Ballchen is required for self-renewal of germline stem cells in Drosophila melanogaster.
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ABSTRACT:
SUBMITTER: Herzig B
PROVIDER: S-EPMC4058086 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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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.
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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.
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Project description:Adults maintain tissue-specific stem cells through niche signals. A model for niche function is the Drosophila melanogaster testis, where a small cluster of cells called the hub produce locally available signals that allow only adjacent cells to self-renew. We show here that the principal signalling pathway implicated in this niche, chemokine activation of STAT, does not primarily regulate self-renewal of germline stem cells (GSCs), but rather governs GSC adhesion to hub cells. In fact, GSC renewal does not require hub cell contact, as GSCs can be renewed solely by contact with the second resident stem cell population, somatic cyst stem cells (CySCs), and this involves BMP signalling. These data suggest a modified paradigm whereby the hub cells function as architects of the stem cell environment, drawing into proximity cellular components necessary for niche function. Self-renewal functions are shared by the hub cells and the CySCs. This work also reconciles key differences in GSC renewal between Drosophila testis and ovary niches, and highlights how a niche can coordinate the production of distinct lineages by having one stem cell type rely on a second.
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Project description:The ability of adult stem cells to maintain their undifferentiated state depends upon residence in their niche. While simple models of a single self-renewal signal are attractive, niche-stem cell interactions are likely to be more complex. Many niches have multiple cell types, and the Drosophila testis is one such complex niche with two stem cell types, germline stem cells (GSCs) and somatic cyst progenitor cells (CPCs). These stem cells require chemokine activation of Jak/STAT signaling for self-renewal. We identified the transcriptional repressor Zfh-1 as a presumptive somatic target of Jak/STAT signaling, demonstrating that it is necessary and sufficient to maintain CPCs. Surprisingly, sustained zfh-1 expression or intrinsic STAT activation in somatic cells caused neighboring germ cells to self-renew outside their niche. In contrast, germline-intrinsic STAT activation was insufficient for GSC renewal. These data reveal unexpected complexity in cell interactions in the niche, implicating CPCs in GSC self-renewal.
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Project description:Germline stem (GS) cells were established from gonocytes and spermatogonia of postnatal mouse testes. GS cells proliferate in the presence of several kinds of cytokines, and a small percentage of GS cells also show spermatogonial stem cell (SSC) activity, i.e., they differentiate into sperm after being transplanted into infertile mouse testes without endogenous spermatogenesis. Interestingly, in GS cell culture, we also found that pluripotent stem cells (multipotent germline stem cells (mGS cells)) could be derived and these mGS cells do not have normal androgenetic genomic imprinting marks that are shown in GS cells, e.g., H19 hypermethylation. A new culture system for fetal male germ cells (embryonic GS (eGS) cells) has also been recently developed. Although these cells exhibited SSC potential, the offspring from cultured cells showed heritable imprinting defects in their DNA methylation patterns. In an attempt to understand the self-renewal machinery in SSCs, we transfected H-Ras and cylin D2 into GS cells, and successfully reconstructed the SSC self-renewal ability without using exogenous cytokines. Although these cells showed SSC activity in germ cell transplantation assays, we also found development of seminomatous tumors, possibly induced by excessive self-renewing signal. These stem cell culture systems are useful tools not only for understanding the mechanisms of self-renewal or epigenetic reprogramming but also for clarifying the mechanism of germ cell tumor development.
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Project description:Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.
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Project description:Self-renewal and differentiation in germline stem cells (GSCs) are tightly regulated by the stem cell niche and via multiple approaches. In our previous study, we screened the novel GSC regulatory gene Srlp in Drosophila testes. However, the underlying mechanistic links between Srlp and the stem cell niche remain largely undetermined. Here, using genetic manipulation of the Drosophila model, we systematically analyze the function and mechanism of Srlp in vivo and in vitro. In Drosophila, Srlp is an essential gene that regulates the self-renewal and differentiation of GSCs in the testis. In the in vitro assay, Srlp is found to control the proliferation ability and cell death in S2 cells, which is consistent with the phenotype observed in Drosophila testis. Furthermore, results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) reveal that RpL6 binds to Srlp. Srlp also regulates the expression of spliceosome and ribosome subunits and controls spliceosome and ribosome function via RpL6 signals. Collectively, our findings uncover the genetic causes and molecular mechanisms underlying the stem cell niche. This study provides new insights for elucidating the pathogenic mechanism of male sterility and the formation of testicular germ cell tumor.
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Project description:Stem cell self-renewal is controlled by concerted actions of extrinsic niche signals and intrinsic factors in a variety of systems. Drosophila ovarian germline stem cells (GSCs) have been one of the most productive systems for identifying the factors controlling self-renewal. The differentiation factor BAM is necessary and sufficient for GSC differentiation, but it still remains expressed in GSCs at low levels. However, it is unclear how its function is repressed in GSCs to maintain self-renewal. Here, we report the identification of the translation initiation factor eIF4A for its essential role in self-renewal by directly inactivating BAM function. eIF4A can physically interact with BAM in Drosophila S2 cells and yeast cells. eIF4A exhibits dosage-specific interactions with bam in the regulation of GSC differentiation. It is required intrinsically for controlling GSC self-renewal and proliferation but not survival. In addition, it is required for maintaining E-cadherin expression but not BMP signaling activity. Furthermore, BAM and BGCN together repress translation of E-cadherin through its 3' UTR in S2 cells. Therefore, we propose that BAM functions as a translation repressor by interfering with translation initiation and eIF4A maintains self-renewal by inhibiting BAM function and promoting E-cadherin expression.
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Project description:The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNPs) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, we demonstrated that Prp3 is essential for male fertility in Drosophila. Prp3 deficiency in germline stem cells (GSCs) and early cyst cells resulted in abnormal structure of testes and maintenance defects of GSCs and cyst stem cells. Knockdown of Prp3 in spermatogonia and early cyst cells mediated tumor formation caused by differentiation defects. Using an in vitro assay, knockdown of Prp3 decreased proliferation and increased cell death, and controlled the spliceosome function via regulating spliceosome subunits expression in Drosophila S2 cells. We also identified two other splicing factors in the Prp complex (Prp19 and Prp8), which mimicked the phenotype of Prp3 in the Drosophila stem cell niche. Our results revealed a significant role of precursor RNA processing factors in male testes, indicating that Prp3, a key spliceosome component in the Prp complex, is essential for male fertility, and germline stem cell self-renewal and differentiation, via regulating the spliceosome function in Drosophila testes.
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