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.     

Headcase Promotes Cell Survival and Niche Maintenance in the Drosophila Testis

At the apical tip of the Drosophila testis, germline and somatic stem cells surround a cluster of somatic cells called the hub. Hub cells produce a self-renewal factor, Unpaired (Upd), that activates the JAK-STAT pathway in adjacent stem cells to regulate stem cell behavior. Therefore, apical hub cells are a critical component of the stem cell niche in the testis. In the course of a screen to identify factors involved in regulating hub maintenance, we identified headcase (hdc). Hub cells depleted for hdc undergo programmed cell death, suggesting that anti-apoptotic pathways play an important role in maintenance of the niche. Using hdc as paradigm, we describe here the first comprehensive analysis on the effects of a progressive niche reduction on the testis stem cell pool. Surprisingly, single hub cells remain capable of supporting numerous stem cells, indicating that although the size and number of niche support cells influence stem cell maintenance, the testis stem cell niche appears to be remarkably robust in the its ability to support stem cells after severe damage.     

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.     

Socs36E Controls Niche Competition by Repressing MAPK Signaling in the Drosophila Testis

The Drosophila testis is a well-established system for studying stem cell self-renewal and competition. In this tissue, the niche supports two stem cell populations, germ line stem cells (GSCs), which give rise to sperm, and somatic stem cells called cyst stem cells (CySCs), which support GSCs and their descendants. It has been established that CySCs compete with each other and with GSCs for niche access, and mutations have been identified that confer increased competitiveness to CySCs, resulting in the mutant stem cell and its descendants outcompeting wild type resident stem cells. Socs36E, which encodes a negative feedback inhibitor of the JAK/STAT pathway, was the first identified regulator of niche competition. The competitive behavior of Socs36E mutant CySCs was attributed to increased JAK/STAT signaling. Here we show that competitive behavior of Socs36E mutant CySCs is due in large part to unbridled Mitogen-Activated Protein Kinase (MAPK) signaling. In Socs36E mutant clones, MAPK activity is elevated. Furthermore, we find that clonal upregulation of MAPK in CySCs leads to their outcompetition of wild type CySCs and of GSCs, recapitulating the Socs36E mutant phenotype. Indeed, when MAPK activity is removed from Socs36E mutant clones, they lose their competitiveness but maintain self-renewal, presumably due to increased JAK/STAT signaling in these cells. Consistently, loss of JAK/STAT activity in Socs36E mutant clones severely impairs their self-renewal. Thus, our results enable the genetic separation of two essential processes that occur in stem cells. While some niche signals specify the intrinsic property of self-renewal, which is absolutely required in all stem cells for niche residence, additional signals control the ability of stem cells to compete with their neighbors. Socs36E is node through which these processes are linked, demonstrating that negative feedback inhibition integrates multiple aspects of stem cell behavior.     

Germ cells commit somatic stem cells to differentiation following priming by PI3K/Tor activity in the Drosophila testis

How and when potential becomes restricted in differentiating stem cell daughters is poorly understood. While it is thought that signals from the niche are actively required to prevent differentiation, another model proposes that stem cells can reversibly transit between multiple states, some of which are primed, but not committed, to differentiate. In the Drosophila testis, somatic cyst stem cells (CySCs) generate cyst cells, which encapsulate the germline to support its development. We find that CySCs are maintained independently of niche self-renewal signals if activity of the PI3K/Tor pathway is inhibited. Conversely, PI3K/Tor is not sufficient alone to drive differentiation, suggesting that it acts to license cells for differentiation. Indeed, we find that the germline is required for differentiation of CySCs in response to PI3K/Tor elevation, indicating that final commitment to differentiation involves several steps and intercellular communication. We propose that CySC daughter cells are plastic, that their fate depends on the availability of neighbouring germ cells, and that PI3K/Tor acts to induce a primed state for CySC daughters to enable coordinated differentiation with the germline.     

Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo

Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. Here, we show that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. We find that the driving force behind such behavior is accelerated proliferation. Our results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co‐opting an underlying homeostatic process.     

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.     

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.     

Hedgehog in the Drosophila testis niche: what does it do there?

Stem cell niche is a specialized microenvironment crucial to self-renewal. The testis in Drosophila contains two different types of stem cells, the germline stem cells and the somatic cyst stem cells that are sustained by their respective niche signals, thus is a good system for studying the interaction between the stem cells and their hosting niche. The JAK-STAT and BMP pathways are known to play critical roles in the self-renewal of different kinds of stem cells, but the roles of several other pathways have emerged recently in a complex signaling network in the testis niche. Reports of independent observations from three research groups have uncovered an important role of Hedgehog (Hh) in the Drosophila testis niche. In this review, we summarize these recent findings and discuss the interplay between the Hh signaling mechanisms and those of the JAK-STAT and BMP pathways. We also discuss directions for further investigation.     

The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals

The stem cell niches at the apex of Drosophila ovaries and testes have been viewed as distinct in two major respects. While both contain germline stem cells, the testis niche also contains "cyst progenitor" stem cells, which divide to produce somatic cells that encase developing germ cells. Moreover, while both niches utilize BMP signaling, the testis niche requires a key JAK/STAT signal. We now show, by lineage marking, that the ovarian niche also contains a second type of stem cell. These "escort stem cells" morphologically resemble testis cyst progenitor cells and their daughters encase developing cysts before undergoing apoptosis at the time of follicle formation. In addition, we show that JAK/STAT signaling also plays a critical role in ovarian niche function, and acts within escort cells. These observations reveal striking similarities in the stem cell niches of male and female gonads, and suggest that they are largely governed by common mechanisms.     

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.     

Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis

Stem cells will undergo self-renewal to produce new stem cells if they are maintained in their niches. The regulatory mechanisms that recruit and maintain stem cells in their niches are not well understood. In Drosophila testes, a group of 12 nondividing somatic cells, called the hub, identifies the stem cell niche by producing the growth factor Unpaired (Upd). Here, we show that Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherin-mediated cell adhesion. Loss of function of a Drosophila Rap-GEF (Gef26) results in loss of both germline and somatic stem cells. The Gef26 mutation specifically impairs adherens junctions at the hub-stem cell interface, which results in the stem cells "drifting away" from the niche and losing stem cell identity. Thus, the Rap signaling/E-cadherin pathway may represent one mechanism that regulates polarized niche formation and stem cell anchoring.     

Efficiency of spermatogonial dedifferentiation during aging

Background

Adult stem cells are critical for tissue homeostasis; therefore, the mechanisms utilized to maintain an adequate stem cell pool are important for the survival of an individual. In Drosophila, one mechanism utilized to replace lost germline stem cells (GSCs) is dedifferentiation of early progenitor cells. However, the average number of male GSCs decreases with age, suggesting that stem cell replacement may become compromised in older flies.

Methodology/Principal Findings

Using a temperature sensitive allelic combination of Stat92E to control dedifferentiation, we found that germline dedifferentiation is remarkably efficient in older males; somatic cells are also effectively replaced. Surprisingly, although the number of somatic cyst cells also declines with age, the proliferation rate of early somatic cells, including cyst stem cells (CySCs) increases.

Conclusions

These data indicate that defects in spermatogonial dedifferentiation are not likely to contribute significantly to an aging-related decline in GSCs. In addition, our findings highlight differences in the ways GSCs and CySCs age. Strategies to initiate or enhance the ability of endogenous, differentiating progenitor cells to replace lost stem cells could provide a powerful and novel strategy for maintaining tissue homeostasis and an alternative to tissue replacement therapy in older individuals.     

Distant activation of Notch signaling induces stem cell niche assembly

Here we show that multiple modes of Notch signaling activation specify the complexity of spatial cellular interactions necessary for stem cell niche assembly. In particular, we studied the formation of the germline stem cell niche in Drosophila ovaries, which is a two-step process whereby terminal filaments are formed first. Then, terminal filaments signal to the adjacent cap cell precursors, resulting in Notch signaling activation, which is necessary for the lifelong acquisition of stem cell niche cell fate. The genetic data suggest that in order to initiate the process of stem cell niche assembly, Notch signaling is activated among non-equipotent cells via distant induction, where germline Delta is delivered to somatic cells located several diameters away via cellular projections generated by primordial germ cells. At the same time, to ensure the robustness of niche formation, terminal filament cell fate can also be induced by somatic Delta via cis- or trans-inhibition. This exemplifies a double security mechanism that guarantees that the germline stem cell niche is formed, since it is indispensable for the adjacent germline precursor cells to acquire and maintain stemness necessary for successful reproduction. These findings contribute to our understanding of the formation of stem cell niches in their natural environment, which is important for stem cell biology and regenerative medicine.     

Stem cells signal to the niche through the Notch pathway in the Drosophila ovary

Stem cells are maintained and retain their capacity to continue dividing because of the influence of a niche. Although niches are important to maintain "stemness" in a wide variety of tissues, control of these niches is poorly understood. The Drosophila germline stem cells (GSCs) reside in a somatic cell niche. We show that Notch activation can induce the expression of niche-cell markers even in an adult fly; overexpression of Delta in the germline, or activated Notch in the somatic cells, results in extra niche cells, up to 10-fold over the normal number. In turn, these ectopic niche cells induce ectopic GSCs. Conversely, when GCSs do not produce functional Notch ligands, Delta and Serrate, the TGF-beta pathway is not activated in the GSCs, and they differentiate and subsequently leave the niche. Importantly, clonal analysis reveals that the receiving end of the Notch pathway is required in the somatic cells. These data show that a feedback loop exists between the stem cells and niche cells. Demonstration that stem cells can contribute to niche function has far-reaching consequences for stem cell therapies and may provide insight into how cancer can spread throughout an organism via populations of cancer stem cells.     

Extreme Divergence of Wolbachia Tropism for the Stem-Cell-Niche in the Drosophila Testis

Microbial tropism, the infection of specific cells and tissues by a microorganism, is a fundamental aspect of host-microbe interactions. The intracellular bacteria Wolbachia have a peculiar tropism for the stem cell niches in the Drosophila ovary, the microenvironments that support the cells producing the eggs. The molecular underpinnings of Wolbachia stem cell niche tropism are unknown. We have previously shown that the patterns of tropism in the ovary show a high degree of conservation across the Wolbachia lineage, with closely related Wolbachia strains usually displaying the same pattern of stem cell niche tropism. It has also been shown that tropism to these structures in the ovary facilitates both vertical and horizontal transmission, providing a strong selective pressure towards evolutionary conservation of tropism. Here we show great disparity in the evolutionary conservation and underlying mechanisms of stem cell niche tropism between male and female gonads. In contrast to females, niche tropism in the male testis is not pervasive, present in only 45% of niches analyzed. The patterns of niche tropism in the testis are not evolutionarily maintained across the Wolbachia lineage, unlike what was shown in the females. Furthermore, hub tropism does not correlate with cytoplasmic incompatibility, a Wolbachia-driven phenotype imprinted during spermatogenesis. Towards identifying the molecular mechanism of hub tropism, we performed hybrid analyses of Wolbachia strains in non-native hosts. These results indicate that both Wolbachia and host derived factors play a role in the targeting of the stem cell niche in the testis. Surprisingly, even closely related Wolbachia strains in Drosophila melanogaster, derived from a single ancestor only 8,000 years ago, have significantly different tropisms to the hub, highlighting that stem cell niche tropism is rapidly diverging in males. These findings provide a powerful system to investigate the mechanisms and evolution of microbial tissue tropism.