ABSTRACT: The germline stem cells (GSCs) of Drosophila melanogaster ovary provide an excellent model system to study the molecular mechanisms of stem cell self-renewal. To reveal novel factors required for Drosophila female GSC maintenance and/or division, we performed a loss-of-function screen in GSCs by using a collection of P-element-induced alleles of essential genes. Mutations in genes of various functional groups were identified to cause defects in GSC self-renewal. Here we report that a group of mutations affecting various ubiquitin-conjugating enzymes cause significant GSCs loss, including Plenty of SH3s (POSH), Ubiquitin-conjugating enzyme 10 (UbcD10), and pineapple eye (pie). Ubiquitin-mediated protein degradation plays a variety of roles in the regulation of many developmental processes, including mediating stem cell division through degradation of cell cycle regulators. We demonstrated that pie, sharing highly conserved RING domains with human E3 ubiquitin ligase G2E3 that are critical for early embryonic development, is specifically required for GSC maintenance, possibly through regulation of bone morphogenetic protein signaling pathway. Despite the previously reported role in imaginal disc cell survival, pie loss-of-function induced GSC loss is not to the result of caspase-involved cell death. Further efforts are needed to elucidate the functions of ubiquitin ligases in GSC maintenance, which will ultimately contribute to a better understanding of how the ubiquitin-conjugating enzymes regulate stem cell biology in mammalian systems.
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:Glioblastoma (GBM) is the most malignant and lethal brain tumor harboring glioma stem cells (GSCs) that promote tumor propagation and therapeutic resistance. GSCs preferentially express several critical cell surface molecules that regulate the pro-survival signaling for maintaining the stem cell-like phenotype. Tetraspanin CD9 has recently been reported as a GSC biomarker that is relevant to the GSC maintenance. However, the underlying molecular mechanisms of CD9 in maintaining GSC property remain elusive. Herein, we report that CD9 stabilizes the IL-6 receptor glycoprotein 130 (gp130) by preventing its ubiquitin-dependent lysosomal degradation to facilitate the STAT3 activation in GSCs. CD9 is preferentially expressed in GSCs of human GBM tumors. Mass spectrometry analysis identified gp130 as an interacting protein of CD9 in GSCs, which was confirmed by immunoprecipitation and immunofluorescent analyses. Disrupting CD9 or gp130 by shRNA significantly inhibited the self-renewal and promoted the differentiation of GSCs. Moreover, CD9 disruption markedly reduced gp130 protein levels and STAT3 activating phosphorylation in GSCs. CD9 stabilized gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote the BMX-STAT3 signaling in GSCs. Importantly, targeting CD9 potently inhibited GSC tumor growth in vivo, while ectopic expression of the constitutively activated STAT3 (STAT3-C) restored the tumor growth impaired by CD9 disruption. Collectively, we uncovered a critical regulatory mechanism mediated by tetraspanin CD9 to maintain the stem cell-like property and tumorigenic potential of GSCs.
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: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.
Project description: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:The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance. SOX2 is required for the maintenance of glioblastoma stem cells (GSCs). Here the authors identify that the RING family E3 ubiquitin ligase TRIM26 promotes SOX2 stability in a non-canonical ligase-independent manner and thus, increases the tumorigenicity of GSCs.
Project description:Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ?25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.
Project description:Stem cell regulation relies on extrinsic signaling from a niche plus intrinsic factors that respond and drive self-renewal within stem cells. <i>A priori</i>, loss of niche signaling and loss of the intrinsic self-renewal factors might be expected to have equivalent stem cell defects. Yet this simple prediction has not been borne out for most stem cells, including <i>Caenorhabditis elegans</i> germline stem cells (GSCs). The central regulators of <i>C. elegans</i> GSCs include extrinsically acting GLP-1/Notch signaling from the niche; intrinsically acting RNA-binding proteins in the PUF family, termed FBF-1 and FBF-2 (collectively FBF); and intrinsically acting PUF partner proteins that are direct Notch targets. Abrogation of either GLP-1/Notch signaling or its targets yields an earlier and more severe GSC defect than loss of FBF-1 and FBF-2, suggesting that additional intrinsic regulators must exist. Here, we report that those missing regulators are two additional PUF proteins, PUF-3 and PUF-11 Remarkably, an <i><i>fbf-1</i></i> <i><i>fbf-2</i></i> ; <i><i>puf-3</i></i> <i><i>puf-11</i></i> quadruple null mutant has a GSC defect virtually identical to that of a <i>glp-1</i> <i>/</i>Notch null mutant. PUF-3 and PUF-11 both affect GSC maintenance, both are expressed in GSCs, and epistasis experiments place them at the same position as FBF within the network. Therefore, action of PUF-3 and PUF-11 explains the milder GSC defect in <i><i>fbf-1</i></i> <i><i>fbf-2</i></i> mutants. We conclude that a "PUF hub," comprising four PUF proteins and two PUF partners, constitutes the intrinsic self-renewal node of the <i>C. elegans</i> GSC RNA regulatory network. Discovery of this hub underscores the significance of PUF RNA-binding proteins as key regulators of stem cell maintenance.
Project description: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.
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.