Stage-specific differences in the requirements for germline stem cell maintenance in the Drosophila ovary.
ABSTRACT: In this study, we uncover a role for microRNAs in Drosophila germline stem cell (GSC) maintenance. Disruption of Dicer-1 function in GSCs during adult life results in GSC loss. Surprisingly, however, loss of Dicer-1 during development does not result in a GSC maintenance defect, although a defect is seen if both Dicer-1 and Dicer-2 function are disrupted. Loss of the bantam microRNA mimics the Dicer-1 maintenance defect when induced in adult GSCs, suggesting that bantam plays a key role in GSC self-renewal. Mad, a component of the TGF-beta pathway, behaves similarly to Dicer-1: adult GSC maintenance requires Mad if it is lost during adult life, but not if it is lost during pupal development. Overall, these results show stage-specific differential sensitivity of GSC maintenance to certain perturbations, and suggest that there may be Dcr-2 dependent redundancy of GSC maintenance mechanisms during development that is lost in later life.
Project description:Animal reproduction responds to nutritional status. During starvation, Drosophila and Caenorhabditis elegans enter a period of reproductive diapause with increase apoptosis, while maintaining a stable pool of germline stem cells (GSCs). How GSCs are protected is not understood. Here, we show that a sisRNA/miRNA axis maintains ovarian GSCs during starvation in Drosophila. Starvation induces the expression of an ovary-enriched sisRNA sisR-2, which negatively regulates GSC maintenance via a fatty acid metabolism gene dFAR1. sisR-2 promotes the expression of bantam, which in turn inhibits the activity of sisR-2, forming a negative feedback loop. Therefore, bantam acts as a buffer to counteract sisR-2 activity to prevent GSC loss during starvation. We propose that the sisR-2/bantam axis confers robustness to GSCs in Drosophila.
Project description:In the Drosophila ovary, bone morphogenetic protein (BMP) signaling activated by the niche promotes germline stem cell (GSC) self-renewal and proliferation, whereas E-cadherin-mediated cell adhesion anchors GSCs in the niche for their continuous self-renewal. Here we show that Lissencephaly-1 (Lis1) regulates BMP signaling and E-cadherin-mediated adhesion between GSCs and their niche and thereby controls GSC self-renewal. Lis1 mutant GSCs are lost faster than control GSCs because of differentiation but not because of cell death, indicating that Lis1 controls GSC self-renewal. The Lis1 mutant GSCs exhibit reduced BMP signaling activity, and Lis1 interacts genetically with the BMP pathway components in the regulation of GSC maintenance. Mechanistically, Lis1 binds directly to and stabilizes the SMAD protein Mothers against decapentaplegic (Mad), facilitates its phosphorylation, and thereby regulates BMP signaling. Finally, the Lis1 mutant GSCs accumulate less E-cadherin in the stem cell-niche junction than do their wild-type counterparts. Germline-specific expression of an activated BMP receptor thickveins (Tkv) or E-cadherin can partially rescue the loss phenotype of Lis1 mutant GSCs. Therefore, this study has revealed a role of Lis1 in the control of Drosophila ovarian GSC self-renewal, at least partly by regulating niche signal transduction and niche adhesion. It has been known that Lis1 controls neural precursor/stem cell proliferation in the developing mammalian brain; this study further suggests that Lis1, which is widely expressed in adult mammalian tissues, could regulate adult tissue stem cells through modulating niche signaling and adhesion.
Project description:Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms.
Project description:Dedifferentiation is an important process to replenish lost stem cells during aging or regeneration after injury to maintain tissue homeostasis. Here, we report that Enhancer of Zeste [E(z)], a component of the Polycomb repression complex 2 (PRC2), is required to maintain a stable pool of germline stem cells (GSCs) within the niche microenvironment. During aging, germ cells with reduced E(z) activity cannot meet that requirement, but the defect arises from neither increased GSC death nor premature differentiation. Instead, we found evidence that the decrease of GSCs upon the inactivation of E(z) in the germline could be attributed to defective dedifferentiation. During recovery from genetically manipulated GSC depletion, E(z) knockdown germ cells also fail to replenish lost GSCs. Taken together, our data suggest that E(z) acts intrinsically in germ cells to activate dedifferentiation and thus replenish lost GSCs during both aging and tissue regeneration.
Project description:Stem cells must proliferate while maintaining 'stemness'; however, much remains to be learned about how factors that control the division of stem cells influence their identity. Multiple stem cell types display cell cycles with short G1 phases, thought to minimize susceptibility to differentiation factors. Drosophila female germline stem cells (GSCs) have short G1 and long G2 phases, and diet-dependent systemic factors often modulate G2. We previously observed that Cyclin E (CycE), a known G1/S regulator, is atypically expressed in GSCs during G2/M; however, it remained unclear whether CycE has cell cycle-independent roles in GSCs or whether it acts exclusively by modulating the cell cycle. In this study, we detected CycE activity during G2/M, reflecting its altered expression pattern, and showed that CycE and its canonical partner, Cyclin-dependent kinase 2 (Cdk2), are required not only for GSC proliferation, but also for GSC maintenance. In genetic mosaics, CycE- and Cdk2-deficient GSCs are rapidly lost from the niche, remain arrested in a G1-like state, and undergo excessive growth and incomplete differentiation. However, we found that CycE controls GSC maintenance independently of its role in the cell cycle; GSCs harboring specific hypomorphic CycE mutations are not efficiently maintained despite normal proliferation rates. Finally, CycE-deficient GSCs have an impaired response to niche bone morphogenetic protein signals that are required for GSC self-renewal, suggesting that CycE modulates niche-GSC communication. Taken together, these results show unequivocally that the roles of CycE/Cdk2 in GSC division cycle regulation and GSC maintenance are separable, and thus potentially involve distinct sets of phosphorylation targets.
Project description:Establishment and maintenance of functional stem cells is critical for organ development and tissue homeostasis. Little is known about the mechanisms underlying stem establishment during organogenesis. Drosophila testes are among the most thoroughly characterized systems for studying stem cell behavior, with germline stem cells (GSCs) and somatic cyst stem cells (CySCs) cohabiting a discrete stem cell niche at the testis apex. GSCs and CySCs are arrayed around hub cells that also comprise the niche and communication between hub cells, GSCs, and CySCs regulates the balance between stem cell maintenance and differentiation. Recent data has shown that functional, asymmetrically dividing GSCs are first established at ?23 h after egg laying during Drosophila testis morphogenesis (Sheng et al., 2009). This process correlates with coalescence of the hub, but development of CySCs from somatic gonadal precursors (SGPs) was not examined. Here, we show that functional CySCs are present at the time of GSC establishment, and that Jak-STAT signaling is necessary and sufficient for CySC maintenance shortly thereafter. Furthermore, hyper-activation of Jak in CySCs promotes expansion of the GSC population, while ectopic Jak activation in the germline induces GSC gene expression in GSC daughter cells but does not prevent spermatogenic differentiation. Together, these observations indicate that, similar to adult testes, Jak-STAT signaling from the hub acts on both GSCs and CySC to regulate their development and differentiation, and that additional signaling from CySCs to the GSCs play a dominant role in controlling GSC maintenance during niche formation.
Project description:It has been suggested that asymmetrically dividing Drosophila germline stem cells (GSCs) maintain their cytoplasmic connection with their daughters throughout most of the G1/S phase. We used live cell imaging to determine the fraction of GSCs sharing cytoplasm with preCB. The alpha-tubulin-tdEOS expressed in GSC was photoconverted in either GSC or preCB (differentiating daughter cell), and the redistribution of signal was followed every 10 seconds for more than130 seconds (Assay1). The percentage of connected pairs and the mass-transfer coefficient for each cases were determined by this study. Similarly, we expressed GFP-Mad protein to determine the GFP-Mad mass-transfer coefficient (Assay2). In the later experiment, we photo-bleached GFP-Mad localizing in entire GSC or preCB and followed recovery signal every 10 seconds for more than130 seconds.
Project description: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.
Project description:Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.
Project description:It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila melanogaster germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. We report that intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, we show that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. We further demonstrate that the miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling.