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Germline Stem Cells: Origin and Destiny

Ruth Lehmann1,2,* 1Howard Hughes Medical Institute 2Skirball Institute, The Helen L. and Martin S. Kimmel Center for Stem Cell Biology, Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA *Correspondence: [email protected] DOI 10.1016/j.stem.2012.05.016

Germline stem cells are key to transmission to future generations. Over recent years, there have been numerous insights into the regulatory mechanisms that govern both specification and the maintenance of the germline in adults. Complex regulatory interactions with both the niche and the environ- ment modulate germline stem cell function. This perspective highlights some examples of this regulation to illustrate the diversity and complexity of the mechanisms involved.

Germ cells have a unique function in the body. They are not by globally repressing RNA Polymerase activity or by more needed for survival or immediate physiological function of the specifically blocking the somatic program. In C. elegans and individual, but rather, are endowed with the ability to contribute Drosophila, PGCs are the first cells separated from the somatic to the next generation. However, while they are ultimately able lineage. In these species, PGCs inherit maternally synthesized to generate all cells in the body, germ cells are unipotent and their germ plasm that is rich in mRNA and RNA binding normal fate is restricted to or . This review will focus on (RBPs). The newly formed PGCs rely entirely on these maternally recent developments in our understanding of how primordial provided factors, since transcription is completely blocked, germ cells (PGCs) are specified during embryogenesis, and how thereby preventing the expression of the zygotic genome. In different strategies, including germline stem cell (GSC) renewal, Drosophila, repression of transcription is achieved by germ- soma-germline interactions, and physiological signals, are used cell-specific translation of a 71 amino acid peptide encoded by to maintain a pool of differentiating, meiotic germ cells in adults. the polar granule component (pgc) gene (Hanyu-Nakamura et al., 2008). Pgc inhibits the recruitment of the Positive Germ Cell Specification Transcription Elongation factor-b (P-TEFb) kinase complex to In most organisms, PGCs are set aside early during embryogen- transcription sites. P-TEFb phosphorylates Ser-2-containing esis. Two distinct mechanisms have been identified in model motifs in the carboxyl-terminal domain (CTD) repeats of RNA organisms that specify germ cells. In flies, worms, fish, and frogs, Polymerase II (RNA PolII) and is responsible for transcriptional maternally synthesized germ plasm is deposited into the egg elongation. Pgc protein is only present in germ cells for a short during . This specialized cytoplasm harbors germline- time and transcriptional activity is observed as soon as germ cells specific, electron-dense RNA-protein particles required for initiate their migration toward the somatic part of the . multiple aspects of germ cell fate. Embryonic nuclei inheriting Robust, high levels of transcription are achieved once germ cells germ plasm are destined to become PGCs. In contrast, in reach the somatic . In C. elegans, transcription in the early mammals and many other species traversing all animal phyla, germline is also repressed via interference with RNA PolII activity, germ cells are specified among the cells of the embryo indepen- but by slightly different mechanisms. During the first two embry- dent of preexisting maternal information. Specification occurs in onic divisions, the Zn-finger proteins Oma1 and Oma2 sequester some species early during development, when germ cell fate is TATA-binding protein associated factor 4 (TAF-4), a subunit of induced in a subset of otherwise pluripotent progenitors cells TFIID required for assembly of the RNA PolII preinitiation such as cells of the early mouse embryo, and in other complex, in the cytoplasm, thereby preventing transcription in species at later embryonic and postembryonic stages, when the entire early embryo (Guven-Ozkan et al., 2008). Subsequently, germ cells can originate from multipotent progenitors within the Pie-1, a CCCH Zn-finger protein, is specifically translated in the mesoderm. Irrespective of these apparent differences in how the germ cell precursors and inhibits transcriptional initiation and germ cell lineage becomes distinct from somatic cells, conserved elongation by interfering with Ser 5 and Ser 2 phosphorylation, molecular principles have emerged (Cinalli et al., 2008). Among respectively, in RNA PolII CTD motifs (Ghosh and Seydoux, 2008). these, global epigenetic regulation of germline gene expression In the mouse, germ cell specification occurs in the proximal and germ-cell-specific posttranscriptional regulation are essential epiblast just prior to gastrulation. A BMP4/8 signal from the both for specifying, maintaining, and protecting the germline extraembryonic initiates the germ-cell-specific pro- during its life cycle and for ensuring transgenerational success. gram by activating the repressor protein Blimp1 (also known as Prdm1)(Lawson et al., 1999; Ohinata et al., 2005; Ying et al., PGC Specification Requires Suppression of the Somatic 2001). Not all cells that receive the BMP signal become PGCs. Program Expression is restricted to the posterior epiblast by antagonizing Transcriptional repression seems to be a necessary component signals from the anterior visceral endoderm and is enhanced by of germline specification (Nakamura and Seydoux, 2008). Lin28, which counteracts let-7 miRNA, a Blimp1 suppressor Depending on the organism this repression is achieved either (Ohinata et al., 2009; West et al., 2009). Blimp1 is responsible

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for the repression of almost all of the genes that are downregu- conversion could be the first step toward demethylation lated in PGCs, but is only required for the activation of some of (He et al., 2011; Ito et al., 2010, 2011; Tahiliani et al., 2009). the genes that are upregulated in germ cells (Kurimoto et al., Subsequent studies have shown that 5hmC marks are enriched 2008). How expression of Stella, Kit, Dnd1/Ter, and Nanos3, in ESCs and are found in the promoter regions of totipotency which play an important part in the specification, migration, genes that are also active in germ cells (Ficz et al., 2011). The and survival of early germ cells, is activated in germ cells is not next steps toward complete demethylation are still unclear, fully understood. Beyond repression of the somatic program, and may involve Tet-mediated oxidation, base excision repair, Blimp1, together with another PR domain protein, Prdm14, and/or passive, replication-dependent demethylation (Hackett also promotes the epigenetic within early germ et al., 2012). cells to regain pluripotency (Yamaji et al., 2008). Indeed, PGCs DNA modifications and their regulation play a less prominent reactivate the pluripotency genes Nanog, Sox2, and Oct4. This role in fly and worm germ cells, but modification of chromatin- finding together with the observation that early PGCs can easily associated histones has important functions in maintaining the give rise to pluripotent embryonic stem cells (called ‘‘EG’’ cells) germ cell fate in later stages of . In led to the hypothesis that PGCs resemble earlier epiblast cells particular, several recent results argue that global chromatin (Matsui et al., 1992). Indeed, recent evidence using activation regulation has a direct impact on the germline-soma fate of a Blimp1 reporter argues that ESC cultures may originate decision. For example, in the synthetic multivulva from PGCs (Chu et al., 2011). However, because Blimp1 activity (synMuv) genes, the worm homologs of the mammalian is not required for ESC derivation, the general implication of proteins retinoblastoma (RB), E2F, HP1, and NuRD complex, these findings remains uncertain. Once PGCs complete their lead to transformation of intestinal cells into germline-like cells migration to the genital ridge and associate with the somatic (Petrella et al., 2011). This complex interacts with l(3)mbt, part of the gonad, they lose the ability to easily revert to the a gene that causes malignant brain tumors when mutated in pluripotent state and reach a germ-cell-specific epigenetic the fly (Lewis et al., 2004). Interestingly, and in contrast to program (Hayashi and Surani, 2009). mutations in other brain tumor genes, l(3)mbt mutant tumor In an effort to reveal the principles of germ cell specification, cells express genes normally restricted to the germline (Janic Hayashi et al. (2011) recently developed a protocol to derive et al., 2010). Chromatin association studies indicate that l(3) germ cells from ESCs and iPSCs. In a stepwise process, plurip- MBT protein binds preferentially to insulator regions and otent stem cells were first cultured into cells resembling pregas- suggest a role for l(3)MBT in global repression of germline trulating epiblast cells, from which in turn PGC-like cells were genes in specific somatic tissues (Richter et al., 2011). Chro- derived. Cells with PGC-like characteristics were able to matin modifiers can also coax the germline into expressing produce functional sperm after being injected into the seminif- somatic programs. Expression of specific neural transcription erous tubules of germ-cell-deprived ( mutant) mice. The effi- factors converts C. elegans germline cells into specific types ciency was similar to that of transplantation of in-vivo-derived of terminally differentiated neurons. This conversion requires PGCs. These new culture conditions will be critical for devel- the removal of the conserved chromatin remodeling protein oping a more complete understanding of germ cell development LIN-53, another component of the synMuv family (Tursun and have a clear application for . et al., 2011). Curiously, germline-to-soma and soma-to-germ- line transformations involve mutations in the same class of A Special Chromatin Program Is Established genes normally associated with a repressive chromatin state. in Germ Cells However, transformation of germline into soma requires Early embryonic germ cells in the mouse are transcriptionally tissue-specific transcription factors, while such factors have active and express the CTD phospho-epitopes characteristic not been identified for soma-to-germline transformations or of active RNA PolII transcription. These marks are no longer for normal germline development. Together these findings point detected during the migration of germ cells to the somatic gonad to the important role of global transcriptional and epigenetic (between E8 and E9) (Seki et al., 2007). In the gonad, epigenetic regulation in the germline. Indeed, evidence is mounting that reprogramming takes place, including global DNA demethy- epigenetic memory in the germline contributes not only to lation, exchange of histone variants, large-scale chromatin germline development but also to patterns of inheritance for remodeling of retrotransposon-linked and imprinted genes, generations to come (Greer et al., 2011; Katz et al., 2009). and reactivation of both X chromosomes (Hajkova et al., 2008). This remodeling is critical for resetting imprint marks and has Conserved RNA Regulators Are Associated with Germ also been proposed to play an important role in the activation Cells throughout the Life Cycle of genes required for early embryonic development in the next While so far no specific, conserved transcriptional regulator for generation (Hayashi and Surani, 2009). For a long time it was germ cell fate has been identified, RBPs play important roles unclear whether DNA demethylation was achieved passively throughout germ cell development. Germline RBPs prevent as a consequence of replication and lack of maintenance somatic differentiation and control many germ cell functions methylation, or whether there were enzymes that actively such as specification of germ cell fate and sexual identity, prolif- removed methylation marks from DNA. Recent findings show eration, survival, migration, and regulation of transposable that the Tet (ten eleven translocation) family of proteins can element activity. RBP families such as Vasa, Nanos, Pumilio, catalyze the conversion of the 5-methylcytosine (5mC) base Dnd1, DAZL, PIWI, and Tudor are broadly conserved from (the methylation mark mostly associated with inactive genes) planarians to humans and are generally found during multiple to 5-hydroxymethylcytosine (5hmC), suggesting that this stages of the germ cell life cycle (Juliano et al., 2010). These

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Figure 1. Drosophila Ovarian Niche (A) Drawing of the ovarian GSC compartment. Each ovary is subdivided into 12–16 ovarioles that generate chains of maturing egg chambers. The niche located at the tip of each ovariole contains three different somatic cell types (terminal filament cells, cap cells, and escort or inner germarial sheath [IGS] cells) and two or three GSCs. Asymmetric division of a GSC perpendicular to the niche (arrow) generates a new GSC and a differentiating daughter that exits the niche. The differentiating daughter, called the cystoblast, subsequently undergoes four rounds of division. Each division is incomplete, generating two, four, eight, and sixteen interconnected germline cysts. One cell within the 16-cell cyst differentiates into the and undergoes , while the other cells become polyploid nurse cells that feed into the growing oocyte and eventually die. The spectrosome and fusome, germline organelles without membrane that contain alpha- spectrin and adducin, play a role in the orientation of the spindle in the GSC and the regulation of cyst division and oocyte determination in the female. The spectrosome has a round appearance and is found in GSCs and cysto- blasts. The fusome extends between dividing cysts. (B) Summary of signaling pathways. Beige: terminal filament. Red: cells of the niche, cap cells, and escort/ IGS cells. Blue: GSCs. Green: differentiating germ cells. Yellow: site of adhesion between GSC and niche. TL, regulation by translation; TS, regulation by transcription; S, regulation of stability. Arrow depicts cell-to-. In addition to transcriptional regulation via the DPP/GBB receptors, RNA regulators such as Nanos and Pumilio as well as the miRNA machinery act within germ cells and are required for the maintenanceof GSCs, likely by repressing RNA translation and stability of germline differentiation genes (Gilboa and Lehmann, 2004; Park et al., 2007; Wang and Lin, 2004). Recent findings propose that in GSCs, Mei-P26 interacts with Nanos and the miRNA machinery to repress RNAs, such as brat, required for differentiation, while in the cystoblast Bam/Bgcn inhibit nanos RNA, thereby favoring Pumilio/Brat interactions, which repress RNAs required for self-renewal and proliferation, such as the BMP-mediated transcription factor Mad and the proliferation factor dMyc (Harris et al., 2011); (Li et al., 2012). Mei-P26 is also required for differentiation and its translation may depend on Vasa (Liu et al., 2009). JAK/STAT signaling is required for GSC maintenance; the ligand Unpaired (UPD) expressed in terminal filament cells activates the Domeless receptor in cap cells, where it activates transcription of dpp (Lo´ pez-Onieva et al., 2008). proteins associate in large RNA-protein complexes referred to as Several RPBs have recently been implicated in the control of P or polar granules, nuage, mitochondrial cloud, and Balbani small RNA pathways. Piwi, Tudor, and Argonaute family body, which have long been recognized as the morphological proteins are part of the ‘‘piwi interacting’’ (pi) small RNA hallmark of germ cells (Voronina et al., 2011). Binding of these pathway and control transposable element abundance specifi- RBPs to their target RNAs regulates RNA stability and translation cally in the germline via the production of small RNAs that and possibly transcription (Richter and Lasko, 2011). It is partic- target transposable elements for destruction (Khurana and ularly intriguing that several of these RBPs are present and Theurkauf, 2010; Malone and Hannon, 2009). Dazl and DND1 employed at multiple stages during the germline life cycle. For in zebrafish counteract the effects of miRNA, thereby prevent- example, Vasa protein is a highly conserved, ATP-dependent ing the degradation of target RNAs in the germline (Kedde helicase with multiple germline functions including germ plasm et al., 2007). The interplay between RBPs and small RNA path- assembly, germ and differentiation, and small- ways provides a mechanism to fine-tune gene expression RNA-mediated transposable element silencing (Lasko and largely independently from instructive gene transcription. This Ashburner, 1990; Liu et al., 2009; Pek and Kai, 2011; Tomancak function may be particularly relevant in the germline to prevent et al., 1998; Vagin et al., 2004). Another example is the conserved somatic differentiation and to protect the germline genome Zn-finger protein Nanos, with a well-defined role in RNA transla- throughout the life cycle. tional repression (Kadyrova et al., 2007; Suzuki et al., 2010). In flies, worm, and mouse, Nanos plays essential roles in specifying GSCs embryonic germ cells and in the maintenance of adult GSCs Analysis of GSC development has greatly influenced the study (Forbes and Lehmann, 1998; Kobayashi et al., 1996; Kraemer of stem cell biology in general and has informed our knowledge et al., 1999; Sada et al., 2009; Saga, 2010; Subramaniam and of human GSC behavior. Because of the early recognition that Seydoux, 1999). Combinatorial interactions between RBPs likely adult contain a self-renewing stem cell population, regulate the affinity and selectivity for cognate RNAs during much is known about the physical nature of the stem cell germline development. This is best illustrated by the interplay compartments and the regulatory networks that keep the between Nanos, the sequence-specific RBP Pumilio, the balance between self-renewal and differentiation. Here, I will TRIM-NHL domain proteins Brat and Mei-P26, and the miRNA summarize the general features of GSC microenvironments in machinery, whose changing associations trigger stage-specific the gonads of some of the best-studied model organisms, translational repression of specific RNA targets during embryo- and I will focus on recent results in building regulator GSC genesis and GSC differentiation in Drosophila (for more detail, networks and observing their response to specific physiolog- please refer to Figure 1)(Harris et al., 2011; Li et al., 2012; ical conditions, such as nutritional state, environment, and Sonoda and Wharton, 2001). aging.

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Figure 2. Drosophila Testis Niche (A) Drawing of the testis GSC compartment. Six to twelve GSCs form a rosette around a single cluster of somatic support cells called the hub. Division is oriented (arrow) by the centrosomes (stars). The mother centrosome remains in the GSC (black star). The hub also maintains somatic cyst cell stem cells (CySCs). Division of the CySCs and GSCs is coordinated such that two cyst cells surround each differentiating germ cell. The differentiating germ cell, called a gonioblast, undergoes four rounds of divi- sion, before each of the interconnected 16-cell cyst undergoes meiosis. GSCs predominately divide asymmetrically but can also divide symmetrically to replenish the stem cell pool (de Cuevas and Matunis, 2011). The round spectrosome is found in GSC and go- nioblast, while the fusome extends equally between dividing cysts. (B) Summary of signaling pathways. Beige: hub cells. Red: cyst stem cells and cyst cells. Blue: GSCs. Green: differ- entiating germ cells. Yellow: site of adhesion between GSC and niche. TL, regulation by translation; TS, regula- tion by transcription; pStat, phosphorylated active Stat. The HOW RNA binding protein binds to bam RNA and represses its translation in stem cells. Ectopic HOW delays BAM protein accumulation and leads to additional spermatogonial divisions (Monk et al., 2010). Once a critical level of BAM is reached spermatogonia cease proliferation and differentiate (Insco et al., 2009).

GSCs Rely on a Specialized Somatic Microenvironment four rounds of division before each spermatocyte of the intercon- Germ cells are only part of the gonad. Throughout development nected 16-cell cyst undergoes meiosis, generating 64 sperm. and during differentiation into , germ cells rely on inter- Asymmetric division of the GSC can be directly linked to spindle actions with specialized somatic cells. For GSCs these interac- orientation, with one centrosome being anchored to the niche via tions take place in specialized somatic gonadal compartment astral microtubules that interact with the adherens junction referred to as the stem cell niche. GSCs have been identified in complex connecting the GSC and the hub. Failure to properly both sexes in C. elegans and Drosophila and in the mouse testis. align the mitotic spindle causes a delay in GSC division, suggest- While some studies suggest that a dormant GSC population ing the existence of a checkpoint that monitors centrosome exists in the female mammalian germline, lineage-tracing exper- orientation prior to (Cheng et al., 2008). Furthermore, iments have not yet been performed in vivo and it remains individual labeling of dividing centrosomes revealed a conserva- possible that these cells represent remnants of an earlier PGC tive mode of centrosome division in males, such that the or gonocyte stage (White et al., 2012). maternal centrosome remains with the stem cell (Yamashita Drosophila et al., 2007). This mode of inheritance is not followed by the Thanks to comparatively simple morphology and ease of genetic dividing chromosome strands, which, with the possible excep- manipulation, the Drosophila ovary and testis have become tion of the Y chromosome, seem to randomly segregate into model tissues for the study of stem cell behavior. GSCs are found the daughter cell (Yadlapalli et al., 2011). In both males and in both male and female gonads where they maintain egg and females, GSCs predominately divide asymmetrically but can sperm production throughout adult life (for detailed description also divide symmetrically to replenish the stem cell pool (de Cue- of the morphology of the Drosophila ovary and testis, please vas and Matunis, 2011). consult Figure 1A and Figure 2A). In the female, each ovary C. elegans contains multiple niches. Each niche is composed of at least Depending on the nutritional state, about 200 mitotically active, three different somatic cell types (terminal filament cells, cap undifferentiated germ cells reside in each of the distal ends of cells, and inner germarial sheath or escort cells), which interact the two gonad arms of the hermaphrodite C. elegans. At the with two or three GSCs and their progeny. Asymmetric division end of each arm, one somatic cell, the distal tip cell (DTC), of the GSC generates a new GSC and a differentiating daughter envelops the gonad tip and serves as a niche (Figure 3A). As cells that exits the niche’s influence. The differentiating daughter, move away from the DTC, they cease division and enter meiosis. also called the cystoblast, subsequently undergoes four rounds Asymmetric divisions have not been observed, but cells closest of division. Each division is incomplete, generating a 16-celled to the DTC form a pool of undifferentiated GSCs, while cells interconnected germline cyst, which will generate the oocyte. further away initiate the transition to meiosis. Long extensions In the Drosophila male, each testis contains 6–12 GSCs that of the DTC encompass the mitotically active stem cell compart- form a rosette around a single cluster of somatic support cells ment (Kimble and Crittenden, 2007). called the hub. The hub also maintains somatic cyst cell stem Mammalian Testis cells (CySCs). Division of the CySC and GSC is coordinated In contrast to Drosophila and C. elegans, where specialized such that two cyst cells surround each differentiating germ somatic cells form a structured niche surrounding the stem cell cell. The differentiating germ cell, called a gonioblast, undergoes compartment, the entire process, starting from self-renewing

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Figure 3. Distal Tip Cell and Stem Cell Compartment of C. elegans Hermaphrodite Gonad (A) Drawing of GSC compartment of one gonad arm. Mitotically active, undifferentiated germ cells reside in each of the distal ends of the two gonad arms. The distal tip cell (DTC) envelops the gonad tip and serves as a niche. As cells move away from the DTC they cease division and enter meiosis. (B) Summary signaling pathways. Beige: ASI neuron. Red: distal tip cell. Blue: GSCs. Green: differentiating germ cells. TL, regulation by translation. Arrow depicts cell-to- cell signaling. The double pointed arrow indicates the random direction of GSC division.

how the self-renewal versus differentiation

decision is achieved for an individual As-A16 . SGPs are found along the basement membrane of the and are preferentially associated with vascula- ture enriched for Leydig and interstitial cells (Yoshida et al., 2007). As spermatogonia undergo differentiation they leave the basal spermatogonia to release of elongated sperm into the lumen of region, suggesting that a vascular niche protects SGPs from the seminiferous tubule, occurs within the environment of large, differentiation. SGPs are highly motile, so one possibility is that somatic Sertoli cells (Figure 4A). As the cell bodies of these cells fluctuations in the interactions between the individual spermato- extend from the stem cell to the spermatocyst compartment, it is gonia and their microenvironment create heterogeneity. unclear how they could provide the type of microenvironment that defines a stem cell niche. However, tight junctions separate Regulatory Networks Control the Balance between the basal compartment, which contains early spermiogenic Self-Renewal, Differentiation, and Regeneration stages, from the adluminal compartment filled with later stages After the initial discovery of specific factors that promote either and could thereby provide a structural separation of function stem cell proliferation and maintenance or differentiation, it has (Oatley and Brinster, 2008). become apparent that there are many levels of regulation. In The existence of stem cells in the mouse testis is well docu- most stem cell systems, one major GSC signaling pathway has mented by cell-lineage and transplantation reconstitution been identified that relies on a signal provided by the niche assays (Brinster and Zimmermann, 1994). Only 0.02%–0.03% and received by the germ cells. Mosaic analysis, tissue-specific of the germ cells in a total testis have reconstitution potential gene expression assays, and transplantation experiments have upon transplantation into a testis devoid of germ cells. This determined the tissue dependence of the respective factors. percentage can be significantly increased if the germ cells are Finally, knockout and overexpression or ectopic expression enriched for undifferentiated spermatogonia (Oatley and Brin- experiments have been used to examine whether a particular ster, 2008). Traditionally it was thought that among the undiffer- signaling pathway plays an instructive role. The major signaling entiated spermatogonia, the single-celled (As) spermatogonia pathways identified include BMP, JAK/STAT, Notch, and constitute the self-renewing population, and that differentiation GDNF. More detailed analysis of these and additional signaling proceeds through a hierarchy of four mitotic divisions producing pathways has provided insight into intricate regulatory networks. sequentially paired (Apr) and Aaligned4-Aal16 spermatogonia As an increasing amount of information emerges, it is becoming connected by intercellular bridges. In this scenario, after the clearer how a balance between self-renewal and differentiation is fourth division the Aal16 spermatogonia would mature into A1 achieved at the molecular level. Because of the sheer complexity spermatogonia, which enter the differentiation path leading to of the present networks and at times incomplete connections, meiosis. In recent years, this hierarchical description of early I will focus on specific examples highlighting recent progress has been revised. Lineage marking and live in our understanding. For a more detailed discussion of specific imaging revealed that Apr-Aal16 spermatogonia can resolve their germline systems, please refer to recent reviews (Kimble, 2011; connecting bridges and thereby contribute to the self-renewing Oatley and Brinster, 2008; Spradling et al., 2011). stem cell population. Furthermore, these experiments sug- Multiple Levels of Control Restrict the Availability gested that As-A8 spermatogonia are also capable of directly of the Self-Renewal Signal and Limit Tissue Response entering differentiation without completing all rounds of division in the Drosophila Ovary (Nakagawa et al., 2007, 2010). Taken together, these experi- In the Drosophila ovary, recent studies have led to a detailed ments revealed an apparent heterogeneity among the undiffer- understanding of how the influence of the GSC signal is entiated spermatogonia independent of the mitotic hierarchy restricted within the somatic niche to prevent ectopic production and suggest that As to Aal16 spermatogonia contribute to the of GSCs and how receptor activity in the GSCs is regulated to spermatogonial progenitor cell (SGP) pool. It remains unclear allow differentiation (Figure 1B). GSCs in the ovary depend on

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Figure 4. Mouse Spermatogonial Niche (A) Drawing of GSC compartment as a quadrant of cross sections of a seminiferous tubule. The entire process, starting from self-renew- ing spermatogonia to release of elongated sperm into the lumen of the seminiferous tubule, occurs within the environment oflarge, somatic Sertolicells. Tight junctions separate the basal compartment, which contains early spermiogenic stages, from the adluminal compartment filled with later stages. (B) Summary of signaling pathways. Beige: blood vessel and Leydig cells, which may contribute additional factors regulatingstem cell maintenance, including Colony stimulating factor 1 (CSF-1) and FGF. Red: Sertoli cells. Note that tight junctions form the blood-testis barrier between the Basal (BC) and Adluminal (AL) compartment, which separates differentiating from undifferentiat- ing SGPs. Blue: SGPs. Green: differentiating germ cells. All depicted interactions are transcriptional. RA: activates KIT receptor during the transition to differentiation. SGPs express high levels of the GDNF receptor GFRa1, the trans- lational repressor Nanos2, and the transcriptional repressor PLZF. Increased levels of the bHLH transcription factors Ngn3 and Nanos3 charac- terize SGPs that are more likely to differentiate. Etv5 expressed in Sertoli cells leads to production of CCL9 ligand, which is recognized by CCR1 receptor in germ cells and attracts SGPs to Sertoli cells (double headed arrow) (Simon et al., 2010). the production of the BMP-type ligands DPP (Decapentaplegic) et al., 2011). (For additional examples of regulation within the and GBB (Glass bottom boat), which are secreted from niche stem cell compartment, please refer to Figure 1B.) cells (Chen et al., 2011; Xie and Spradling, 1998). The DPP/ Experimental Models Distinguish between GBB signal is received in germ cells by the Thickveins (TKV) Germ-Cell-Autonomous Functions and Intercellular and Saxophone (SAX) receptors. Receptor activation leads to Competition in the Drosophila Testis transcriptional repression of the bag of marbles (bam) gene in In the male gonad, genetic analysis had pointed to a role for the the GSCs. As a consequence, bam expression is restricted to JAK/STAT pathway in GSC self-renewal (Kiger et al., 2001; the GSC daughter, the cystoblast, and its progeny (Song et al., Tulina and Matunis, 2001). Recent studies revealed, however, 2004). Bam is an instructive factor for germ cell differentiation: that this function is dispensable as long as STAT levels are loss of bam causes accumulation of undifferentiated germ cells kept uniform among GSCs. However, in genetic mosaics of and ectopic expression of bam causes GSC differentiation and wild-type and stat mutant GSCs, cells with higher STAT levels germline depletion (Ohlstein and McKearin, 1997). will outcompete mutant cells and take over the niche (Leather- Multiple levels of control restrict the availability of the BMP man and Dinardo, 2010). Uniform loss of JAK/STAT signaling in signal and the activity of its receptor to the stem cell compart- GSCs causes a weak GSC maintenance phenotype due to ments (Chen et al., 2011). Niche cells produce the BMP ligand reduced adhesion between the GSCs and the hub. In contrast DPP. Its diffusion is limited by the heparin sulfate proteoglycan to this apparent minor role in GSCs, the JAK/STAT pathway DALLY, ensuring that a high level of BMP remains close to the plays an indispensable and instructive role in maintenance of cap cells that are located directly adjacent to the GSCs (Guo the CySCs (Issigonis et al., 2009). It does so through activation and Wang, 2009). dally expression itself is negatively regulated of two downstream transcription factors, ZFHI and CHINMO in by the activity of the Drosophila EGF receptor (EGFR) in the CySCs, which are required for CySC self-renewal. Conversely, somatic escort cells, which intermingle and send fine extensions overexpression of either ZFHI or CHINMO, or constitutive activa- between the dividing, differentiating germ cells (Liu et al., 2010). tion of JAK, leads to a CySC expansion. Activation of the JAK/ Differentiating germ cells express and process the ligand for the STAT pathway in the somatic cells controls GSC maintenance, EGFR, thereby providing a negative feedback loop to limit the and overexpression of ZFHI and CHINMO causes accumulation GSC signal. In addition to Dally, Collagen IV directly binds to of undifferentiated germ cells (Flaherty et al., 2010; Leatherman and antagonizes DPP, which further restricts its reach (Wang and Dinardo, 2008). How the somatic cells signal back to the et al., 2008). Germ-cell-intrinsic mechanisms control the activity GSCs remains unclear, but could be directly by controlling the of the receptor by regulating its expression within GSCs and expression, production, or secretion of the BMP ligands GBB promoting its degradation by a complex of the E3 ubiquitin ligase and DPP, which have been implicated in GSC maintenance Smurf and the serine-threonine kinase Fused (Xia et al., 2010). in the testis, or indirectly by preventing GSC differentiation This regulation, together with posttranscriptional repression of (Shivdasani and Ingham, 2003). These new results point to an the downstream transcription factor Mad by the RNA regulator interesting experimental dilemma in the analysis of stem cell complex Pumilio/Brat in cystoblasts, ensures that the GSC behavior: most signaling pathways are used at multiple times signal is only transmitted to a small group of GSCs (Harris during development and in multiple tissues. Thus, analysis of

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mutant phenotypes caused by the loss of a particular factor is 2012). When specifically removed from the germline, these often limited to conditional knockout approaches. These genes affect SGP proliferation, self-renewal, and survival. One approaches in general produce mosaics of wild-type and mutant critical transcription factor that is regulated apparently indepen- cells within the same tissue. Such mosaics do not allow one to dently of GDNF in SGPs is PLZF. This Zn-finger transcriptional distinguish between a bona fide, tissue-specific requirement repressor forms a complex with the Spalt-like protein SALL4, for a gene product and a more indirect effect due to the compe- and the stoichiometry of SALL4 to PLZF in germ cells influences tition between cells with different levels of the respective factor. the self-renewal-to-differentiation decision (Hobbs et al., 2012). Transplantation assays as used in the mammalian testis; tissue- Consistent with the opposing effects of these two genes, specific complementation of the gene in question in the soma, SALL4 deletion affects spermatogonial differentiation, while but not the germline; or germline-specific RNAi knockout can PLZF deletion causes loss of stem cell self-renewal. A high be utilized to create genetically uniform cell populations. PLZF-to-SALL4 ratio determines self-renewal, at least in part, An RNA Regulatory Network Maintains GSCs and via expression of SAL1, while a high SALL4-to-PLFZ ratio favors Promotes the Mitosis-to-Meiosis Decision in C. elegans differentiation possibly via kit expression (Filipponi et al., 2007). The mitosis-to-meiosis decision in C. elegans is dependent on SALL4 is not only active in SPGs but also in embryonic germ signals produced from the DTC (Figure 3A). The DTC produces cells, where PLZF levels are low. It has been proposed that the Notch ligand, and activation of the Notch receptor, GLP-1, SALL4 preserves the undifferentiated state of the germ cell, in the germline is sufficient to maintain a proliferating stem cell possibly by acting through the KIT receptor, which is required population, while loss of GLP-1 activity leads to entry into meiosis for PGC survival and proliferation. Later in the adult SALL4 again (Cinquin et al., 2010). The regulatory network downstream of regulates kit expression, but this time, to promote differentiation Notch/Glp-1 is one of the best-understood models of how the (Hobbs et al., 2012). balance between stem cell self-renewal and proliferation versus differentiation and meiosis is established and maintained Adhesion between GSCs and Niche Control Asymmetry (Figure 3B). Central to this network is the crosstalk between of GSC Division and Tissue Regenerative Potential several RNA regulatory factors. In the stem cell compartment, Application of live imaging and differential labeling techniques Notch transcriptionally activates fbf-1 and fbf-2 (Lamont et al., are beginning to reveal intriguing aspects of GSC regulation 2004). These homologs of the conserved RBP Pumilio repress (Cheng et al., 2008; Morris and Spradling, 2011). One critical translation of another set of RNA regulators, GLD1, GLD2, and aspect is the role of adhesion between GSCs and the niche. In GLD3, in the stem cell compartment. GLD1, a member of the the Drosophila ovary, GSCs are anchored to the cap cells of STAR family of RBPs, in turn inhibits stem cell maintenance and the niche via gap junctions and E-cadherin-rich adherens junc- proliferation by repressing Notch/Glp-1 and Cyclin E translation, tions. E-cadherin downregulation favors GSC differentiation, respectively (Biedermann et al., 2009; Marin and Evans, 2003). while loss of communication causes the death of GLD2 and GLD3 are part of a cytoplasmic polyA-polymerase differentiating germ cells (Jin et al., 2008; Tazuke et al., 2002). translational activator complex that promotes translation of In the male, adherens junctions between the hub and GSCs gld-1 RNA, thereby reinforcing the meiosis decision (Suh et al., orient the mitotic spindle and may directly contribute to keeping 2006). The regulatory pathway downstream of Notch demon- the mother centrosome with the GSC (Inaba et al., 2010). As in strates the important role that RNA regulatory networks play in the female, E-cadherin mutants rapidly lose GSCs. Interestingly, germ cell development. Indeed, reporter studies have revealed a recent study suggests that BMP receptor activation in the male that independent of a specific transcriptional input, 30UTRs are is localized to the adherens junctions. In this model trafficking of the primary determinants regulating spatial gene expression in the BMP ligand together with adherens junction components the C. elegans germline (Merritt et al., 2008). provides a short-range source for BMP receptor activation (Mi- Opposing Functions of Transcription Factors Define chel et al., 2011). Rather than E-cadherin, spermatogonial the Germline Progenitor Pool in the Mammalian Testis stem cells in the mouse require beta-1-integrin to home and The molecular mechanisms underlying self-renewal and differ- adhere to their niche, and beta-1-integrin expression in germ entiation of SGPs are beginning to be understood due to the cells is necessary for normal spermatogenesis (Kanatsu-Shino- establishment of culture conditions to grow, select, and hara et al., 2008). Taken together, these studies indicate that increase the SGP population, as well as to tissue-specific adhesion molecules are required to anchor stem cells to their knockout strategies. A critical factor in the maintenance of niche and play a role in orienting stem cell divisions. Further- a self-renewing SGP population is the TGFb-related growth more, regulation of the strength of adhesion by environmental factor GDNF, which is produced by Sertoli cells (Figure 4B). factors or during aging can influence the balance between self- GDNF supports maintenance of a stem cell population in vitro renewal and differentiation and thereby affect the regenerative and in vivo, and overexpression of GDNF in Sertoli cells leads ability of the tissue (Pan et al., 2007; Yamashita, 2010). to an increase in As and Apr spermatogonia (Meng et al., 2000). Two GDNF receptors, GFRa1 and c-RET, are required Environmental and Systemic Factors Regulate Gonad in spermatogonia for stem cell maintenance (Naughton et al., Homeostasis 2006; Oatley et al., 2007). Downstream effectors expressed in Cooperation between GSCs and their niches maintains homeo- SGPs in response to GDNF include a number of transcription stasis by setting the balance between self-renewal and differen- factors, such as Oct4, Bcl6b, and Taf4b, but also the RNA regu- tiation. This balance has to be flexible to adapt to changes in lator NANOS-2, which, upon forced expression, can partially environmental conditions like food availability or to systemic rescue stem cell loss in the absence of GDNF (Sada et al., changes caused by hormonal fluctuation and aging. During

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Drosophila larval growth, levels of the ecdysone coor- sensitive to environmental influence. Furthermore, the ability of dinate the development and differentiation of the niche and mTORC1 to downregulate GDNF receptors could entice SGPs GSCs in the ovaries. Ecdysone prevents precocious maturation to leave their niche and initiate the differentiation program. The of the niche and PGC differentiation during early larval develop- balanced interactions of PLZF with SALL4 and mTORC1 nicely ment, but promotes niche morphogenesis and germ cell differ- exemplify how small, local changes in the levels of regulators entiation during later larval stages (Gancz et al., 2011). In the of cell growth and differentiation can contribute to the heteroge- adult, niche signals, GSC-to-niche adhesion, GSC proliferation, neity and potential of the SGP population. and are affected by both external and internal condi- tions. Aging is associated with delay or complete failure in Summary and Outlook GSC self-renewal in both males and females. The effects of Systematic analysis of germ cell development in the embryo aging can be overcome by the expression of niche factors, and stem cell homeostasis in the adult gonad has brought insight such as the ligands of the BMP and JAK/STAT pathways, the into the molecular mechanisms that establish and regulate adhesion molecule E-cadherin, or the cell cycle regulator niche-stem cell interactions. In contrast to all other stem cell STRING, a Cdc25 homolog (Inaba et al., 2011; Pan et al., systems in the body, homeostasis of the gonadal system is 2007). Interestingly, with aging the number of GSCs decreases directly related to reproductive success. Thus specific mecha- less than expected, likely due to replacement of lost stem cells nisms such as global epigenetic regulation of the germ cell by symmetrical division, leading to clonal expansion of a subset program and the need for a complete reprogramming of the of GSCs (Cheng et al., 2008; Wallenfang et al., 2006). If a similar germ cell genome during development are features of essential process occurs in mammals, such a clonal expansion could importance for germ cells to give rise to a new generation. contribute to the accumulation of genomic defects in the How transgenerational inheritance is transmitted at the gene progeny of aged parents. and genome level is still largely unclear. Nutrition has a dramatic effect on egg production in The regulatory networks controlling GSC self-renewal and Drosophila. Flies that are fed a protein-rich diet produce up to differentiation are beginning to be unraveled. These include 15 times more progeny than those on a protein-poor diet (Drum- germ-cell-specific RNA regulators that are conserved between mond-Barbosa and Spradling, 2001). In addition to decreased species. The targets of these regulators are still largely unknown, cell death during oogenesis, GSC proliferation rate increases but in some cases they control strikingly similar processes, such with a high-protein diet. These effects are in part mediated by as the role of Nanos family proteins in PGC specification and Insulin and Tor, which regulate niche size, GSC-niche adhesion, GSC maintenance in worms, flies, and mice. These RNA regula- and germline cyst differentiation (Hsu and Drummond-Barbosa, tors, together with their targets, which include small regulatory 2011; Sun et al., 2010). Environmental factors such as food RNAs, are packaged into intracellular particles that change in abundance and population density also influence the rate of composition and cellular location during the germ cell life cycle. proliferation and the mitosis-to-meiosis decision in C. elegans. Compartmentalization may allow the same RBP to entertain Limited food availability restricts the size of the stem cell pool multiple regulatory relationships during different stages of germ- during larval growth, and starvation leads to a dramatic resorp- line development. tion of the germline. Even under these conditions, about 30 Niche-stem cell interactions play a crucial role for GSC stem cells remain, which, if conditions improve, can regenerate homeostasis. These interactions protect GSCs from differentia- the entire germline (Angelo and Van Gilst, 2009). The Insulin/ tion, regulate proliferation, and, in some cases, orient the GSC- IGF and Tor/S6K pathways regulate germline proliferation during daughter cell division. In Drosophila, the path from GSC to larval stages (Korta et al., 2012; Michaelson et al., 2010). Favor- differentiated germline cyst was considered to be rather hierar- able conditions are also sensed by a group of neurons located chical until the discovery that male and female GSCs can regen- b close to the pharynx, triggering activation of the TGF receptor erate from advanced, interconnected germline cysts (Brawley in the DTC and promoting GSC proliferation (Dalfo et al., 2012). and Matunis, 2004; Kai and Spradling, 2004; Wong and Jones, b In addition to proliferation, Tor/S6K and TGF also affect the 2012). In the mouse testis, heterogeneity among SPGs seems b mitosis-to- meiosis decision. Insulin-, Tor-, and TGF -pathway to be the rule rather than the exception, since more advanced mutants exacerbate the glp-1 phenotype, suggesting that these cysts routinely contribute to the precursor pool. The functional pathways exert their function at least in part independently of the and hereditary consequences of this plasticity are not under- Notch regulatory network. How these sensors and mediators of stood. environmental conditions are integrated at the level of germ cell Germ cells, through their potential to differentiate into sperm self-renewal and differentiation is not understood. and egg, have the ability to create a new organism. Analysis of Environmental stimuli may also regulate the SGP pool in the the regulatory networks that control germ cell specification, mouse testis. Recent studies show that PLZF and mTORC1, self-renewal, and differentiation may ultimately lead to a better one of two Tor complexes that mediate cell growth in response understanding of the control mechanisms that balance the to nutrients, growth factors, and cellular stress in mammals, need for genomic fidelity with the opportunity for evolutionary are engaged in a negative feedback loop that can shift the self- change. renewal versus differentiation balance depending on environ- mental conditions. PLZF represses mTORC1 via activation of ACKNOWLEDGMENTS Redd1, an mTORC1 inhibitor; mTORC1 activation in turn leads to downregulation of the GDNF receptors (Hobbs et al., 2010). I would like to thank members of my lab and my colleagues in the Develop- Thus, PLZF expression in SPGs may render these cells less mental Genetics Program for discussion, and specifically Drs. Daria Siekhaus

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and Ryan Cinalli for critically reading the manuscript. I apologize to those Guo, Z., and Wang, Z. (2009). The glypican Dally is required in the niche for the colleagues whose work I was unable to properly cite due to space restrictions. maintenance of germline stem cells and short-range BMP signaling in the Research on germ cell development in my lab is supported by the NIH Drosophila ovary. Development 136, 3627–3635. (R01HD041900), the HHMI, and the Kimmel Center for Stem Cell Biology. Guven-Ozkan, T., Nishi, Y., Robertson, S.M., and Lin, R. (2008). Global tran- scriptional repression in C. elegans germline precursors by regulated seques- REFERENCES tration of TAF-4. Cell 135, 149–160.

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