Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary

All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. Here we show that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (beta-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs through which E-cadherin transits are tightly associated with the fusome. We propose that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology.     

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.     

Stem cell aging is controlled both intrinsically and extrinsically in the Drosophila ovary

It is widely postulated that tissue aging could be, at least partially, caused by reduction of stem cell number, activity, or both. However, the mechanisms of controlling stem cell aging remain largely a mystery. Here, we use Drosophila ovarian germline stem cells (GSCs) as a model to demonstrate that age-dependent decline in the functions of stem cells and their niche contributes to overall stem cell aging. BMP signaling activity from the niche significantly decreases with age, and increasing BMP signaling can prolong GSC life span and promote their proliferation. In addition, the age-dependent E-cadherin decline in the stem cell-niche junction also contributes to stem cell aging. Finally, overexpression of SOD, an enzyme that helps eliminate free oxygen species, in either GSCs or their niche alone can prolong GSC life span and increase GSC proliferation. Therefore, this study demonstrates that stem cell aging is controlled extrinsically and intrinsically in the Drosophila ovary.     

The Drosophila ovary: an active stem cell community

Only a small number of cells in adult tissues (the stem cells) possess the ability to self-renew at every cell division,while producing differentiating daughter cells to maintain tissue homeostasis for an organism's lifetime.The Drosophilaovary harbors three different types of stem cell populations (germline stem cell (GSC),somatic stem cell (SSC) andescort stem cell (ESC)) located in a simple anatomical structure known as germarium,rendering it one of the best modelsystems for studying stem cell biology due to reliable stem cell identification and available sophisticated genetic toolsfor manipulating gene functions.Particularly,the niche for the GSC is among the first and best studied ones,and studieson the GSC and its niche have made many unique contributions to a better understanding of relationships between stemcells and their niche.So far,both the GSC and the SSC have been shown to be regulated by extrinsic factors originatingfrom their niche and intrinsic factors functioning within.Multiple signaling pathways are required for controlling GSCand SSC self-renewal and differentiation,which provide unique opportunities to investigate how multiple signals fromthe niche are interpreted in the stem cell.Since the Drosophila ovary contains three types of stem cells,it also providesoutstanding opportunities to study how multiple stem cells in a given tissue work collaboratively to contribute to tissuefunction and maintenance.This review highlights recent major advances in studying Drosophila ovarian stem cells andalso discusses future directions and challenges.     

Differentiation-defective stem cells outcompete normal stem cells for niche occupancy in the Drosophila ovary

Rapid progress has recently been made regarding how the niche controls stem cell function, but little is yet known about how stem cells in the same niche interact with one another. In this study, we show that differentiation-defective Drosophila ovarian germline stem cells (GSCs) can outcompete normal ones for niche occupancy in a cadherin-dependent manner. The differentiation-defective bam or bgcn mutant GSCs invade the niche space of neighboring wild-type GSCs and gradually push them out of the niche by upregulating E-cadherin expression. Furthermore, the bam/bgcn-mediated GSC competition requires E-cadherin and normal GSC division, but not the self-renewal-promoting BMP niche signal, while different E-cadherin levels can sufficiently stimulate GSC competition. Therefore, we propose that GSCs have a competitive relationship for niche occupancy, which may serve as a quality control mechanism to ensure that accidentally differentiated stem cells are rapidly removed from the niche and replaced by functional ones.     

Niche-associated activation of rac promotes the asymmetric division of Drosophila female germline stem cells

Background

Drosophila female germline stem cells (GSCs) reside adjacent to a cellular niche that secretes Bone Morphogenetic Protein (BMP) ligands and anchors the GSCs through adherens junctions. The GSCs divide asymmetrically such that one daughter remains in the niche as a GSC, while the other is born away from the niche and differentiates. However, given that the BMP signal can be diffusible, it remains unclear how a local extracellular asymmetry is sufficient to result in a robust pattern of asymmetric division.

Methods and Findings

Here we show that GSCs are polarized with respect to the cellular niche. We first use a modified biosensor to demonstrate that the small GTPase Rac is asymmetrically activated within the GSC at the niche-GSC interface. Experiments using loss-of-function and gain-of-function mutations in Rac indicate that asymmetric Rac activity both localizes the microtubule binding protein Apc2 to orient one GSC centrosome at the niche-GSC interface during interphase and activates the Jun N-terminal kinase pathway to increase the ability of the GSC to respond to BMP ligands. Other processes act in concert with each function of Rac. Specifically, we demonstrate that the GSC cell cycle arrests at prometaphase if centrosomes are misoriented.

Conclusions

Thus, the GSCs, an adult stem cell present in a cellular niche, have a niche-associated polarity that couples control of the division plane with increased response to an extracellular maintenance signal. Other processes work in parallel with the Rac-mediated polarity to ensure a robust pattern of asymmetric division. We suggest that all adult stem cells likely employ multiple, independently acting mechanisms to ensure asymmetric division to maintain tissue homeostasis.     

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.     

Self-maintained escort cells form a germline stem cell differentiation niche

Stem cell self-renewal is controlled by concerted actions of niche signals and intrinsic factors in a variety of systems. In the Drosophila ovary, germline stem cells (GSCs) in the niche continuously self-renew and generate differentiated germ cells that interact physically with escort cells (ECs). It has been proposed that escort stem cells (ESCs), which directly contact GSCs, generate differentiated ECs to maintain the EC population. However, it remains unclear whether the differentiation status of germ cells affects EC behavior and how the interaction between ECs and germ cells is regulated. In this study, we have found that ECs can undergo slow cell turnover regardless of their positions, and the lost cells are replenished by their neighboring ECs via self-duplication rather than via stem cells. ECs extend elaborate cellular processes that exhibit extensive interactions with differentiated germ cells. Interestingly, long cellular processes of ECs are absent when GSC progeny fail to differentiate, suggesting that differentiated germ cells are required for the formation or maintenance of EC cellular processes. Disruption of Rho functions leads to the disruption of long EC cellular processes and the accumulation of ill-differentiated single germ cells by increasing BMP signaling activity outside the GSC niche, and also causes gradual EC loss. Therefore, our findings indicate that ECs interact extensively with differentiated germ cells through their elaborate cellular processes and control proper germ cell differentiation. Here, we propose that ECs form a niche that controls GSC lineage differentiation and is maintained by a non-stem cell mechanism.     

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.     

小分子RNA决定果蝇生殖干细胞命运的分子机制研究

生殖干细胞是具有自我更新能力的一群生殖细胞,充当配子生成的源泉。果蝇生殖干细胞的特征在于通过不对称分裂产生两个子代细胞,一个通过自我更新维持干细胞特性,另一个则进行分化。生殖干细胞的命运受其周围的微环境——"干细胞niche"控制,而"niche"的功能又通过干细胞的外源和内源信号间的相互作用来完成。小分子RNA通过复杂的RNAi途径调控基因的表达。大量证据表明生殖干细胞的维持和分化需要小分子RNA参与,小分子RNA生成的紊乱会导致干细胞的"丢失"或"未分化"。该文综述了小分子RNA对果蝇生殖干细胞命运调控的研究进展,并讨论新发现的小分子RNA在生殖干细胞命运决定中的相关功能。     

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.     

Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation

The transition from a Drosophila ovarian germline stem cell (GSC) to its differentiated daughter cell, the cystoblast, is controlled by both niche signals and intrinsic factors. piwi and pumilio (pum) are essential for GSC self-renewal, whereas bag-of-marbles (bam) is required for cystoblast differentiation. We demonstrate that Piwi and Bam proteins are expressed independently of each other in reciprocal patterns in GSCs and cystoblasts. However, overexpression of either one antagonizes the other in these cells. Furthermore, piwi;bam double mutants phenocopy the bam mutant. This epistasis reflects the niche signaling function of piwi because depleting piwi from niche cells in bam mutant ovaries also phenocopies bam mutants. Thus, bam is epistatic to niche Piwi, but not germline Piwi function. Despite this, bam- ovaries lacking germline Piwi contain approximately 4-fold fewer germ cells than bam- ovaries, consistent with the role of germline Piwi in promoting GSC mitosis by 4-fold. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. We propose that niche Piwi maintains GSCs by repressing bam expression in GSCs, which consequently prevents Bam from downregulating Pum/Nos function in repressing the translation of differentiation genes and germline Piwi function in promoting germ cell division.     

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.