Novel roles for RNA binding proteins squid, hephaesteus, and Hrb27C in Drosophila oogenesis.

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.

Nuclear receptors linking physiology and germline stem cells in Drosophila.

Maternal nutrition and physiology are intimately associated with reproductive success in diverse organisms. Despite decades of study, the molecular mechanisms linking maternal diet to the production and quality of oocytes remain poorly defined. Nuclear receptors (NRs) link nutritional signals to cellular responses and are essential for oocyte development. The fruit fly, Drosophila melanogaster, is an excellent genetically tractable model to study the relationship between NR signaling and oocyte production. In this review, we explore how NRs in Drosophila regulate the earliest stages of oocyte development. Long-recognized as an essential mediator of developmental transitions, we focus on the intrinsic roles of the Ecdysone Receptor and its ligand, ecdysone, in oogenesis. We also review recent studies suggesting broader roles for NRs as regulators of maternal physiology and their impact specifically on oocyte production. We propose that NRs form the molecular basis of a broad physiological surveillance network linking maternal diet with oocyte production. Given the functional conservation between Drosophila and humans, continued experimental investigation into the molecular mechanisms by which NRs promote oogenesis will likely aid our understanding of human fertility.

Spargel/dPGC-1 is essential for oogenesis and nutrient-mediated ovarian growth in Drosophila.

Dietary proteins are crucial for oogenesis. The Target of Rapamycin (TOR) is a major nutrient sensor controlling organismal growth and fertility, but the downstream effectors of TOR signaling remain largely uncharacterized. We previously identified Drosophila Spargel/dPGC-1 as a terminal effector of the TOR-TSC pathway, and now report that Spargel connects nutrition to oogenesis. We found that Spargel is expressed predominantly in the ovaries of adult flies, and germline spargel knockdown inhibits cyst growth, ultimately leading to egg chamber degeneration and female sterility. In situ staining demonstrated nuclear localization of Spargel in the nurse cells and follicle cells of the ovariole. Furthermore, Spargel/dPGC-1 expression is influenced by dietary yeast concentration and TOR signaling, suggesting Spargel/dPGC-1 might transmit nutrient-mediated signals into ovarian growth. We propose that potentiating Spargel/dPGC-1 expression in the ovary is instrumental in nutrient-mediated regulation of oogenesis.

RNAi-Based Techniques for the Analysis of Gene Function in Drosophila Germline Stem Cells.

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.

A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis.

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.

Functional assessment of pharmacological telomerase activators in human T cells.

Telomeres are structures at the ends of chromosomes that shorten during cell division and eventually signal an irreversible state of growth arrest known as cellular senescence. To delay this cellular aging, human T cells, which are critical in the immune control over infections and cancer, activate the enzyme telomerase, which binds and extends the telomeres. Several different extracts from the Astragalus membranaceus root have been documented to activate telomerase activity in human T cells. The objective of this research was to compare two extracts from Astragalus membranaceus, TA-65 and HTA, for their effects on both telomerase and proliferative activity of human CD4 and CD8 T cells. Our results demonstrate that, TA-65 increased telomerase activity significantly (1.3 to 3.3-fold relative to controls) in T cell cultures from six donors tested, whereas HTA only increased telomerase levels in two out of six donors. We also demonstrate that TA-65 activates telomerase by a MAPK- specific pathway. Finally, we determine that during a three-day culture period, only the T cells treated with the TA-65 extract showed a statistically significant increase in proliferative activity. Our results underscore the importance of comparing multiple telomerase activators within the same experiment, and of including functional assays in addition to measuring telomerase activity.