in vivo stem cell system. In Stem Cell Biology, ed. DR Marshak, RL Gardner, D Gottlieb, pp. 129 48. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press 104. Xie T, Spradling AC. 1998. decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 94:251 60 105. Xie T, Spradling AC. 2000. A niche maintaining germ line stem cells in the Drosophila ovary. Science 290:328 30 106. Yamashita Y, Fuller M, Jones DL. 2005. Signaling in stem cell niches: lessons from the Drosophila germline. J. Cell Sci. 15:665 72 107. Yamashita YM, Jones DL, Fuller MT. 2003. Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301:1547 50 108. Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S, Kunisada T, et al. 1991. Role of ckit in mouse spermatogenesis: identi cation of spermatogonia as a speci c site of c-kit expression and function. Development 113:689 99 Wong Jin Xie 109. Zhang B, Gallegos M, Puoti A, Durkin E, Fields S, et al. 1997. A conserved RNAbinding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature 390:477 84 110. Zhao G, Chen YX, Liu XM, Xu Z, Qi X. 2001. Mutation in Bmp7 exacerbates the phenotype of Bmp8a mutants in spermatogenesis and epididymis. Dev. Biol. 240:212 22 105. The rst study directly demonstrating that GSCs are situated in the niche in the Drosophila ovary. A GSC can undergo symmetric cell division to produce two GSCs if both daughters remain in the niche. 107. This study demonstrates that regulation of GSC spindle orientation is critical for asymmetric GSC division in the Drosophila testis. The authors also show that APC-1, APC-2 and Centrosomin play crucial roles in orienting the GSC spindle. Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. www.annualreviews.org Germline Stem Cell Regulation 195 Contents John Maynard Smith Richard E. Michod p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Annual Review of Genetics Volume 39, 2005 The Genetics of Hearing and Balance in Zebra sh Teresa Nicolson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 9 Immunoglobulin Gene Diversi cation Nancy Maizels p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p23 Complexity in Regulation of Tryptophan Biosynthesis in Bacillus subtilis Paul Gollnick, Paul Babitzke, Alfred Antson, and Charles Yanofsky p p p p p p p p p p p p p p p p p p p p p p p47 Cell-Cycle Control of Gene Expression in Budding and Fission Yeast J rg B hler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p69 a Comparative Developmental Genetics and the Evolution of Arthropod Body Plans David R. Angelini and Thomas C. Kaufman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p95 Concerted and Birth-and-Death Evolution of Multigene Families Masatoshi Nei and Alejandro P. Rooney p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 121 Drosophila as a Model for Human Neurodegenerative Disease Julide Bilen and Nancy M. Bonini p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 153 Molecular Mechanisms of Germline Stem Cell Regulation Marco D. Wong, Zhigang Jin, and Ting Xie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 173 Molecular Signatures of Natural Selection Rasmus Nielsen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 197 T-Box Genes in Vertebrate Development L.A. Naiche, Zachary Harrelson, Robert G. Kelly, and Virginia E. Papaioannou p p p p p p 219 Connecting Mammalian Genome with Phenome by ENU Mouse Mutagenesis: Gene Combinations Specifying the Immune System Peter Papathanasiou and Christopher C. Goodnow p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 241 Evolutionary Genetics of Reproductive Behavior in Drosophila: Connecting the Dots Patrick M. O Grady and Therese Anne Markow p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 263 v Sex Determination in the Teleost Medaka, Oryzias latipes Masura Matsuda p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 293 Orthologs, Paralogs, and Evolutionary Genomics Eugene V. Koonin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 309 The Moss Physcomitrella patens David Cove p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 339 A Mitochondrial Paradigm of Metabolic and Degenerative Diseases, Aging, and Cancer: A Dawn for Evolutionary Medicine Douglas C. Wallace p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 359 Annu. Rev. Genet. 2005.39:173-195. Downloaded from arjournals.annualreviews.org by University of California - San Diego on 09/16/06. For personal use only. Switches in Bacteriophage Lambda Development Amos B. Oppenheim, Oren Kobiler, Joel Stavans, Donald L. Court, and Sankar Adhya p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 409 Nonhomologous End Joining in Yeast James M. Daley, Phillip L. Palmbos, Dongliang Wu, and Thomas E. Wilson p p p p p p p p p p 431 Plasmid Segregation Mechanisms Gitte Ebersbach and Kenn Gerdes p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 453 Use of the Zebra sh System to Study Primitive and De nitive Hematopoiesis Jill L.O. de Jong and Leonard I. Zon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 481 Mitochondrial Morphology and Dynamics in Yeast and Multicellular Eukaryotes Koji Okamoto and Janet M. Shaw p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 503 RNA-Guided DNA Deletion in Tetrahymena: An RNAi-Based Mechanism for Programmed Genome Rearrangements Meng-Chao Yao and Ju-Lan Chao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 537 Molecular Genetics of Axis Formation in Zebra sh Alexander F. Schier and William S. Talbot p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 561 Chromatin Remodeling in Dosage Compensation John C. Lucchesi, William G. Kelly, and Barbara Panning p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 615 INDEXES Subject Index p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 653 ERRATA An online log of corrections to Annual Review of Genetics chapters may be found at http://genet.annualreviews.org/errata.shtml vi Contents

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