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The self-renewing mechanism of stem cells in the germline
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The self-renewing mechanism of stem cells in the germline Haifan Lin Germline stem cells (GSCs) are a self-renewing population of germ cells that serve as the source of gametes in diverse organisms. Current research suggests that the self-renewing division of GSCs is controlled both by somatic signaling and by intracellular mechanisms such as differential gene expression, asymmetric cytoskeletal organization, and the cell cycle machinery. These findings provide a framework for the further study of GSCs and stem cell renewal in general. Addresses Department of Cell Biology, Box 3709, Duke UniversityMedical Center, Durham, NC 27710; e-mail:[email protected] Current Opinion in Cell Biology 1998, 10:687-693 http://biomednet.com/elecref/0955067401000687 © Current BiologyLtd ISSN 0955-0674 Abbreviations CDK cyclin-dependentkinase DAZ deletedin azoospermia dpc days post coitum DPP decapentaplegic DTC distal tip cell GSC germlinestem cell mad mothersagainst dpp Med Medea TF terminalfilament Introduction
Central to gametogenesis in m a n y organisms are germline stem cells (GSCs), which serve as a source for the continuous production of gametes. A striking feature of GSCs, very much like any other stem cells, is their ability to self-renew while remaining capable of generating n u m e r o u s differentiated daughter cells. T h e existence of stem cells in the germline was proposed over a century ago [1]. Decades of research on spermatogenesis in mammalian systems has yielded a wealth of cytological information as well as interesting hypotheses pertinent to GSC division (reviewed in [2,3]). Recent genetic and molecular analyses in Caenorhabditis elegans, Drosophila, and the mouse have provided further insight into the mechanisms underlying GSC division and maintenance. This article reviews the latest progress in understanding GSC division, with emphasis on Drosophila since its study represents most of the progress in the past two years. For a more comprehensive review on GSCs, please refer to [4] (see also de Rooij and Grootegoed, this issue pp 694-701). S t e m c e l l s e x i s t in d i v e r s e f o r m s in t h e g e r m l i n e
Stem cells possess two fundamental properties: the ability to self-renew and the ability to produce numerous differentiated progeny. According to this definition, stem cells are present in the germline of diverse organisms, including hydrazoan polyps [5,6], nematodes [7], arthropods [8-11],
amphibians [12], birds [13], fishes [14], reptiles [12], and mammals [15,16]. Notably, the ovaries of some vertebrates, especially that of mammals, do not contain self-renewing GSCs [17]. GSCs can be classified into two types according to how they self-renew. T h e first type, herein called stereotypic GSCs, self-renew strictly by asymmetric divisions that produce a daughter GSC and a differentiated daughter cell. For example in the Drosophila ovary each GSC division produces a daughter GSC that remains in contact with a cluster of apical somatic cells called the terminal filament (TF), and a cystoblast that is one cell away from the T F [18,19 °°] (Figure 1). Laser ablation of the GSCs ceases the production of new egg chambers revealing them as the exclusive source for germline renewal [11]. Stereotypic GSCs also exist in the Drosophila testis and probably also in vertebrate testis [13-16,20-22]. In the Drosophila testis, between five and nine GSCs divide asymmetrically in a defined orientation with respect to a group of nonmitotic somatic hub cells, in a similar fashion to the division of ovarian GSCs [20,22]. In mammalian testes, various hypotheses have been proposed to describe the exact identity of GSCs and their pattern of division, most of which point to a subset of spermatogonia (Type A or a subtype of Type A) as GSCs (reviewed in [4]). Despite this, it has not been unequivocally shown whether GSCs in the mammalian testis divide asymmetrically like those in Drosophila or alternatively divide symmetrically and should be classified as the second type of GSCs (see below). T h e second type of GSCs, herein called populational GSCs, divide symmetrically to produce two daughter cells each of which has an equal probability of differentiating. T h e y selfperpetuate only at the populational level whereby the collective behavior of mitotic cells in the gonad maintains a steady source of germline cells. For example, in C. elegans, the mitotically active germline nuclei share a common cytoplasm at the distal end of the gonad and serve as a stem cell population for gametogenesis throughout adulthood by virtue of their proximity to a somatic signaling cell, the distal tip cell (DTC) [23,24]). Laser ablation of between five and ten of the most distal germ cells in third larval stage gonads does not eliminate the proliferative germ cell population [25°]. It is possible that these nuclei show a gradient of mitotic ability, with those closest to the D T C being most mitotically active and those more than 20 nuclei away eventually entering meiosis to become terminally differentiated. Populational GSCs may also exist in Hydra oligactis [5] and Hydra magnipapillata [6]. T h e known mechanism for the self-renewal of populational GSCs differs from that of stereotypic GSCs (see below). At present, it is too early to determine whether this difference is significant.
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Figure 1 (a)
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Current Opinion in Cell Biology
Intracellular mechanisms underlying asymmetric germline stem cell division Current studies in C. elegans, Drosophila, and mammals indicate that GSC divisions are controlled by both intracellular mechanisms and cell-cell interactions (reviewed in [4]). For stereotypic GSCs, intracellular mechanisms should include both a basic cell-cycle machinery as well as a mechanism for generating divisional asymmetry. This asymmetric mechanism should in turn further consist of asymmetrically-segregated cell-fate regulators and a cellular machinery responsible for their asymmetric segregation or for establishing an asymmetric exposure of the two daughter cells to extrinsic signals.
Cell cycle machinery T h e role of celt cycle regulators in controlling GSC division has not been systematically analyzed. Recent studies are starting to reveal the expression patterns of these molecules in GSCs. In mice, the G 1 cyclins D1, D2, and D3, the G z cyclin A2, as well as CDK4, and Wee-1 are expressed in spermatogonia, with some of them also expressed in later stages of spermatogenesis [26-30]. Cdc25A is also weakly expressed in the spermatogonia. T h e PCNA (proliferating cell nuclear antigen) protein, involved in DNA synthesis and repair, is detected in proliferating spermatogonia but not in meiotic spermatocytes [26]. In Drosophila, R O U G H E X and cyclin A regulate the normal progression of meiosis while
The asymmetric division of germline stem cells (GSC) in the Drosophila ovary (modified from [4]). (a) An ovariole with a string of developing egg chambers produced by germarium. (b) A magnified view of the apical region of the germarium (boxed area in [a]). There are usually two GSCs (gsc) in the germarium, in contact with apical somatic cells expressing YB, PlWl and HEDGEHOG (HH). Boldface type indicates a high concentration of the named protein. Although YB- and PlWlmediated signalling are required for GSC maintenance, HH is only known to regulate the division and differentiation of somatic stem cells (ssc) two to five cells away from the terminal filament (TF) [6g]. In GSCs, spectrosomes (shaded spheres) containing spectrin (Sp) and HULITAISHAO (Hts) proteins, reside in the apical region of the cytoplasm both at interphase and during mitosis, apposed to the signaling somatic cells. During mitosis, the spectrosome anchors one pole of the spindle so that the divisional plane is approximately perpendicular to the apico-basal axis of the germarium. As a result, the daughter GSC remains in contact with the TF while the cystoblast (cb) becomes one cell away from the somatic cells. The PUM protein is present at a high level in GSCs and at a low level in cystoblasts and cysts, NOS protein shows the opposite distribution profile, while the cytoplasmic form of BAM is only present in cystoblasts and cysts. The source of DPP has not been identified.
cyclin E controls the nurse cell endocycle. Their role in GSC division, however, has not been addressed [31,32]. Similarly, in C. elegans, a subset of cyclins are mostly expressed in the germline [33]. These expression patterns provide a basis for functional analysis of these molecules in GSC division. Asymmetrically-segregated cell-fate regulator
Asymmetrically-segregating cell-fate determinants, such as N U M B and P R O S P E R O in Drosophila neuroblasts (reviewed in [18]), have not been identified in GSCs. But three Drosophila genes have recently been shown to be required cell-autonomously for either the maintenance of female GSCs or the proper differentiation of cystoblasts. Among them, pumilio (pure) is essential for GSC maintenance. Females with pure mutations contain normal numbers of GSCs at the onset of oogenesis but fail to maintain them [34°°,35 °] and the often appear to differentiate. Since PUM is a R N A binding protein that mediates translational repression in the embryo [36,37] and that it is present at a high level in GSCs but a low level in cystoblasts [35 °] (Figure 1), it is tempting to speculate that PUM is an asymmetrically-expressed cellfate regulator that selectively suppresses the translation of certain RNAs in the GSC to prevent differentiation. A pum homolog has been identified in mammals [38], suggesting that a pum-like m e c h a n i s m may exist in mammalian GSCs as well.
The self-renewing mechanism of stem cells in the germline Lin 689
In addition to pure, nanos (nos) may be involved in GSC maintenance since in flies with nos mutations 50% of the newly eclosed adult ovaries are germlineless [35°]. This defect, however, could also be due to an earlier requirement of nos for germline development prior to oogenesis. During oogenesis, nos also appears to function in the differentiation of cystoblasts and germline cysts, the interconnected daughters of cystoblasts, since nos mutations block germline cyst development in adult ovaries without the immediate loss of GSCs [35°]. Consistent with this second role, NOS is abundant in cysts but is barely detectable in GSCs and cystoblasts (Figure 1). This difference in the protein profile between NOS and PUM as well as the difference in their GSC phenotype suggest that NOS and PUM may not interact during early oogenesis as they do in embryos. Complementary to the role of pure, bag of marbles (barn) is required cell-autonomously to switch germ cells from GSC fate to a differentiating daughter fate in both males and females [39,40,41°]. Germ cells with barn mutations fail to differentiate, but instead proliferate like stem cells or ill-differentiated germline cells [39,41",42]. Heat-shockinduced ectopic expression of barn is sufficient to extinguish GSC divisions [40"]. Differential accumulation of barn m R N A and proteins in cystoblast versus GSCs [39,42] suggest, that BAM could be a differentially expressed cell-autonomous regulator for the cystoblast fate. In mammals, a promising candidate for an intrinsic factor required for GSC self-renewal is the human deleted in azoospermia (DAZ) gene cluster in the azoospermiafactor (AZF) region on the Y chromosome [43]. DAZ, a candidate for the AZF gene, encodes a putative RNA binding protein with strong homology to the Drosophila B O U L E which is protein required for meiosis during spermatogenesis [44]. DAZ is expressed specifically in the germ cells of the adult human testis and most abundantly in spermatogonia [45"]. T h e mouse homolog of DAZ, Dazh (also known as Dazla), is also expressed in germ cells in embryonic gonads before germ cell sex differentiation, starting at 12.5 days post coitum (dpc) [46--48]. T h e Dazh level decreases in female embryos following the entry of oogonia into meiosis but persists in the male gonad and is present in day 1 neonatal male mice whose germ cells are gonocytes, the immediate precursors of spermatogonia. Subsequently, Dazh expression increases as spermatogonial stem cells appear, reaches a peak as spermatogenic cells first enter meiosis and persists at this level thereafter [46]. hnportant information on Dazh function comes from the analysis of Dazh-knockout mice, whose embryonic gonads are normal up to 15 dpc, but by 19 dpc germ cells in seminiferous tubules (and in ovaries as well) are significantly reduced in both heterozygous and homozygous Dazh- mice [49"]. This result suggests that Dazh is required quantitatively for the initial maintenance of gonocytes which are either mitotically arrested GSCs or the immediate precursors of GSCs [4].
Cellular machinery for asymmetric localization/orientation
It is well established that cytoskeletal systems play a crucial role in the localization and segregation of cytoplasmic components and in spindle orientation during mitosis [18,50-52]. Recent work on Drosophila ovarian GSCs has revealed the importance of the cytoskeleton in the asymmetric GSC divisions [19"°,34",53-56]. In the Drosophila ovary, the asymmetry of GSC division is cytologically manifested both by the relative position of the two daughter cells to the "FF and by the differential segregation of a cytoplasmic structure called the spectrosome [53,54,57"] (Figure 1). The spectrosome resides in the apical region of the GSC and contains membrane vesicles and membrane skeletal proteins such as ~- and 13-spectrin, ankyrin, and the adducin-like H U L I T A I S H A O (HTS) protein [53-55]. It also contains regulatory molecules such as a BAM isoform and cyclin A ([39]; M Lilly, M de Cuevas, AC Spradling, personal communication) and is associated with a centrosome [53]. During mitosis, the spectrosome is associated with the pole of the mitotic spindle located near basal T F cells ([19"',34")1; Figure lb). At telophase, it grows in size and elongates towards the future cystoblast and eventually becomes asymmetrically bisected by delayed cytokinesis occurring at the next cell cycle [19"',34",57"]. Delayed cytokinesis may be a common feature of GSC division, since it is also seen during Drosophila and mammalian spermatogonial GSC divisions. T h e spectrosome may have a dual function in establishing the asymmetry of GSC division. First, it anchors the mitotic spindle to define the orientation of GSC division so that, during each division, one daughter cell will remain in contact with the T F and may thus remain as a GSC while the other daughter will become one cell away from the signal and may thus differentiate. This function can be inferred from the observations that spectrosome disassembly causes randomized spindle orientation and that germline cysts fail to develop properly in the absence of the spectrosome and its derivative structure, the fusome [19°°]. Second, the spectrosome may localize molecules important for stem cell fate as a means of selectively retaining them in the daughter GSC. This localization function is suggested by the ability of the spcctrosome to localize a BAM isoform and cyclin A (see above) and the ability of spectrosomelike structures in mammalian cells to localize protein kinase C [58]. Despite the above two potential functions, the spectrosome, however, does not regulate the rate of GSC division [34"]. It is important to point out that, even if the spectrosome is essential for the self-renewing asymmetric division of GSCs, such division may not be essential for GSC maintenance in the Drosophila ovary. In the absence of the spectrosome and fusome, a normal number of egg chambers, though ill-differentiated, are still produced [34"']. This possibly reflects the fact that, although GSC divisions are now randomly oriented, the topology of the germarium
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still allows about 50% of the daughter cells to contact the T E Consequently, a self-renewing population of germ cells is still maintained, even though cystoblasts may not properly develop possibly due to the failure to localize cellfate regulators in the spectrosome. If this hypothesis is true, the functional significance of the spectrosome mechanism is to convert populational GSCs to stereotypic GSCs. Nevertheless, studies on the spectrosome have revealed the critical role of membrane skeletal proteins in establishing a divisional asymmetry which may be important in other stem cell systems. What then links the spindle pole to the spectrosome? What orients the spectrosome towards the TF? A recent study suggests that the spindle-spectrosome linkage is mediated by cytoplasmic dynein [56]. In germline cysts, cytoplasmic dynein is required to link one pole of the mitotic spindle to the fusome. Given the structural similarities between the fusome and the spectrosome, it is likely that cytoplasmic dynein plays the same role in GSCs.
Signaling mechanisms controlling asymmetric GSC division Extrinsic signaling has long been implicated in regulating stem cell division, even though relatively little is known about its specific role in GSC division (reviewed in [4]). Extrinsic signaling is mediated by hormones, growth factors, cytokines, and short-range cell-cell signaling pathways such as those involving Steel and c-kit in mammals or leg-2 and glp-1 in C. elegans. These cell-cell interactions have been extensively reviewed [4]. T h e past two years have witnessed exciting progress in the genetic dissection of the signaling mechanisms that regulate GSC regulation in Drosophila, which will be the focus of this section. In Drosophila, the role of somatic cells in regulating GSC division was first indicated by the laser ablation of the apical portion of the terminal filament, which increased the rate of GSC division [11]. Although the partial ablation did not address other functions of the T F in regulating GSC division, such functions have been revealed by the genetic analysis of piwi and fs(1)Yb (Yb) genes [34°°]; J King, L Chang, H Lin, unpublished data). Loss-of-function mutations in piwi or Yb cause the failure of GSC maintenance. In flies with mutations in piwi or Yb GSCs often differentiate into germline cysts without divisions. Interestingly, Yb is specifically expressed in the T F (J King, Lin H, unpublished data); removing Yb function in the germline does not affect GSC division ([59];J King, H Lin, unpublished data). PIWI is expressed in both the T F and in the germline; removing piwi from the germline does not affect GSC division (D Cox, H Lin, unpublished data). Thus, the study of Yb and piwi reveals the essential role of a TF-mediated somatic signaling mechanism in regulating GSC division. T h e Yb-mediated signaling may be female-specific since severe loss-of-function Yb mutations only affect female GSC maintenance, while piwai-mediated signaling is required for both male and
female GSCs. Yb and piwi encode novel proteins. T h e PIWI-like proteins are found from C. elegans to humans and to Arabidopsis with apparently conserved functions. Further molecular analysis of Yb and piwi and their interacting genes should lead to the identification of molecular pathways involved in this somatic signaling. Although the molecular nature of somatic signaling is currently unknown, a recent study revealed the essential role of the Drosophila homolog of BMP2/4 decapentaplegic(dpp) signaling pathway in GSC division and maintenance [60"°]. Loss of dpp function causes Yb-like GSC maintenance defects, while over-expressing dpp produces GSC tumors. Genetic clonal analyses of the receptors and downstream transducers of the DPP signal, such as punt (encoding a type II receptor) and mad/Med(encodinga dimeric transcription activator), shows that the DPP signal is directly received by GSCs. It is possible that the DPP signal emanates from the surrounding somatic cells. Alternatively, DPP could be produced as a paracrine factor in differentiated germline cells or even as an autocrine factor in GSCs. Indeed, in mouse testis BMP8b, a DPP-like molecule, is expressed only in germline cells, including spermatogonia, for the initiation and maintenance of spermatogenesis [61]. It will be interesting to see if Drosophila dpp expression is germline- or soma-dependent and whether DPP homologs BMP2/4 also play a role in regulating GSC division in mammals. T h e dpp signaling pathway appears to play a different role in the Drosophila testis [62°]. First, the dpp signaling in the testis is not required for GSC division or maintenance, but restricts the proliferation of the differentiated daughter. Second, the receptors and downstream transducers of the dpp signal are not required in the germline but in the somatic cyst cells that flank each GSC and its differentiated daughter cells. T h e loss of punt and schnurti (shn) function in the somatic cyst cells results in overproliferation of the differentiated daughter of the GSC, where shn encodes a zinc finger transcription factor (a MBP1/2 homolog) that acts downstream of the dpp signal to regulate the expression of dpp-responsive genes. T h e dpp-equivalent signaling pathway is also involved in mammalian spermatogenesis, but again with a different function. For example, raadr-1, a mouse homolog of the Drosophila mad gene, is expressed mainly in the pachytene spermatocytes and round spermatids. In Bmp8b homozygous mutants, the madr-l-expressing pachytene spermatocytes are the first cells to show increased apoptosis. T h e s e data suggest that MADR1 serves as a downstream component of the BMP8b signaling pathway during the differentiation of meiotic male germ cells [63°]. Madr-2, on the other hand, is present mainly in spermatogonia and early meiotic cells and Sertoli cells [61], and thus may be involved in GSC division. In addition to the dpp signaling pathway, it has been long established that lag-2/glp-1 signaling pathway plays a key
The self-renewing mechanism of stem cells in the germline Lin
role in germline maintenance in C. elegans, where the LAG-2 somatic signal on the surface of the D T C interacts directly with the GLP-1 receptor (reviewed in [64]). LAG-2 is a transmembrane protein homologous to Delta (DI) and Serrate (Ser) in Drosophila, while GLP-1 is homologous to Notch, the receptor of DI and Ser. T h e GLP-1 mediated signal is transduced in the germ cells through interaction with the LAG-1 protein, a homolog of the Drosophila DNA binding protein Suppressor of Hairless (Su(H)) and the mammalian DNA binding protein CBF1, which are downstream interactors of the Notch protein in Drosophila and mammals, respectively [65]. Recent work further suggests that lag-1 maintains a mitotic population of germ nuclei by suppressing the function ofgld-I and gld-2 which promotes meiosis [66"]. Thus, g/d-1 and gld-2 may be a key differentiation factor in the C. elegans germline. Although the lag-2/glp-1 signaling pathway is conserved between C. e/egans and Drosophila, it appears to have no obvious function in regulating Drosophila GSC division [67,68]. This dichotomy again reflects the divergence of the signaling mechanisms employed by different GSC systems.
Conclusion Recent work on GSCs has clearly revealed both intracellular and intercellular mechanisms for GSC maintenance. Some important intrinsic factors and cell-cell signaling molecules have been identified, and their regulatory relationship has begun to be explored in the context of gene expression, cell cycle progression, cytoskeletal organization, and mitotic behavior of GSCs. Current research supports the idea that the formation and the self-renewing division of GSCs are largely under the control of extrinsic signals which regulate the mitotic and differential segregation mechanisms inside the stern cells. Elucidation of the mechanisms for the maintenance of GSCs is now occurring at an exciting rate. New genetic screens will identify more genes required for GSC division and maintenance. Meanwhile, further analysis of known GSC mechanisms will reveal exactly how cell-cell signaling and intracellular events occur in an orchestrated manner to ensure the stem cell property. These genetic and molecular analyses will also reveal how GSC division and maintenance are related to general cell cycle and mitotic mechanisms. T h e knowledge gained from these studies should provide valuable insight not only into the GSC division mechanism but also the mechanisms of stem cell division and germline development in general.
Note a d d e d in proof A recent paper [70] shows that the Drosophiliapiwi gene is required for the assymetric division of GSCs to produce and maintain a daughter GSC but it is not essential for the further differentiation of the cystoblast. It also describes genetic mosaic and RNA in situ analyses which suggest that piwi expression in adjacent somatic cells regulates GSC division . In addition, this paper reports the cloning of Drosophilia piwi and its homologs in C. elegans and
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Arabidopsispiwi-like genes known to be required for meristern cell maintenance. Decreasing C. elegans piwi expression reduces the proliferation of GSC-equivalent cells. Thus, piwi represents a novel class of genes required for GSC division in diverse organisms.
Acknowledgements I thank D McKearin, C Berg, A Chao, A Szackmary, W Deng, and T Srinivsan for critical reading of the manuscript. This work was supported by a National Institutes of Hcahh grant (R01HD33760), a David and Lucile Packard Fellowship, an American Cancer Society Junior Faculty Research Award and a March of Dimes Basil O'Connor Award.
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19. Deng W, Lin H: Spectrosomes and fusomes are essential for *= anchoring mitotic spindles during asymmetric germ cell divisions and for the microtubule-based RNA transport during oocyte specification in Drosophila. Dev Biol 1997, 189:79-94. The first description of the Drosophila germline stem cell (GSC) asymmetric mitotic cycle and the independent mitotic behavior of different GSCs in a germarium. This paper also provide the first demonstration that the spectrosome is required to anchor the mitotic spindle to define the orientation of GSC division. In addition, it systematically illustrates various roles of the fusome in germline cyst formation and in intracyst RNA transport that leads to oocyte differentiation. 20. Hardy RW, Tokuyasu KT, Lindsley DL, Garavito M: The germinal proliferation center in the testis of Drosophila melanogaster. J Ultrastruct Res 1979, 69:180-190. 21. Brinster RL, Avarbock MR: Germline transmission of donor haplotype following spermatogonial transplantation. Proc Nat/ Acad Sci USA 1994, 91:11303-1130?. 22. Gonczy P, DiNardo S: The germ line regulates somatic cyst cell proliferation and fate during Drosophila spermatogenesis. Development 1996, 122:2437-2447. 23. Hirsh D, Oppenheim D, Klass M: Development of the reproductive system of Caenorrhabditis elegans. Dev Bio/1976, 49:200-219. 24. Kimble J, White J: On the control of germ cell development in Caenorhabditis elegans. Dev Bio/1981, 81:208-219. 25. McCarter J, Bartlett B, Dang T, Schedl T: Soma-germ cell • interactions in Caenorhabditis elegans: multiple events of hermaphrodite germline development require the somatic sheath and spermathecal lineages. Dev Bio/199?, 181:121-143. Laser ablation studies reveal the requirement of the somatic sheath and spermathecal cells for multiple events of germline development as well as the dispensablility of the most apical germline nuclei as a source of stem cells. 26. Chapman DL, Wolgemuth DJ: Expression of proliferating cell nuclear antigen in the mouse germ line and surrounding somatic cells suggests both proliferation-dependent and independent modes of function./nt J Dev Bio/1994, 38:491-497. 27. RavnikSE, Rhee K, Wolgemuth DJ: Distinct patterns of expression of the D-type cyclins during testicular development in the mouse. Dev Genet 1995, 16:171-178. 28. Wu S, Wolgemuth DJ: The distinct and developmentally regulated patterns of expression of members of the mouse Cdc25 gene family suggest differential functions during gametogenesis. Dev Biol 1995, 170:195-206. 29. RavnikSE, Wolgemuth DJ: The developmentally restricted pattern of expression in the male germ line of a routine cyclin A, cyclin A2, suggests roles in both mitotic and meiotic cell cycles. Oev Bio/1996, 173:69-78. 30. Sweeney C, Murphy M, Kubelka M, Ravnik SE, Hawkins CF, Wolgemuth DJ, Carrington M: A distinct cyclin A is expressed in germ cells in the mouse. Development 1996, 122:53-64. 31. Gonczy P, Thomas BJ, DiNardo S: Roughex is a dose-dependent regulator of the second meiotic division during Drosophila spermatogenesis. Ce//1994, 77:1015-25. 32. Lilly M, Spradlin9 AC: The Drosophila endocycle is controlled by cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev 1996, 10:2514-2526. 33. KreutzerMA, Richards JP, De Silva-Udawatta MN, Temenak JJ, Knoblich JA, Lehner CF, Bennett KL: C,aenorhabditis elegans cyclin A- and B-type genes: a cyclin A multigene family, an ancestral cyclin B3 and differential germline expression. J Ceil Sci 1995, 108:2415-2424. 34. Lin H, Spradling AC: A novel group of pumilio mutations affects • - the asymmetric division of germline stem cells in the Drosophila ovary. Development 199?, 124:2463-2476. The first description of the asymmetric 9ermline stem cell division in the Drosophila ovary, with respect to the spectrosome and to the terminal filament. It also demonstrates the essential roles of pure and piwi in germline stem cell maintenance.
35. Forbes A, Lehmann R: Nanos and Pumilio have critical roles in the • development and function of Drosophila germline stem cells. Development 1998, 125:679-690. This paper shows the role of nos in cystoblast differentiation and confirms the role of pure in germline stem cell maintenance reported earlier in [34"]. It also shows the germline dependence of nos and purn function. 36. Murata Y, Wharton RP: Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell 1995, 80:747-756. 37. Wharton RP, Sonoda J, Lee T, Pastternson M, Murata Y: The pumilio RNA-binding domain is also a transcriptional regulator. Mo/Ceil 1998, 1:863-872. 38. Nagase T, Seki N, Ishikawa K, Ohira M, Kawarrabayasi Y, Ohara O, Tanaka A, Kotani H, Miyajima N, Nomura N: Prediction of the coding sequences of unidenUfed human genes. VI. The coding sequences of 80 new genes (KIAA0201-KIAA0280) deduced by analysis of cDNA clones from cell line KG-1 and brain. DNA Res 1996, 3:321-329. 39. McKearin D, Ohlstein B: A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells. Development 1995, 121:293?-2947. 40. Ohlstein B, McKearin D: Ectopic expression of the Drosophila Bam • protein eliminates oogenic germline stem cells. Development 1997, 124:3651-3662. A nice demonstration that heat-shock-induced ectopic bam expression converts germline stem cells to cystoblasts. 41. Gonczy P, Matunis E, DiNardo S: bag-of-marble and benign genial • cell neoplasm act in the germ line to restrict proliferation during Drosophila spermatogenesis. Development 1997, 124:4361-4371. This paper shows that barn and bgcn act cell-autonomously in the male germline to restrict the mitotic potential of the differentiated daughter cells of germline stem cells. 42. McKearin DM, Spradling AC: Bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis. Genes Dev 1990, 4:2242-2251. 43. Saxena R, Brown LG, Hawkins T, Alagappan RK, Skaletsky H, Reeve MP, Reijo R, Rozen S, Dinulos MB, Disteche CM, Page DC: The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned. Nat Genet 1996,14:292-299. 44. Eberhart CG, Maines JZ, Wasserman SA: Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia. Nature 1996, 381:783-785. 45. Menke DB, Mutter GL, Page DC: Expression of DAZ, an • azoospermia factor candidate, in human spermatogonia. Am J Hum Genet 1997, 60:237-241. This paper shows that DAZ is expressed mostly in spermatogonia in human testes. 46. Reijo R, Seligman J, Dinulos MB, Jaffe T, Brown LG, Disteche CM, Page DC: Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics 1996, 35:346-352. 47. Cooke HJ, Lee M, Kerr S, Ruggiu M: A murine homologue of the human DAZgene is autosomal and expressed only in male and female gonads, Hum Mo/Genet 1996, 5:513-516. 48. Seligman J, Page DC: The Dazh gene is expressed in male and female embryonic gonads before germ cell sex differentiation. Biochem Biophy Res Corn 1998, 245:878-882. 49. Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F, Saunders P, *Dorin J, Cooke HJ: The mouse Dalza gene encodes a cytoplasmic protein essential for gametogenesis. Nature 199'7, 389:73-77. This is the first demonstration, by knockout experiments, that the Dazl gene is essential for the maintenance of gonoeytes, the precursor of active spermatogonia, in the embryonic testis in mice. It also shows that DAZL is a cytoplasmic protein present at low level in the spermatogonia of the adult testis and at high level in spermatocytes. 50. Strome S: Determination of cleavage planes. Ceil 1993, 72:3-6. 51. Amon A: Mother and daughter are doing fine: asymmetric cell division in yeast. Ceil 1996, 84:651-654. 52. Guo S, Kemphues KJ: Molecular genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo. Curt Opin Genet Dev 1996, 6:408-415.
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53. Lin H, Yue L, Spradling AS: The Drosophila fusome, a germline-specific organelle, contains membrane skeletal proteins and functions in cyst formation. Development 1994, 120:947-956.
a somatically-derived signal which restricts germ cell proliferation. Development 1997, 124:4361-4371. An elegant genetic clonal analysis on the role of the dpp signaling pathway in spermatogenesis.
54. Lin H, Spradling A: Fusome asymmetry and oocyte determination. Dev Genet 1995, 16:6-12.
63.
55. de Cuevas M, Lee J, Spradling AC: a-spectrin is required for germline cell division and differentiation in the Drosophila ovary. Development 1996, 122:3959-3966. 56. McGrail M, Hays TS: The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila. Development f 997, 124:2409-2419. 57. de Cuevas M, Spradling AC: Morphogenesis of the Drosophila • fusome and its implications for oocyte specification. Development
1998,125:2781-2769. An interesting description of spectrosome regeneration at the end of germline stem cell division and fusome growth during germline cyst formation. 56. Gregorio CC, Kubo RT, Bankert RB, Repasky F_A:Translocation of spectrin and protein kinase C to a cytoplasmic aggregate upon lymphocyte activation. Proc Nat/Acad Sci USA 1992, 89:494? 4951. 59. Johnson E, Wayne S, Nagoshi R: fs(1)Yb is required for ovary follicle cell differentiation in Drosophila melanogaster and has genetic interactions with the Notch group of neurogenic genes. Genetics 1995, 140:207-217. 60. Xie T, Spradling AC: decepentaplegic is essential for the ee maintenance and division of germline stem cells in the Drosophila ovary. Ceil 1998, 94:251-260. An elegant demonstration that the dpp signaling pathway is involved in Drosophila germline stem cell division and maintenance. 61. Zhao GQ, Deng K, Labosky PA, Liaw L, Hogan BL: The gene encoding bone morphogeneUc protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse. Genes Dev 1996, 10:1657-1669, 62. Matunis E, Tran J, Gonczy P, DiNardo S: punt and schnurri control • progression through the germ line stem cell lineage by regulating
Zhao GQ, Hogan BLM: Evidence that mother-against-dpp-related 1 (madrl) plays a role in the initiation and maintenance of spermatogenesis in the mouse. Mech Dev 1997, 61:63-73. Describes the cloning of a murine homolog of the Drosophila mad gene and the examination of its expression pattern during embryogenesis and spermatogenesis.
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64. Kimble J, Simpson P: The UN-12/Notch signaling pathway and its regulation. Ann Rev CeU Dev Bio11997, 13:333-361. 65. Kopan R, Turner DL: The Notch pathway - democracy and aristocracy during the selection of cell fate. Curr Opin Neurobiol 1996, 6:594-601. 66. Kadyk LC, Kimble J: Genetic regulation of entry into meiosis in oe '" Caenorhabdifis elegans. Development 1998, 125:1803-1813. A genetic study that identified gld-2 as a gene required for the normal progression of meiosis and that suggests gld-1 and gld-2 act in two independent pathways, each of which is sufficient for entry into meiosis. This study also showed that gld-1 and gld-2 are negatively controlled by the glp-1 signaling pathway. 67.
Ruohota H, Bremer KA, Baker D, Swedlow JR, Jan LY, Jan YN: Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila. Cell 1991, 66:433-449.
68. Xu T, Caron I_A, Fehon RG, Artavanis-TsakonasS: The involvement of the Notch locus in Drosophila oogenesis. Development 1992, 115:913-922. 69. ForbesA J, Lin H, Ingharn PW, Spradling AC: hedgehog is required for the proliferation and specification of ovarian somatic cells prior to egg chamber formation in Drosophila. Development 1996, 122:1125-1135. 70. Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H: A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev, 1998, in press.
The self-renewing mechanism of stem cells in the germline Haifan Lin Germline stem cells (GSCs) are a self-renewing population of germ cells that serve as the source of gametes in diverse organisms. Current research suggests that the self-renewing division of GSCs is controlled both by somatic signaling and by intracellular mechanisms such as differential gene expression, asymmetric cytoskeletal organization, and the cell cycle machinery. These findings provide a framework for the further study of GSCs and stem cell renewal in general. Addresses Department of Cell Biology, Box 3709, Duke UniversityMedical Center, Durham, NC 27710; e-mail:[email protected] Current Opinion in Cell Biology 1998, 10:687-693 http://biomednet.com/elecref/0955067401000687 © Current BiologyLtd ISSN 0955-0674 Abbreviations CDK cyclin-dependentkinase DAZ deletedin azoospermia dpc days post coitum DPP decapentaplegic DTC distal tip cell GSC germlinestem cell mad mothersagainst dpp Med Medea TF terminalfilament Introduction
Central to gametogenesis in m a n y organisms are germline stem cells (GSCs), which serve as a source for the continuous production of gametes. A striking feature of GSCs, very much like any other stem cells, is their ability to self-renew while remaining capable of generating n u m e r o u s differentiated daughter cells. T h e existence of stem cells in the germline was proposed over a century ago [1]. Decades of research on spermatogenesis in mammalian systems has yielded a wealth of cytological information as well as interesting hypotheses pertinent to GSC division (reviewed in [2,3]). Recent genetic and molecular analyses in Caenorhabditis elegans, Drosophila, and the mouse have provided further insight into the mechanisms underlying GSC division and maintenance. This article reviews the latest progress in understanding GSC division, with emphasis on Drosophila since its study represents most of the progress in the past two years. For a more comprehensive review on GSCs, please refer to [4] (see also de Rooij and Grootegoed, this issue pp 694-701). S t e m c e l l s e x i s t in d i v e r s e f o r m s in t h e g e r m l i n e
Stem cells possess two fundamental properties: the ability to self-renew and the ability to produce numerous differentiated progeny. According to this definition, stem cells are present in the germline of diverse organisms, including hydrazoan polyps [5,6], nematodes [7], arthropods [8-11],
amphibians [12], birds [13], fishes [14], reptiles [12], and mammals [15,16]. Notably, the ovaries of some vertebrates, especially that of mammals, do not contain self-renewing GSCs [17]. GSCs can be classified into two types according to how they self-renew. T h e first type, herein called stereotypic GSCs, self-renew strictly by asymmetric divisions that produce a daughter GSC and a differentiated daughter cell. For example in the Drosophila ovary each GSC division produces a daughter GSC that remains in contact with a cluster of apical somatic cells called the terminal filament (TF), and a cystoblast that is one cell away from the T F [18,19 °°] (Figure 1). Laser ablation of the GSCs ceases the production of new egg chambers revealing them as the exclusive source for germline renewal [11]. Stereotypic GSCs also exist in the Drosophila testis and probably also in vertebrate testis [13-16,20-22]. In the Drosophila testis, between five and nine GSCs divide asymmetrically in a defined orientation with respect to a group of nonmitotic somatic hub cells, in a similar fashion to the division of ovarian GSCs [20,22]. In mammalian testes, various hypotheses have been proposed to describe the exact identity of GSCs and their pattern of division, most of which point to a subset of spermatogonia (Type A or a subtype of Type A) as GSCs (reviewed in [4]). Despite this, it has not been unequivocally shown whether GSCs in the mammalian testis divide asymmetrically like those in Drosophila or alternatively divide symmetrically and should be classified as the second type of GSCs (see below). T h e second type of GSCs, herein called populational GSCs, divide symmetrically to produce two daughter cells each of which has an equal probability of differentiating. T h e y selfperpetuate only at the populational level whereby the collective behavior of mitotic cells in the gonad maintains a steady source of germline cells. For example, in C. elegans, the mitotically active germline nuclei share a common cytoplasm at the distal end of the gonad and serve as a stem cell population for gametogenesis throughout adulthood by virtue of their proximity to a somatic signaling cell, the distal tip cell (DTC) [23,24]). Laser ablation of between five and ten of the most distal germ cells in third larval stage gonads does not eliminate the proliferative germ cell population [25°]. It is possible that these nuclei show a gradient of mitotic ability, with those closest to the D T C being most mitotically active and those more than 20 nuclei away eventually entering meiosis to become terminally differentiated. Populational GSCs may also exist in Hydra oligactis [5] and Hydra magnipapillata [6]. T h e known mechanism for the self-renewal of populational GSCs differs from that of stereotypic GSCs (see below). At present, it is too early to determine whether this difference is significant.
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Cell differentiation
Figure 1 (a)
F--I
(b)
TF ~ l l
Germarium
Egg chambers
Oviduct ~
Current Opinion in Cell Biology
Intracellular mechanisms underlying asymmetric germline stem cell division Current studies in C. elegans, Drosophila, and mammals indicate that GSC divisions are controlled by both intracellular mechanisms and cell-cell interactions (reviewed in [4]). For stereotypic GSCs, intracellular mechanisms should include both a basic cell-cycle machinery as well as a mechanism for generating divisional asymmetry. This asymmetric mechanism should in turn further consist of asymmetrically-segregated cell-fate regulators and a cellular machinery responsible for their asymmetric segregation or for establishing an asymmetric exposure of the two daughter cells to extrinsic signals.
Cell cycle machinery T h e role of celt cycle regulators in controlling GSC division has not been systematically analyzed. Recent studies are starting to reveal the expression patterns of these molecules in GSCs. In mice, the G 1 cyclins D1, D2, and D3, the G z cyclin A2, as well as CDK4, and Wee-1 are expressed in spermatogonia, with some of them also expressed in later stages of spermatogenesis [26-30]. Cdc25A is also weakly expressed in the spermatogonia. T h e PCNA (proliferating cell nuclear antigen) protein, involved in DNA synthesis and repair, is detected in proliferating spermatogonia but not in meiotic spermatocytes [26]. In Drosophila, R O U G H E X and cyclin A regulate the normal progression of meiosis while
The asymmetric division of germline stem cells (GSC) in the Drosophila ovary (modified from [4]). (a) An ovariole with a string of developing egg chambers produced by germarium. (b) A magnified view of the apical region of the germarium (boxed area in [a]). There are usually two GSCs (gsc) in the germarium, in contact with apical somatic cells expressing YB, PlWl and HEDGEHOG (HH). Boldface type indicates a high concentration of the named protein. Although YB- and PlWlmediated signalling are required for GSC maintenance, HH is only known to regulate the division and differentiation of somatic stem cells (ssc) two to five cells away from the terminal filament (TF) [6g]. In GSCs, spectrosomes (shaded spheres) containing spectrin (Sp) and HULITAISHAO (Hts) proteins, reside in the apical region of the cytoplasm both at interphase and during mitosis, apposed to the signaling somatic cells. During mitosis, the spectrosome anchors one pole of the spindle so that the divisional plane is approximately perpendicular to the apico-basal axis of the germarium. As a result, the daughter GSC remains in contact with the TF while the cystoblast (cb) becomes one cell away from the somatic cells. The PUM protein is present at a high level in GSCs and at a low level in cystoblasts and cysts, NOS protein shows the opposite distribution profile, while the cytoplasmic form of BAM is only present in cystoblasts and cysts. The source of DPP has not been identified.
cyclin E controls the nurse cell endocycle. Their role in GSC division, however, has not been addressed [31,32]. Similarly, in C. elegans, a subset of cyclins are mostly expressed in the germline [33]. These expression patterns provide a basis for functional analysis of these molecules in GSC division. Asymmetrically-segregated cell-fate regulator
Asymmetrically-segregating cell-fate determinants, such as N U M B and P R O S P E R O in Drosophila neuroblasts (reviewed in [18]), have not been identified in GSCs. But three Drosophila genes have recently been shown to be required cell-autonomously for either the maintenance of female GSCs or the proper differentiation of cystoblasts. Among them, pumilio (pure) is essential for GSC maintenance. Females with pure mutations contain normal numbers of GSCs at the onset of oogenesis but fail to maintain them [34°°,35 °] and the often appear to differentiate. Since PUM is a R N A binding protein that mediates translational repression in the embryo [36,37] and that it is present at a high level in GSCs but a low level in cystoblasts [35 °] (Figure 1), it is tempting to speculate that PUM is an asymmetrically-expressed cellfate regulator that selectively suppresses the translation of certain RNAs in the GSC to prevent differentiation. A pum homolog has been identified in mammals [38], suggesting that a pum-like m e c h a n i s m may exist in mammalian GSCs as well.
The self-renewing mechanism of stem cells in the germline Lin 689
In addition to pure, nanos (nos) may be involved in GSC maintenance since in flies with nos mutations 50% of the newly eclosed adult ovaries are germlineless [35°]. This defect, however, could also be due to an earlier requirement of nos for germline development prior to oogenesis. During oogenesis, nos also appears to function in the differentiation of cystoblasts and germline cysts, the interconnected daughters of cystoblasts, since nos mutations block germline cyst development in adult ovaries without the immediate loss of GSCs [35°]. Consistent with this second role, NOS is abundant in cysts but is barely detectable in GSCs and cystoblasts (Figure 1). This difference in the protein profile between NOS and PUM as well as the difference in their GSC phenotype suggest that NOS and PUM may not interact during early oogenesis as they do in embryos. Complementary to the role of pure, bag of marbles (barn) is required cell-autonomously to switch germ cells from GSC fate to a differentiating daughter fate in both males and females [39,40,41°]. Germ cells with barn mutations fail to differentiate, but instead proliferate like stem cells or ill-differentiated germline cells [39,41",42]. Heat-shockinduced ectopic expression of barn is sufficient to extinguish GSC divisions [40"]. Differential accumulation of barn m R N A and proteins in cystoblast versus GSCs [39,42] suggest, that BAM could be a differentially expressed cell-autonomous regulator for the cystoblast fate. In mammals, a promising candidate for an intrinsic factor required for GSC self-renewal is the human deleted in azoospermia (DAZ) gene cluster in the azoospermiafactor (AZF) region on the Y chromosome [43]. DAZ, a candidate for the AZF gene, encodes a putative RNA binding protein with strong homology to the Drosophila B O U L E which is protein required for meiosis during spermatogenesis [44]. DAZ is expressed specifically in the germ cells of the adult human testis and most abundantly in spermatogonia [45"]. T h e mouse homolog of DAZ, Dazh (also known as Dazla), is also expressed in germ cells in embryonic gonads before germ cell sex differentiation, starting at 12.5 days post coitum (dpc) [46--48]. T h e Dazh level decreases in female embryos following the entry of oogonia into meiosis but persists in the male gonad and is present in day 1 neonatal male mice whose germ cells are gonocytes, the immediate precursors of spermatogonia. Subsequently, Dazh expression increases as spermatogonial stem cells appear, reaches a peak as spermatogenic cells first enter meiosis and persists at this level thereafter [46]. hnportant information on Dazh function comes from the analysis of Dazh-knockout mice, whose embryonic gonads are normal up to 15 dpc, but by 19 dpc germ cells in seminiferous tubules (and in ovaries as well) are significantly reduced in both heterozygous and homozygous Dazh- mice [49"]. This result suggests that Dazh is required quantitatively for the initial maintenance of gonocytes which are either mitotically arrested GSCs or the immediate precursors of GSCs [4].
Cellular machinery for asymmetric localization/orientation
It is well established that cytoskeletal systems play a crucial role in the localization and segregation of cytoplasmic components and in spindle orientation during mitosis [18,50-52]. Recent work on Drosophila ovarian GSCs has revealed the importance of the cytoskeleton in the asymmetric GSC divisions [19"°,34",53-56]. In the Drosophila ovary, the asymmetry of GSC division is cytologically manifested both by the relative position of the two daughter cells to the "FF and by the differential segregation of a cytoplasmic structure called the spectrosome [53,54,57"] (Figure 1). The spectrosome resides in the apical region of the GSC and contains membrane vesicles and membrane skeletal proteins such as ~- and 13-spectrin, ankyrin, and the adducin-like H U L I T A I S H A O (HTS) protein [53-55]. It also contains regulatory molecules such as a BAM isoform and cyclin A ([39]; M Lilly, M de Cuevas, AC Spradling, personal communication) and is associated with a centrosome [53]. During mitosis, the spectrosome is associated with the pole of the mitotic spindle located near basal T F cells ([19"',34")1; Figure lb). At telophase, it grows in size and elongates towards the future cystoblast and eventually becomes asymmetrically bisected by delayed cytokinesis occurring at the next cell cycle [19"',34",57"]. Delayed cytokinesis may be a common feature of GSC division, since it is also seen during Drosophila and mammalian spermatogonial GSC divisions. T h e spectrosome may have a dual function in establishing the asymmetry of GSC division. First, it anchors the mitotic spindle to define the orientation of GSC division so that, during each division, one daughter cell will remain in contact with the T F and may thus remain as a GSC while the other daughter will become one cell away from the signal and may thus differentiate. This function can be inferred from the observations that spectrosome disassembly causes randomized spindle orientation and that germline cysts fail to develop properly in the absence of the spectrosome and its derivative structure, the fusome [19°°]. Second, the spectrosome may localize molecules important for stem cell fate as a means of selectively retaining them in the daughter GSC. This localization function is suggested by the ability of the spcctrosome to localize a BAM isoform and cyclin A (see above) and the ability of spectrosomelike structures in mammalian cells to localize protein kinase C [58]. Despite the above two potential functions, the spectrosome, however, does not regulate the rate of GSC division [34"]. It is important to point out that, even if the spectrosome is essential for the self-renewing asymmetric division of GSCs, such division may not be essential for GSC maintenance in the Drosophila ovary. In the absence of the spectrosome and fusome, a normal number of egg chambers, though ill-differentiated, are still produced [34"']. This possibly reflects the fact that, although GSC divisions are now randomly oriented, the topology of the germarium
690 Celldifferentiation
still allows about 50% of the daughter cells to contact the T E Consequently, a self-renewing population of germ cells is still maintained, even though cystoblasts may not properly develop possibly due to the failure to localize cellfate regulators in the spectrosome. If this hypothesis is true, the functional significance of the spectrosome mechanism is to convert populational GSCs to stereotypic GSCs. Nevertheless, studies on the spectrosome have revealed the critical role of membrane skeletal proteins in establishing a divisional asymmetry which may be important in other stem cell systems. What then links the spindle pole to the spectrosome? What orients the spectrosome towards the TF? A recent study suggests that the spindle-spectrosome linkage is mediated by cytoplasmic dynein [56]. In germline cysts, cytoplasmic dynein is required to link one pole of the mitotic spindle to the fusome. Given the structural similarities between the fusome and the spectrosome, it is likely that cytoplasmic dynein plays the same role in GSCs.
Signaling mechanisms controlling asymmetric GSC division Extrinsic signaling has long been implicated in regulating stem cell division, even though relatively little is known about its specific role in GSC division (reviewed in [4]). Extrinsic signaling is mediated by hormones, growth factors, cytokines, and short-range cell-cell signaling pathways such as those involving Steel and c-kit in mammals or leg-2 and glp-1 in C. elegans. These cell-cell interactions have been extensively reviewed [4]. T h e past two years have witnessed exciting progress in the genetic dissection of the signaling mechanisms that regulate GSC regulation in Drosophila, which will be the focus of this section. In Drosophila, the role of somatic cells in regulating GSC division was first indicated by the laser ablation of the apical portion of the terminal filament, which increased the rate of GSC division [11]. Although the partial ablation did not address other functions of the T F in regulating GSC division, such functions have been revealed by the genetic analysis of piwi and fs(1)Yb (Yb) genes [34°°]; J King, L Chang, H Lin, unpublished data). Loss-of-function mutations in piwi or Yb cause the failure of GSC maintenance. In flies with mutations in piwi or Yb GSCs often differentiate into germline cysts without divisions. Interestingly, Yb is specifically expressed in the T F (J King, Lin H, unpublished data); removing Yb function in the germline does not affect GSC division ([59];J King, H Lin, unpublished data). PIWI is expressed in both the T F and in the germline; removing piwi from the germline does not affect GSC division (D Cox, H Lin, unpublished data). Thus, the study of Yb and piwi reveals the essential role of a TF-mediated somatic signaling mechanism in regulating GSC division. T h e Yb-mediated signaling may be female-specific since severe loss-of-function Yb mutations only affect female GSC maintenance, while piwai-mediated signaling is required for both male and
female GSCs. Yb and piwi encode novel proteins. T h e PIWI-like proteins are found from C. elegans to humans and to Arabidopsis with apparently conserved functions. Further molecular analysis of Yb and piwi and their interacting genes should lead to the identification of molecular pathways involved in this somatic signaling. Although the molecular nature of somatic signaling is currently unknown, a recent study revealed the essential role of the Drosophila homolog of BMP2/4 decapentaplegic(dpp) signaling pathway in GSC division and maintenance [60"°]. Loss of dpp function causes Yb-like GSC maintenance defects, while over-expressing dpp produces GSC tumors. Genetic clonal analyses of the receptors and downstream transducers of the DPP signal, such as punt (encoding a type II receptor) and mad/Med(encodinga dimeric transcription activator), shows that the DPP signal is directly received by GSCs. It is possible that the DPP signal emanates from the surrounding somatic cells. Alternatively, DPP could be produced as a paracrine factor in differentiated germline cells or even as an autocrine factor in GSCs. Indeed, in mouse testis BMP8b, a DPP-like molecule, is expressed only in germline cells, including spermatogonia, for the initiation and maintenance of spermatogenesis [61]. It will be interesting to see if Drosophila dpp expression is germline- or soma-dependent and whether DPP homologs BMP2/4 also play a role in regulating GSC division in mammals. T h e dpp signaling pathway appears to play a different role in the Drosophila testis [62°]. First, the dpp signaling in the testis is not required for GSC division or maintenance, but restricts the proliferation of the differentiated daughter. Second, the receptors and downstream transducers of the dpp signal are not required in the germline but in the somatic cyst cells that flank each GSC and its differentiated daughter cells. T h e loss of punt and schnurti (shn) function in the somatic cyst cells results in overproliferation of the differentiated daughter of the GSC, where shn encodes a zinc finger transcription factor (a MBP1/2 homolog) that acts downstream of the dpp signal to regulate the expression of dpp-responsive genes. T h e dpp-equivalent signaling pathway is also involved in mammalian spermatogenesis, but again with a different function. For example, raadr-1, a mouse homolog of the Drosophila mad gene, is expressed mainly in the pachytene spermatocytes and round spermatids. In Bmp8b homozygous mutants, the madr-l-expressing pachytene spermatocytes are the first cells to show increased apoptosis. T h e s e data suggest that MADR1 serves as a downstream component of the BMP8b signaling pathway during the differentiation of meiotic male germ cells [63°]. Madr-2, on the other hand, is present mainly in spermatogonia and early meiotic cells and Sertoli cells [61], and thus may be involved in GSC division. In addition to the dpp signaling pathway, it has been long established that lag-2/glp-1 signaling pathway plays a key
The self-renewing mechanism of stem cells in the germline Lin
role in germline maintenance in C. elegans, where the LAG-2 somatic signal on the surface of the D T C interacts directly with the GLP-1 receptor (reviewed in [64]). LAG-2 is a transmembrane protein homologous to Delta (DI) and Serrate (Ser) in Drosophila, while GLP-1 is homologous to Notch, the receptor of DI and Ser. T h e GLP-1 mediated signal is transduced in the germ cells through interaction with the LAG-1 protein, a homolog of the Drosophila DNA binding protein Suppressor of Hairless (Su(H)) and the mammalian DNA binding protein CBF1, which are downstream interactors of the Notch protein in Drosophila and mammals, respectively [65]. Recent work further suggests that lag-1 maintains a mitotic population of germ nuclei by suppressing the function ofgld-I and gld-2 which promotes meiosis [66"]. Thus, g/d-1 and gld-2 may be a key differentiation factor in the C. elegans germline. Although the lag-2/glp-1 signaling pathway is conserved between C. e/egans and Drosophila, it appears to have no obvious function in regulating Drosophila GSC division [67,68]. This dichotomy again reflects the divergence of the signaling mechanisms employed by different GSC systems.
Conclusion Recent work on GSCs has clearly revealed both intracellular and intercellular mechanisms for GSC maintenance. Some important intrinsic factors and cell-cell signaling molecules have been identified, and their regulatory relationship has begun to be explored in the context of gene expression, cell cycle progression, cytoskeletal organization, and mitotic behavior of GSCs. Current research supports the idea that the formation and the self-renewing division of GSCs are largely under the control of extrinsic signals which regulate the mitotic and differential segregation mechanisms inside the stern cells. Elucidation of the mechanisms for the maintenance of GSCs is now occurring at an exciting rate. New genetic screens will identify more genes required for GSC division and maintenance. Meanwhile, further analysis of known GSC mechanisms will reveal exactly how cell-cell signaling and intracellular events occur in an orchestrated manner to ensure the stem cell property. These genetic and molecular analyses will also reveal how GSC division and maintenance are related to general cell cycle and mitotic mechanisms. T h e knowledge gained from these studies should provide valuable insight not only into the GSC division mechanism but also the mechanisms of stem cell division and germline development in general.
Note a d d e d in proof A recent paper [70] shows that the Drosophiliapiwi gene is required for the assymetric division of GSCs to produce and maintain a daughter GSC but it is not essential for the further differentiation of the cystoblast. It also describes genetic mosaic and RNA in situ analyses which suggest that piwi expression in adjacent somatic cells regulates GSC division . In addition, this paper reports the cloning of Drosophilia piwi and its homologs in C. elegans and
human
as
well
as
the
identification
of
691
two
Arabidopsispiwi-like genes known to be required for meristern cell maintenance. Decreasing C. elegans piwi expression reduces the proliferation of GSC-equivalent cells. Thus, piwi represents a novel class of genes required for GSC division in diverse organisms.
Acknowledgements I thank D McKearin, C Berg, A Chao, A Szackmary, W Deng, and T Srinivsan for critical reading of the manuscript. This work was supported by a National Institutes of Hcahh grant (R01HD33760), a David and Lucile Packard Fellowship, an American Cancer Society Junior Faculty Research Award and a March of Dimes Basil O'Connor Award.
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