Germline Stem Cells: Origin and Destiny

Abstract

Germline stem cells are key to genome transmission to future generations. Over recent years, there have been numerous insights into the regulatory mechanisms that govern both germ cell specification and the maintenance of the germline in adults. Complex regulatory interactions with both the niche and the environment 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 needed for survival or immediate physiological function of the individual, but rather, are endowed with the ability to contribute to the next generation. However, while they are ultimately able to generate all cells in the body, germ cells are unipotent and their normal fate is restricted to sperm or egg. This review will focus on recent developments in our understanding of how primordial germ cells (PGCs) are specified during embryogenesis, and how different strategies, including germline stem cell (GSC) renewal, soma-germline interactions, and physiological signals, are used to maintain a pool of differentiating, meiotic germ cells in adults.

Germ Cell Specification

In most organisms, PGCs are set aside early during embryogenesis. Two distinct mechanisms have been identified in model organisms that specify germ cells. In flies, worms, fish, and frogs, maternally synthesized germ plasm is deposited into the egg during oogenesis. This specialized cytoplasm harbors germline-specific, electron-dense RNA-protein particles required for multiple aspects of germ cell fate. Embryonic nuclei inheriting germ plasm are destined to become PGCs. In contrast, in mammals and many other species traversing all animal phyla, germ cells are specified among the cells of the embryo independent of preexisting maternal information. Specification occurs in some species early during development, when germ cell fate is induced in a subset of otherwise pluripotent progenitors cells such as epiblast cells of the early mouse embryo, and in other species at later embryonic and postembryonic stages, when germ cells can originate from multipotent progenitors within the mesoderm. Irrespective of these apparent differences in how the germ cell lineage becomes distinct from somatic cells, conserved molecular principles have emerged (Cinalli et al., 2008). Among these, global epigenetic regulation of germline gene expression and germ-cell-specific posttranscriptional regulation are essential both for specifying, maintaining, and protecting the germline during its life cycle and for ensuring transgenerational success.

PGC Specification Requires Suppression of the Somatic Program

Transcriptional repression seems to be a necessary component of germline specification (Nakamura and Seydoux, 2008). Depending on the organism this repression is achieved either by globally repressing RNA Polymerase activity or by more specifically blocking the somatic program. In C. elegans and Drosophila, PGCs are the first cells separated from the somatic lineage. In these species, PGCs inherit maternally synthesized germ plasm that is rich in mRNA and RNA binding proteins (RBPs). The newly formed PGCs rely entirely on these maternally provided factors, since transcription is completely blocked, thereby preventing the expression of the zygotic genome. In Drosophila, repression of transcription is achieved by germ-cell-specific translation of a 71 amino acid peptide encoded by the polar granule component (pgc) gene (Hanyu-Nakamura et al., 2008). Pgc protein inhibits the recruitment of the Positive Transcription Elongation factor-b (P-TEFb) kinase complex to transcription sites. P-TEFb phosphorylates Ser-2-containing motifs in the carboxyl-terminal domain (CTD) repeats of RNA Polymerase II (RNA PolII) and is responsible for transcriptional elongation. Pgc protein is only present in germ cells for a short time and transcriptional activity is observed as soon as germ cells initiate their migration toward the somatic part of the embryo. Robust, high levels of transcription are achieved once germ cells reach the somatic gonad. In C. elegans, transcription in the early germline is also repressed via interference with RNA PolII activity, but by slightly different mechanisms. During the first two embryonic divisions, the Zn-finger proteins Oma1 and Oma2 sequester TATA-binding protein associated factor 4 (TAF-4), a subunit of TFIID required for assembly of the RNA PolII preinitiation complex, in the cytoplasm, thereby preventing transcription in the entire early embryo (Guven-Ozkan et al., 2008). Subsequently, Pie-1, a CCCH Zn-finger protein, is specifically translated in the germ cell precursors and inhibits transcriptional initiation and elongation by interfering with Ser 5 and Ser 2 phosphorylation, respectively, in RNA PolII CTD motifs (Ghosh and Seydoux, 2008).

In the mouse, germ cell specification occurs in the proximal epiblast just prior to gastrulation. A BMP4/8 signal from the extraembryonic ectoderm initiates the germ-cell-specific program by activating the repressor protein Blimp1 (also known as Prdm1) (Lawson et al., 1999; Ohinata et al., 2005; Ying et al., 2001). Not all cells that receive the BMP signal become PGCs. Expression is restricted to the posterior epiblast by antagonizing signals from the anterior visceral endoderm and is enhanced by Lin28, which counteracts let-7 miRNA, a Blimp1 suppressor (Ohinata et al., 2009; West et al., 2009). Blimp1 is responsible for the repression of almost all of the genes that are downregulated in PGCs, but is only required for the activation of some of the genes that are upregulated in germ cells (Kurimoto et al., 2008). How expression of Stella, Kit, Dnd1/Ter, and Nanos3, which play an important part in the specification, migration, and survival of early germ cells, is activated in germ cells is not fully understood. Beyond repression of the somatic program, Blimp1, together with another PR domain protein, Prdm14, also promotes the epigenetic reprogramming within early germ cells to regain pluripotency (Yamaji et al., 2008). Indeed, PGCs reactivate the pluripotency genes Nanog, Sox2, and Oct4. This finding together with the observation that early PGCs can easily give rise to pluripotent embryonic stem cells (called “EG” cells) led to the hypothesis that PGCs resemble earlier epiblast cells (Matsui et al., 1992). Indeed, recent evidence using activation of a Blimp1 reporter argues that ESC cultures may originate from PGCs (Chu et al., 2011). However, because Blimp1 activity is not required for ESC derivation, the general implication of these findings remains uncertain. Once PGCs complete their migration to the genital ridge and associate with the somatic part of the gonad, they lose the ability to easily revert to the pluripotent state and reach a germ-cell-specific epigenetic program (Hayashi and Surani, 2009).

In an effort to reveal the principles of germ cell specification, Hayashi et al. (2011) recently developed a protocol to derive germ cells from ESCs and iPSCs. In a stepwise process, pluripotent stem cells were first cultured into cells resembling pregastrulating epiblast cells, from which in turn PGC-like cells were derived. Cells with PGC-like characteristics were able to produce functional sperm after being injected into the seminiferous tubules of germ-cell-deprived (kit mutant) mice. The efficiency was similar to that of transplantation of in-vivo-derived PGCs. These new culture conditions will be critical for developing a more complete understanding of germ cell development and have a clear application for reproductive medicine.

A Special Chromatin Program Is Established in Germ Cells

Early embryonic germ cells in the mouse are transcriptionally active and express the CTD phospho-epitopes characteristic of active RNA PolII transcription. These marks are no longer detected during the migration of germ cells to the somatic gonad (between E8 and E9) (Seki et al., 2007). In the gonad, epigenetic reprogramming takes place, including global DNA demethylation, exchange of histone variants, large-scale chromatin remodeling of retrotransposon-linked and imprinted genes, and reactivation of both X chromosomes (Hajkova et al., 2008). This remodeling is critical for resetting imprint marks and has also been proposed to play an important role in the activation of genes required for early embryonic development in the next generation (Hayashi and Surani, 2009). For a long time it was unclear whether DNA demethylation was achieved passively as a consequence of replication and lack of maintenance methylation, or whether there were enzymes that actively removed methylation marks from DNA. Recent findings show that the Tet (ten eleven translocation) family of proteins can catalyze the conversion of the 5-methylcytosine (5mC) base (the methylation mark mostly associated with inactive genes) to 5-hydroxymethylcytosine (5hmC), suggesting that this conversion could be the first step toward demethylation (He et al., 2011; Ito et al., 2010, 2011; Tahiliani et al., 2009). Subsequent studies have shown that 5hmC marks are enriched in ESCs and are found in the promoter regions of totipotency genes that are also active in germ cells (Ficz et al., 2011). The next steps toward complete demethylation are still unclear, and may involve Tet-mediated oxidation, base excision repair, and/or passive, replication-dependent demethylation (Hackett et al., 2012).

DNA modifications and their regulation play a less prominent role in fly and worm germ cells, but modification of chromatin-associated histones has important functions in maintaining the germ cell fate in later stages of germline development. In particular, several recent results argue that global chromatin regulation has a direct impact on the germline-soma fate decision. For example, mutations in the synthetic multivulva (synMuv) genes, the worm homologs of the mammalian proteins retinoblastoma (RB), E2F, HP1, and NuRD complex, lead to transformation of intestinal cells into germline-like cells (Petrella et al., 2011). This complex interacts with l(3)mbt, a gene that causes malignant brain tumors when mutated in the fly (Lewis et al., 2004). Interestingly, and in contrast to mutations in other brain tumor genes, l(3)mbt mutant tumor cells express genes normally restricted to the germline (Janic et al., 2010). Chromatin association studies indicate that l(3) MBT protein binds preferentially to insulator regions and suggest a role for l(3)MBT in global repression of germline genes in specific somatic tissues (Richter et al., 2011). Chromatin modifiers can also coax the germline into expressing somatic programs. Expression of specific neural transcription factors converts C. elegans germline cells into specific types of terminally differentiated neurons. This conversion requires the removal of the conserved chromatin remodeling protein LIN-53, another component of the synMuv family (Tursun et al., 2011). Curiously, germline-to-soma and soma-to-germline transformations involve mutations in the same class of genes normally associated with a repressive chromatin state. However, transformation of germline into soma requires tissue-specific transcription factors, while such factors have not been identified for soma-to-germline transformations or for normal germline development. Together these findings point to the important role of global transcriptional and epigenetic regulation in the germline. Indeed, evidence is mounting that epigenetic memory in the germline contributes not only to germline development but also to patterns of inheritance for generations to come (Greer et al., 2011; Katz et al., 2009).

Conserved RNA Regulators Are Associated with Germ Cells throughout the Life Cycle

While so far no specific, conserved transcriptional regulator for germ cell fate has been identified, RBPs play important roles throughout germ cell development. Germline RBPs prevent somatic differentiation and control many germ cell functions such as specification of germ cell fate and sexual identity, proliferation, survival, migration, and regulation of transposable element activity. RBP families such as Vasa, Nanos, Pumilio, Dnd1, DAZL, PIWI, and Tudor are broadly conserved from planarians to humans and are generally found during multiple stages of the germ cell life cycle (Juliano et al., 2010). These proteins associate in large RNA-protein complexes referred to as P or polar granules, nuage, mitochondrial cloud, and Balbani body, which have long been recognized as the morphological hallmark of germ cells (Voronina et al., 2011). Binding of these RBPs to their target RNAs regulates RNA stability and translation and possibly transcription (Richter and Lasko, 2011). It is particularly intriguing that several of these RBPs are present and employed at multiple stages during the germline life cycle. For example, Vasa protein is a highly conserved, ATP-dependent helicase with multiple germline functions including germ plasm assembly, germ cell proliferation and differentiation, and small-RNA-mediated transposable element silencing (Lasko and Ashburner, 1990; Liu et al., 2009; Pek and Kai, 2011; Tomancak et al., 1998; Vagin et al., 2004). Another example is the conserved Zn-finger protein Nanos, with a well-defined role in RNA translational repression (Kadyrova et al., 2007; Suzuki et al., 2010). In flies, worm, and mouse, Nanos plays essential roles in specifying embryonic germ cells and in the maintenance of adult GSCs (Forbes and Lehmann, 1998; Kobayashi et al., 1996; Kraemer et al., 1999; Sada et al., 2009; Saga, 2010; Subramaniam and Seydoux, 1999). Combinatorial interactions between RBPs likely regulate the affinity and selectivity for cognate RNAs during germline development. This is best illustrated by the interplay between Nanos, the sequence-specific RBP Pumilio, the TRIM-NHL domain proteins Brat and Mei-P26, and the miRNA machinery, whose changing associations trigger stage-specific translational repression of specific RNA targets during embryogenesis and GSC differentiation in Drosophila (for more detail, please refer to Figure 1) (Harris et al., 2011; Li et al., 2012; Sonoda and Wharton, 2001).

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 oocyte and undergoes meiosis, 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 cystoblasts. 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-cell signaling. 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 maintenance of 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 (López-Onieva et al., 2008).

Several RPBs have recently been implicated in the control of small RNA pathways. Piwi, Tudor, and Argonaute family proteins are part of the “piwi interacting” (pi) small RNA pathway and control transposable element abundance specifically in the germline via the production of small RNAs that target transposable elements for destruction (Khurana and Theurkauf, 2010; Malone and Hannon, 2009). Dazl and DND1 in zebrafish counteract the effects of miRNA, thereby preventing the degradation of target RNAs in the germline (Kedde et al., 2007). The interplay between RBPs and small RNA pathways provides a mechanism to fine-tune gene expression largely independently from instructive gene transcription. This function may be particularly relevant in the germline to prevent somatic differentiation and to protect the germline genome throughout the life cycle.

GSCs

Analysis of GSC development has greatly influenced the study of stem cell biology in general and has informed our knowledge of human GSC behavior. Because of the early recognition that adult gonads contain a self-renewing stem cell population, much is known about the physical nature of the stem cell compartments and the regulatory networks that keep the balance between self-renewal and differentiation. Here, I will summarize the general features of GSC microenvironments in the gonads of some of the best-studied model organisms, and I will focus on recent results in building regulator GSC networks and observing their response to specific physiological conditions, such as nutritional state, environment, and aging.

GSCs Rely on a Specialized Somatic Microenvironment

Germ cells are only part of the gonad. Throughout development and during differentiation into gametes, germ cells rely on interactions with specialized somatic cells. For GSCs these interactions take place in specialized somatic gonadal compartment referred to as the stem cell niche. GSCs have been identified in both sexes in C. elegans and Drosophila and in the mouse testis. While some studies suggest that a dormant GSC population exists in the female mammalian germline, lineage-tracing experiments have not yet been performed in vivo and it remains possible that these cells represent remnants of an earlier PGC or gonocyte stage (White et al., 2012).

Drosophila

Thanks to comparatively simple morphology and ease of genetic manipulation, the Drosophila ovary and testis have become model tissues for the study of stem cell behavior. GSCs are found in both male and female gonads where they maintain egg and sperm production throughout adult life (for detailed description of the morphology of the Drosophila ovary and testis, please consult Figure 1A and Figure 2A). In the female, each ovary contains multiple niches. Each niche is composed of at least three different somatic cell types (terminal filament cells, cap cells, and inner germarial sheath or escort cells), which interact with two or three GSCs and their progeny. Asymmetric division of the GSC generates a new GSC and a differentiating daughter that exits the niche’s influence. The differentiating daughter, also called the cystoblast, subsequently undergoes four rounds of division. Each division is incomplete, generating a 16-celled interconnected germline cyst, which will generate the oocyte.

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 division, before each spermatocyte 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 gonioblast, 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: differentiating germ cells. Yellow: site of adhesion between GSC and niche. TL, regulation by translation; TS, regulation 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).

In the Drosophila male, each testis contains 6–12 GSCs that form a rosette around a single cluster of somatic support cells called the hub. The hub also maintains somatic cyst cell stem cells (CySCs). Division of the CySC and GSC is coordinated such that two cyst cells surround each differentiating germ cell. The differentiating germ cell, called a gonioblast, undergoes four rounds of division before each spermatocyte of the interconnected 16-cell cyst undergoes meiosis, generating 64 sperm. Asymmetric division of the GSC can be directly linked to spindle orientation, with one centrosome being anchored to the niche via astral microtubules that interact with the adherens junction complex connecting the GSC and the hub. Failure to properly align the mitotic spindle causes a delay in GSC division, suggesting the existence of a checkpoint that monitors centrosome orientation prior to mitosis (Cheng et al., 2008). Furthermore, individual labeling of dividing centrosomes revealed a conservative mode of centrosome division in males, such that the maternal centrosome remains with the stem cell (Yamashita et al., 2007). This mode of inheritance is not followed by the dividing chromosome strands, which, with the possible exception of the Y chromosome, seem to randomly segregate into the daughter cell (Yadlapalli et al., 2011). In both males and females, GSCs predominately divide asymmetrically but can also divide symmetrically to replenish the stem cell pool (de Cuevas and Matunis, 2011).

C. elegans

Depending on the nutritional state, about 200 mitotically active, undifferentiated germ cells reside in each of the distal ends of the two gonad arms of the hermaphrodite C. elegans. At the end of each arm, one somatic cell, the distal tip cell (DTC), envelops the gonad tip and serves as a niche (Figure 3A). As cells move away from the DTC, they cease division and enter meiosis. Asymmetric divisions have not been observed, but cells closest to the DTC form a pool of undifferentiated GSCs, while cells further away initiate the transition to meiosis. Long extensions of the DTC encompass the mitotically active stem cell compartment (Kimble and Crittenden, 2007).

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.

Mammalian Testis

In contrast to Drosophila and C. elegans, where specialized somatic cells form a structured niche surrounding the stem cell compartment, the entire process, starting from self-renewing spermatogonia to release of elongated sperm into the lumen of the seminiferous tubule, occurs within the environment of large, somatic Sertoli cells (Figure 4A). As the cell bodies of these cells extend from the stem cell to the spermatocyst compartment, it is unclear how they could provide the type of microenvironment that defines a stem cell niche. However, tight junctions separate the basal compartment, which contains early spermiogenic stages, from the adluminal compartment filled with later stages and could thereby provide a structural separation of function (Oatley and Brinster, 2008).

Figure 4. Mouse Spermatogonial Niche.

(A) Drawing of GSC compartment as a quadrant of cross sections of a seminiferous tubule. The entire spermiogenesis process, starting from self-renewing spermatogonia to release of elongated sperm into the lumen of the seminiferous tubule, occurs within the environment of large, somatic Sertoli cells. 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 regulating stem 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 spermatocytes from undifferentiating SGPs. Blue: SGPs. Green: differentiating germ cells. All depicted interactions are transcriptional. RA: retinoic acid activates KIT receptor during the transition to differentiation. SGPs express high levels of the GDNF receptor GFRα1, the translational repressor Nanos2, and the transcriptional repressor PLZF. Increased levels of the bHLH transcription factors Ngn3 and Nanos3 characterize 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 existence of stem cells in the mouse testis is well documented by cell-lineage and transplantation reconstitution assays (Brinster and Zimmermann, 1994). Only 0.02%–0.03% of the germ cells in a total testis have reconstitution potential upon transplantation into a testis devoid of germ cells. This percentage can be significantly increased if the germ cells are enriched for undifferentiated spermatogonia (Oatley and Brinster, 2008). Traditionally it was thought that among the undifferentiated spermatogonia, the single-celled (A_s) spermatogonia constitute the self-renewing population, and that differentiation proceeds through a hierarchy of four mitotic divisions producing sequentially paired (A_pr) and A_aligned4-A_al16 spermatogonia connected by intercellular bridges. In this scenario, after the fourth division the A_al16 spermatogonia would mature into A1 spermatogonia, which enter the differentiation path leading to meiosis. In recent years, this hierarchical description of early spermatogenesis has been revised. Lineage marking and live imaging revealed that A_pr-A_al16 spermatogonia can resolve their connecting bridges and thereby contribute to the self-renewing stem cell population. Furthermore, these experiments suggested that A_s-A₈ spermatogonia are also capable of directly entering differentiation without completing all rounds of division (Nakagawa et al., 2007, 2010). Taken together, these experiments revealed an apparent heterogeneity among the undifferentiated spermatogonia independent of the mitotic hierarchy and suggest that A_s to A_al16 spermatogonia contribute to the spermatogonial progenitor cell (SGP) pool. It remains unclear how the self-renewal versus differentiation decision is achieved for an individual A_s-A₁₆ spermatogonium. SGPs are found along the basement membrane of the seminiferous tubule and are preferentially associated with vasculature enriched for Leydig and interstitial cells (Yoshida et al., 2007). As spermatogonia undergo differentiation they leave the basal region, suggesting that a vascular niche protects SGPs from differentiation. SGPs are highly motile, so one possibility is that fluctuations in the interactions between the individual spermatogonia and their microenvironment create heterogeneity.

Regulatory Networks Control the Balance between Self-Renewal, Differentiation, and Regeneration

After the initial discovery of specific factors that promote either stem cell proliferation and maintenance or differentiation, it has become apparent that there are many levels of regulation. In most stem cell systems, one major GSC signaling pathway has been identified that relies on a signal provided by the niche and received by the germ cells. Mosaic analysis, tissue-specific gene expression assays, and transplantation experiments have determined the tissue dependence of the respective factors. Finally, knockout and overexpression or ectopic expression experiments have been used to examine whether a particular signaling pathway plays an instructive role. The major signaling pathways identified include BMP, JAK/STAT, Notch, and GDNF. More detailed analysis of these and additional signaling pathways has provided insight into intricate regulatory networks. As an increasing amount of information emerges, it is becoming clearer how a balance between self-renewal and differentiation is achieved at the molecular level. Because of the sheer complexity of the present networks and at times incomplete connections, I will focus on specific examples highlighting recent progress in our understanding. For a more detailed discussion of specific germline systems, please refer to recent reviews (Kimble, 2011; Oatley and Brinster, 2008; Spradling et al., 2011).

Multiple Levels of Control Restrict the Availability of the Self-Renewal Signal and Limit Tissue Response in the Drosophila Ovary

In the Drosophila ovary, recent studies have led to a detailed understanding of how the influence of the GSC signal is restricted within the somatic niche to prevent ectopic production of GSCs and how receptor activity in the GSCs is regulated to allow differentiation (Figure 1B). GSCs in the ovary depend on the production of the BMP-type ligands DPP (Decapentaplegic) and GBB (Glass bottom boat), which are secreted from niche cells (Chen et al., 2011; Xie and Spradling, 1998). The DPP/GBB signal is received in germ cells by the Thickveins (TKV) and Saxophone (SAX) receptors. Receptor activation leads to transcriptional repression of the bag of marbles (bam) gene in the GSCs. As a consequence, bam expression is restricted to the GSC daughter, the cystoblast, and its progeny (Song et al., 2004). Bam is an instructive factor for germ cell differentiation: loss of bam causes accumulation of undifferentiated germ cells and ectopic expression of bam causes GSC differentiation and germline depletion (Ohlstein and McKearin, 1997).

Multiple levels of control restrict the availability of the BMP signal and the activity of its receptor to the stem cell compartments (Chen et al., 2011). Niche cells produce the BMP ligand DPP. Its diffusion is limited by the heparin sulfate proteoglycan DALLY, ensuring that a high level of BMP remains close to the cap cells that are located directly adjacent to the GSCs (Guo and Wang, 2009). dally expression itself is negatively regulated by the activity of the Drosophila EGF receptor (EGFR) in the somatic escort cells, which intermingle and send fine extensions between the dividing, differentiating germ cells (Liu et al., 2010). Differentiating germ cells express and process the ligand for the EGFR, thereby providing a negative feedback loop to limit the GSC signal. In addition to Dally, Collagen IV directly binds to and antagonizes DPP, which further restricts its reach (Wang et al., 2008). Germ-cell-intrinsic mechanisms control the activity of the receptor by regulating its expression within GSCs and promoting its degradation by a complex of the E3 ubiquitin ligase Smurf and the serine-threonine kinase Fused (Xia et al., 2010). This regulation, together with posttranscriptional repression of the downstream transcription factor Mad by the RNA regulator complex Pumilio/Brat in cystoblasts, ensures that the GSC signal is only transmitted to a small group of GSCs (Harris et al., 2011). (For additional examples of regulation within the stem cell compartment, please refer to Figure 1B.)

Experimental Models Distinguish between Germ-Cell-Autonomous Functions and Intercellular Competition in the Drosophila Testis

In the male gonad, genetic analysis had pointed to a role for the JAK/STAT pathway in GSC self-renewal (Kiger et al., 2001; Tulina and Matunis, 2001). Recent studies revealed, however, that this function is dispensable as long as STAT levels are kept uniform among GSCs. However, in genetic mosaics of wild-type and stat mutant GSCs, cells with higher STAT levels will outcompete mutant cells and take over the niche (Leatherman and Dinardo, 2010). Uniform loss of JAK/STAT signaling in GSCs causes a weak GSC maintenance phenotype due to reduced adhesion between the GSCs and the hub. In contrast to this apparent minor role in GSCs, the JAK/STAT pathway plays an indispensable and instructive role in maintenance of the CySCs (Issigonis et al., 2009). It does so through activation of two downstream transcription factors, ZFHI and CHINMO in CySCs, which are required for CySC self-renewal. Conversely, overexpression of either ZFHI or CHINMO, or constitutive activation of JAK, leads to a CySC expansion. Activation of the JAK/STAT pathway in the somatic cells controls GSC maintenance, and overexpression of ZFHI and CHINMO causes accumulation of undifferentiated germ cells (Flaherty et al., 2010; Leatherman and Dinardo, 2008). How the somatic cells signal back to the GSCs remains unclear, but could be directly by controlling the expression, production, or secretion of the BMP ligands GBB and DPP, which have been implicated in GSC maintenance in the testis, or indirectly by preventing GSC differentiation (Shivdasani and Ingham, 2003). These new results point to an interesting experimental dilemma in the analysis of stem cell behavior: most signaling pathways are used at multiple times during development and in multiple tissues. Thus, analysis of mutant phenotypes caused by the loss of a particular factor is often limited to conditional knockout approaches. These approaches in general produce mosaics of wild-type and mutant cells within the same tissue. Such mosaics do not allow one to distinguish between a bona fide, tissue-specific requirement for a gene product and a more indirect effect due to the competition between cells with different levels of the respective factor. Transplantation assays as used in the mammalian testis; tissue-specific complementation of the gene in question in the soma, but not the germline; or germline-specific RNAi knockout can be utilized to create genetically uniform cell populations.

An RNA Regulatory Network Maintains GSCs and Promotes the Mitosis-to-Meiosis Decision in C. elegans

The mitosis-to-meiosis decision in C. elegans is dependent on signals produced from the DTC (Figure 3A). The DTC produces the Notch ligand, and activation of the Notch receptor, GLP-1, in the germline is sufficient to maintain a proliferating stem cell population, while loss of GLP-1 activity leads to entry into meiosis (Cinquin et al., 2010). The regulatory network downstream of Notch/Glp-1 is one of the best-understood models of how the balance between stem cell self-renewal and proliferation versus differentiation and meiosis is established and maintained (Figure 3B). Central to this network is the crosstalk between several RNA regulatory factors. In the stem cell compartment, Notch transcriptionally activates fbf-1 and fbf-2 (Lamont et al., 2004). These homologs of the conserved RBP Pumilio repress translation of another set of RNA regulators, GLD1, GLD2, and GLD3, in the stem cell compartment. GLD1, a member of the STAR family of RBPs, in turn inhibits stem cell maintenance and proliferation by repressing Notch/Glp-1 and Cyclin E translation, respectively (Biedermann et al., 2009; Marin and Evans, 2003). GLD2 and GLD3 are part of a cytoplasmic polyA-polymerase translational activator complex that promotes translation of gld-1 RNA, thereby reinforcing the meiosis decision (Suh et al., 2006). The regulatory pathway downstream of Notch demonstrates the important role that RNA regulatory networks play in germ cell development. Indeed, reporter studies have revealed that independent of a specific transcriptional input, 3′UTRs are the primary determinants regulating spatial gene expression in the C. elegans germline (Merritt et al., 2008).

Opposing Functions of Transcription Factors Define the Germline Progenitor Pool in the Mammalian Testis

The molecular mechanisms underlying self-renewal and differentiation of SGPs are beginning to be understood due to the establishment of culture conditions to grow, select, and increase the SGP population, as well as to tissue-specific knockout strategies. A critical factor in the maintenance of a self-renewing SGP population is the TGFβ-related growth factor GDNF, which is produced by Sertoli cells (Figure 4B). GDNF supports maintenance of a stem cell population in vitro and in vivo, and overexpression of GDNF in Sertoli cells leads to an increase in A_s and A_pr spermatogonia (Meng et al., 2000). Two GDNF receptors, GFRα1 and c-RET, are required in spermatogonia for stem cell maintenance (Naughton et al., 2006; Oatley et al., 2007). Downstream effectors expressed in SGPs in response to GDNF include a number of transcription factors, such as Oct4, Bcl6b, and Taf4b, but also the RNA regulator NANOS-2, which, upon forced expression, can partially rescue stem cell loss in the absence of GDNF (Sada et al., 2012). When specifically removed from the germline, these genes affect SGP proliferation, self-renewal, and survival. One critical transcription factor that is regulated apparently independently of GDNF in SGPs is PLZF. This Zn-finger transcriptional repressor forms a complex with the Spalt-like protein SALL4, and the stoichiometry of SALL4 to PLZF in germ cells influences the self-renewal-to-differentiation decision (Hobbs et al., 2012). Consistent with the opposing effects of these two genes, SALL4 deletion affects spermatogonial differentiation, while PLZF deletion causes loss of stem cell self-renewal. A high PLZF-to-SALL4 ratio determines self-renewal, at least in part, via expression of SAL1, while a high SALL4-to-PLFZ ratio favors differentiation possibly via kit expression (Filipponi et al., 2007). SALL4 is not only active in SPGs but also in embryonic germ cells, where PLZF levels are low. It has been proposed that SALL4 preserves the undifferentiated state of the germ cell, possibly by acting through the KIT receptor, which is required for PGC survival and proliferation. Later in the adult SALL4 again regulates kit expression, but this time, to promote differentiation (Hobbs et al., 2012).

Adhesion between GSCs and Niche Control Asymmetry of GSC Division and Tissue Regenerative Potential

Application of live imaging and differential labeling techniques are beginning to reveal intriguing aspects of GSC regulation (Cheng et al., 2008; Morris and Spradling, 2011). One critical aspect is the role of adhesion between GSCs and the niche. In the Drosophila ovary, GSCs are anchored to the cap cells of the niche via gap junctions and E-cadherin-rich adherens junctions. E-cadherin downregulation favors GSC differentiation, while loss of gap junction communication causes the death of differentiating germ cells (Jin et al., 2008; Tazuke et al., 2002). In the male, adherens junctions between the hub and GSCs orient the mitotic spindle and may directly contribute to keeping the mother centrosome with the GSC (Inaba et al., 2010). As in the female, E-cadherin mutants rapidly lose GSCs. Interestingly, a recent study suggests that BMP receptor activation in the male is localized to the adherens junctions. In this model trafficking of the BMP ligand together with adherens junction components provides a short-range source for BMP receptor activation (Michel et al., 2011). Rather than E-cadherin, spermatogonial stem cells in the mouse require beta-1-integrin to home and adhere to their niche, and beta-1-integrin expression in germ cells is necessary for normal spermatogenesis (Kanatsu-Shinohara et al., 2008). Taken together, these studies indicate that adhesion molecules are required to anchor stem cells to their niche and play a role in orienting stem cell divisions. Furthermore, regulation of the strength of adhesion by environmental factors or during aging can influence the balance between self-renewal and differentiation and thereby affect the regenerative ability of the tissue (Pan et al., 2007; Yamashita, 2010).

Environmental and Systemic Factors Regulate Gonad Homeostasis

Cooperation between GSCs and their niches maintains homeostasis by setting the balance between self-renewal and differentiation. This balance has to be flexible to adapt to changes in environmental conditions like food availability or to systemic changes caused by hormonal fluctuation and aging. During Drosophila larval growth, levels of the hormone ecdysone coordinate the development and differentiation of the niche and GSCs in the ovaries. Ecdysone prevents precocious maturation of the niche and PGC differentiation during early larval development, but promotes niche morphogenesis and germ cell differentiation during later larval stages (Gancz et al., 2011). In the adult, niche signals, GSC-to-niche adhesion, GSC proliferation, and apoptosis are affected by both external and internal conditions. Aging is associated with delay or complete failure in GSC self-renewal in both males and females. The effects of aging can be overcome by the expression of niche factors, such as the ligands of the BMP and JAK/STAT pathways, the adhesion molecule E-cadherin, or the cell cycle regulator STRING, a Cdc25 homolog (Inaba et al., 2011; Pan et al., 2007). Interestingly, with aging the number of GSCs decreases less than expected, likely due to replacement of lost stem cells by symmetrical division, leading to clonal expansion of a subset of GSCs (Cheng et al., 2008; Wallenfang et al., 2006). If a similar process occurs in mammals, such a clonal expansion could contribute to the accumulation of genomic defects in the progeny of aged parents.

Nutrition has a dramatic effect on egg production in Drosophila. Flies that are fed a protein-rich diet produce up to 15 times more progeny than those on a protein-poor diet (Drummond-Barbosa and Spradling, 2001). In addition to decreased cell death during oogenesis, GSC proliferation rate increases with a high-protein diet. These effects are in part mediated by Insulin and Tor, which regulate niche size, GSC-niche adhesion, and germline cyst differentiation (Hsu and Drummond-Barbosa, 2011; Sun et al., 2010). Environmental factors such as food abundance and population density also influence the rate of proliferation and the mitosis-to-meiosis decision in C. elegans. Limited food availability restricts the size of the stem cell pool during larval growth, and starvation leads to a dramatic resorption of the germline. Even under these conditions, about 30 stem cells remain, which, if conditions improve, can regenerate the entire germline (Angelo and Van Gilst, 2009). The Insulin/IGF and Tor/S6K pathways regulate germline proliferation during larval stages (Korta et al., 2012; Michaelson et al., 2010). Favorable conditions are also sensed by a group of neurons located close to the pharynx, triggering activation of the TGFb receptor in the DTC and promoting GSC proliferation (Dalfo et al., 2012). In addition to proliferation, Tor/S6K and TGFβ also affect the mitosis-to-meiosis decision. Insulin-, Tor-, and TGFβ-pathway mutants exacerbate the glp-1 phenotype, suggesting that these pathways exert their function at least in part independently of the Notch regulatory network. How these sensors and mediators of environmental conditions are integrated at the level of germ cell self-renewal and differentiation is not understood.

Environmental stimuli may also regulate the SGP pool in the mouse testis. Recent studies show that PLZF and mTORC1, one of two Tor complexes that mediate cell growth in response to nutrients, growth factors, and cellular stress in mammals, are engaged in a negative feedback loop that can shift the self-renewal versus differentiation balance depending on environmental conditions. PLZF represses mTORC1 via activation of Redd1, an mTORC1 inhibitor; mTORC1 activation in turn leads to downregulation of the GDNF receptors (Hobbs et al., 2010). Thus, PLZF expression in SPGs may render these cells less sensitive to environmental influence. Furthermore, the ability of mTORC1 to downregulate GDNF receptors could entice SGPs to leave their niche and initiate the differentiation program. The balanced interactions of PLZF with SALL4 and mTORC1 nicely exemplify how small, local changes in the levels of regulators of cell growth and differentiation can contribute to the heterogeneity and potential of the SGP population.

Summary and Outlook

Systematic analysis of germ cell development in the embryo and stem cell homeostasis in the adult gonad has brought insight into the molecular mechanisms that establish and regulate niche-stem cell interactions. In contrast to all other stem cell systems in the body, homeostasis of the gonadal system is directly related to reproductive success. Thus specific mechanisms such as global epigenetic regulation of the germ cell program and the need for a complete reprogramming of the germ cell genome during development are features of essential importance for germ cells to give rise to a new generation. How transgenerational inheritance is transmitted at the gene and genome level is still largely unclear.

The regulatory networks controlling GSC self-renewal and differentiation are beginning to be unraveled. These include germ-cell-specific RNA regulators that are conserved between species. The targets of these regulators are still largely unknown, but in some cases they control strikingly similar processes, such as the role of Nanos family proteins in PGC specification and GSC maintenance in worms, flies, and mice. These RNA regulators, together with their targets, which include small regulatory RNAs, are packaged into intracellular particles that change in composition and cellular location during the germ cell life cycle. Compartmentalization may allow the same RBP to entertain multiple regulatory relationships during different stages of germline development.

Niche-stem cell interactions play a crucial role for GSC homeostasis. These interactions protect GSCs from differentiation, regulate proliferation, and, in some cases, orient the GSC-daughter cell division. In Drosophila, the path from GSC to differentiated germline cyst was considered to be rather hierarchical until the discovery that male and female GSCs can regenerate from advanced, interconnected germline cysts (Brawley and Matunis, 2004; Kai and Spradling, 2004; Wong and Jones, 2012). In the mouse testis, heterogeneity among SPGs seems to be the rule rather than the exception, since more advanced cysts routinely contribute to the precursor pool. The functional and hereditary consequences of this plasticity are not understood.

Germ cells, through their potential to differentiate into sperm and egg, have the ability to create a new organism. Analysis of the regulatory networks that control germ cell specification, self-renewal, and differentiation may ultimately lead to a better understanding of the control mechanisms that balance the need for genomic fidelity with the opportunity for evolutionary change.