Development of the male germline stem cell niche in Drosophila

Stem cells are found in specialized microenvironments, or "niches", which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. Here, we investigate the formation of the testis GSC niche, or "hub", during the late stages of embryogenesis. By morphological and molecular criteria, we identify and follow the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad.     

The receptor tyrosine phosphatase Lar regulates adhesion between Drosophila male germline stem cells and the niche

The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. Here we show that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions.     

Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis

Germline stem cells (GSCs) in Drosophila are descendants of primordial germ cells (PGCs) specified during embryogenesis. The precise timing of GSC establishment in the testis has not been determined, nor is it known whether mechanisms that control GSC maintenance in the adult are involved in GSC establishment. Here, we determine that PGCs in the developing male gonad first become GSCs at the embryo to larval transition. This coincides with formation of the embryonic hub; the critical signaling center that regulates adult GSC behavior within the stem cell microenvironment (niche). We find that the Jak-STAT signaling pathway is activated in a subset of PGCs that associate with the newly-formed embryonic hub. These PGCs express GSC markers and function like GSCs, while PGCs that do not associate with the hub begin to differentiate. In the absence of Jak-STAT activation, PGCs adjacent to the hub fail to exhibit the characteristics of GSCs, while ectopic activation of the Jak-STAT pathway prevents differentiation. These findings show that stem cell formation is closely linked to development of the stem cell niche, and suggest that Jak-STAT signaling is required for initial establishment of the GSC population in developing testes.     

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.     

Maintenance of Stem Cell Niche Integrity by a Novel Activator of Integrin Signaling

Stem cells depend critically on the surrounding microenvironment, or niche, for their maintenance and self-renewal. While much is known about how the niche regulates stem cell self-renewal and differentiation, mechanisms for how the niche is maintained over time are not well understood. At the apical tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells share a common niche formed by hub cells. Here we demonstrate that a novel protein named Shriveled (Shv) is necessary for the maintenance of hub/niche integrity. Depletion of Shv protein results in age-dependent deterioration of the hub structure and loss of GSCs, whereas upregulation of Shv preserves the niche during aging. We find Shv is a secreted protein that modulates DE-cadherin levels through extracellular activation of integrin signaling. Our work identifies Shv as a novel activator of integrin signaling and suggests a new integration model in which crosstalk between integrin and DE-cadherin in niche cells promote their own preservation by maintaining the niche architecture.     

magu is required for germline stem cell self-renewal through BMP signaling in the Drosophila testis

Understanding how stem cells are maintained in their microenvironment (the niche) is vital for their application in regenerative medicine. Studies of Drosophila male germline stem cells (GSCs) have served as a paradigm in niche-stem cell biology. It is known that the BMP and JAK-STAT pathways are necessary for the maintenance of GSCs in the testis (Kawase et al., 2004; Kiger et al., 2001; Schulz et al., 2004; Shivdasani and Ingham, 2003; Tulina and Matunis, 2001). However, our recent work strongly suggests that BMP signaling is the primary pathway leading to GSC self-renewal (Leatherman and DiNardo, 2010). Here we show that magu controls GSC maintenance by modulating the BMP pathway. We found that magu was specifically expressed from hub cells, and accumulated at the testis tip. Testes from magu mutants exhibited a reduced number of GSCs, yet maintained a normal population of somatic stem cells and hub cells. Additionally, BMP pathway activity was reduced, whereas JAK-STAT activation was retained in mutant testes. Finally, GSC loss caused by the magu mutation could be suppressed by overactivating the BMP pathway in the germline.     

Dynamics of the male germline stem cell population during aging of Drosophila melanogaster

Drosophila melanogaster has emerged as an important model system for the study of both stem cell biology and aging. Much is known about how molecular signals from the somatic niche regulate adult stem cells in the germline, and a variety of environmental factors as well as single point mutations have been shown to affect lifespan. Relatively little is known, however, about how aging affects specific populations of cells, particularly adult stem cells that may be susceptible to aging-related damage. Here we show that male germline stem cells (GSCs) are lost from the stem cell niche during aging, but are efficiently replaced to maintain overall stem cell number. We also find that the division rate of GSCs slows significantly during aging, and that this slowing correlates with a reduction in the number of somatic hub cells that contribute to the stem cell niche. Interestingly, slowing of stem cell division rate was not observed in long-lived methuselah mutant flies. We finally investigated whether two mechanisms that are thought to be used in other adult stem cell types to minimize the effects of aging were operative in this system. First, in many adult tissues stem cells exhibit markedly fewer cell cycles relative to transit-amplifying cells, presumably protecting the stem cell pool from replication-associated damage. Second, at any given time not all stem cells actively cycle, leading to 'clonal succession' from the reserve pool of initially quiescent stem cells. We find that neither of these mechanisms is used in Drosophila male GSCs.     

Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event we term 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.     

Germline stem cells in the Drosophila ovary descend from pole cells in the anterior region of the embryonic gonad

A fundamental yet unexplored question in stem cell biology is how the fate of tissue stem cells is initially determined during development. In Drosophila, germline stem cells (GSCs) descend from a subset of primordial germ cells (PGCs) at the onset of oogenesis. GSC determination may occur at the onset of oogenesis when a subset of PGCs is induced to become GSCs by contacting niche cells. Alternatively, the GSC fate could be predetermined for a subset of PGCs before oogenesis, due to either their interaction with specific somatic cells in the embryonic/larval gonads, or their inherently heterogeneous potential in becoming GSCs, or both. Here, we show that anterior somatic cells in the embryonic gonad already differ from posterior somatic cells and are likely to be the precursors of niche cells in the adult ovary. Furthermore, only pole cells in the anterior half of the embryonic gonad give rise to the PGCs that frequently acquire contact with nascent niche cells in the late larval ovary. Eventually, only these contacting PGCs become GSCs, whereas non-contacting PGCs directly differentiate into cystoblasts. The strong preference of these 'anterior PGCs' towards contacting niche cells does not require DE-cadherin-mediated adhesion and is not correlated with either orientation or rate of their divisions. These data suggest that the GSC fate is predetermined before oogenesis. The predetermination probably involves soma/pole-cell interaction in the anterior half of the embryonic gonad, followed by an active homing mechanism during PGC proliferation to maintain the contact between the 'anterior PGCs' and anterior somatic cells.     

Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis

Stem cells are responsible for replacing damaged or dying cells in various adult tissues throughout a lifetime. They possess great potential for future regenerative medicine and gene therapy. However, the mechanisms governing stem cell regulation are poorly understood. Germline stem cells (GSCs) in the Drosophila testis have been shown to reside in niches, and thus these represent an excellent system for studying relationships between niches and stem cells. Here we show that Bmp signals from somatic cells are essential for maintaining GSCs in the Drosophila testis. Somatic cyst cells and hub cells express two Bmp molecules, Gbb and Dpp. Our genetic analysis indicates that gbb functions cooperatively with dpp to maintain male GSCs, although gbb alone is essential for GSC maintenance. Furthermore, mutant clonal analysis shows that Bmp signals directly act on GSCs and control their maintenance. In GSCs defective in Bmp signaling, expression of bam is upregulated, whereas forced bam expression in GSCs causes the GSCs to be lost. This study demonstrates that Bmp signals from the somatic cells maintain GSCs, at least in part, by repressing bam expression in the Drosophila testis. dpp signaling is known to be essential for maintaining GSCs in the Drosophila ovary. This study further suggests that both Drosophila male and female GSCs use Bmp signals to maintain GSCs.     

The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche

Maintenance of adult stem cells is largely dependent on the balance between their self-renewal and differentiation. The Drosophila ovarian germline stem cells (GSCs) provide a powerful in vivo system for studying stem cell fate regulation. It has been shown that maintaining the GSC population involves both genetic and epigenetic mechanisms. Although the role of epigenetic regulation in this process is evident, the underlying mechanisms remain to be further explored. In this study, we find that Enoki mushroom (Enok), a Drosophila putative MYST family histone acetyltransferase controls GSC maintenance in the ovary at multiple levels. Removal or knockdown of Enok in the germline causes a GSC maintenance defect. Further studies show that the cell-autonomous role of Enok in maintaining GSCs is not dependent on the BMP/Bam pathway. Interestingly, molecular studies reveal an ectopic expression of Bruno, an RNA binding protein, in the GSCs and their differentiating daughter cells elicited by the germline Enok deficiency. Misexpression of Bruno in GSCs and their immediate descendants results in a GSC loss that can be exacerbated by incorporating one copy of enok mutant allele. These data suggest a role for Bruno in Enok-controlled GSC maintenance. In addition, we observe that Enok is required for maintaining GSCs non-autonomously. Compromised expression of enok in the niche cells impairs the niche maintenance and BMP signal output, thereby causing defective GSC maintenance. This is the first demonstration that the niche size control requires an epigenetic mechanism. Taken together, studies in this paper provide new insights into the GSC fate regulation.     

Steroid signaling promotes stem cell maintenance in the Drosophila testis

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). Here, we show that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which we previously showed maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.     

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. Here we show that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. Our observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated.     

Cease and desist: modulating short-range Dpp signalling in the stem-cell niche

Drosophila ovarian germline stem cells (GSCs) are maintained by the extracellular BMP2/4 orthologue Dpp, which is produced from the surrounding somatic niche. The Dpp signal has a short range; it induces a response in GSCs within the niche, but is rapidly extinguished in their progeny only one cell-diameter away. To ensure the correct balance between stem-cell maintenance and differentiation, several regulatory mechanisms that modulate the Dpp signal at many stages of the pathway have been described. Here, we discuss the nature of the ovarian Dpp signal and review the catalogue of mechanisms that regulate it, demonstrating how the exquisite modulation of Dpp signalling in this context can result in precise and robust control of stem-cell fate. This modulation is applicable to other stem-cell environments that use BMPs as a niche signal, and the regulatory mechanisms are conceptually relevant to several other stem-cell systems.     

Notch signaling controls germline stem cell niche formation in the Drosophila ovary

Stem cells, which can self-renew and generate differentiated cells, have been shown to be controlled by surrounding microenvironments or niches in several adult tissues. However, it remains largely unknown what constitutes a functional niche and how niche formation is controlled. In the Drosophila ovary, germline stem cells (GSCs), which are adjacent to cap cells and two other cell types, have been shown to be maintained in the niche. In this study, we show that Notch signaling controls formation and maintenance of the GSC niche and that cap cells help determine the niche size in the Drosophila ovary. Expanded Notch activation causes the formation of more cap cells and bigger niches, which support more GSCs, whereas compromising Notch signaling during niche formation decreases the cap cell number and niche size and consequently the GSC number. Furthermore, the niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs. Finally, loss of Notch function in adults results in rapid loss of the GSC niche, including cap cells and thus GSCs. Our results indicate that Notch signaling is important for formation and maintenance of the GSC niche, and that cap cells help determine niche size and function.     

lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis

To function properly, tissue-specific stem cells must reside in a niche. The Drosophila testis niche is one of few niches studied in vivo. Here, a single niche, comprising ten hub cells, maintains both germline stem cells (GSC) and somatic stem cells (CySC). Here, we show that lines is an essential CySC factor. Surprisingly, lines-depleted CySCs adopted several characteristics of hub cells, including the recruitment of new CySCs. This led us to examine the developmental relationship between CySCs and hub cells. In contrast to a previous report, we did not observe significant conversion of steady-state CySC progeny to hub fate. However, we found that these two cell types derive from a common precursor pool during gonadogenesis. Furthermore, lines mutant embryos exhibited gonads containing excess hub cells, indicating that lines represses hub cell fate during gonadogenesis. In many tissues, lines acts antagonistically to bowl, and we found that this is true for hub specification, establishing bowl as a positively acting factor in the development of the testis niche.     

Scratching the niche that controls Caenorhabditis elegans germline stem cells

The Caenorhabditis elegans gonad provides a well-defined model for a stem cell niche and its control of self-renewal and differentiation. The distal tip cell (DTC) forms a mesenchymal niche that controls germline stem cells (GSCs), both to generate the germline tissue during development and to maintain it during adulthood. The DTC uses GLP-1/Notch signaling to regulate GSCs; germ cells respond to Notch signaling with a network of RNA regulators to control the decision between self-renewal and entry into the meiotic cell cycle.     

Clonal expansion of ovarian germline stem cells during niche formation in Drosophila

Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. We report that signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Our lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic (dpp), the Drosophila homolog of human bone morphogenetic protein 2/4, is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential dpp receptor, loses the ability to clonally populate a niche. Therefore, dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches.