Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.
Elucidating the full repertoire of molecular mechanisms that promote stem cell maintenance requires sophisticated techniques for identifying and characterizing gene function in stem cells in their native environment. Ovarian germline stem cells in the fruit fly, Drosophila melanogaster, are an ideal model to study the complex molecular mechanisms driving stem cell function in vivo. A variety of new genetic tools make RNAi a useful complement to traditional genetic mutants for the investigation of the molecular mechanisms guiding ovarian germline stem cell function. Here, we provide a detailed guide for using targeted RNAi knockdown for the discovery of gene function in ovarian germline stem cells and their progeny.
Background Reproductive capacity in many organisms is maintained by germline stem cells (GSCs). A complex regulatory network influences stem cell fate, including intrinsic factors, local signals, and hormonal and nutritional cues. Posttranscriptional regulatory mechanisms ensure proper cell fate transitions, promoting germ cell differentiation to oocytes. As essential RNA binding proteins with constitutive functions in RNA metabolism, heterogeneous nuclear ribonucleoproteins (hnRNPs) have been implicated in GSC function and axis specification during oocyte development. HnRNPs support biogenesis, localization, maturation, and translation of nascent transcripts. Whether and individual hnRNPs specifically regulate GSC function has yet to be explored. Results We demonstrate that hnRNPs are expressed in distinct patterns in the Drosophila germarium. We show that three hnRNPs, squid, hephaestus, and Hrb27C are cell‐autonomously required in GSCs for their maintenance. Although these hnRNPs do not impact adhesion of GSCs to adjacent cap cells, squid and hephaestus (but not Hrb27C) are necessary for proper bone morphogenetic protein signaling in GSCs. Moreover, Hrb27C promotes proper GSC proliferation, whereas hephaestus promotes cyst division. Conclusions We find that hnRNPs are independently and intrinsically required in GSCs for their maintenance in adults. Our results support the model that hnRNPs play unique roles in stem cells essential for their self‐renewal and proliferation.
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