Characterization of Factors Controlling Germline Stem Cell Maintenance in Drosophila Melanogaster

During Drosophila oogenesis, a germline stem cell (GSC) divides asymmetrically to produce a renewed stem cell and a differentiated daughter cystoblast (CB) that will progress through the 14 stages of oogenesis to produce a mature egg. A myriad of factors regulate GSC maintenance; extrinsic signals from the somatic niche integrate with intrinsic GSC factors to control CB differentiation pathway. In particular, studies of a key differentiation factor, bam, underscored the important paradigm of maintaining GSC by preventing the initiation of the CB differentiation pathway. The Dpp/BMP signaling pathway from the GSC niche promotes quiescence of bam transcription in the GSC. In the differentiating CB, bam transcription is initiated and Bam protein, together with its protein partner Bgcn, functions to antagonize the translational repression mediated by Pumilio-Nanos (Pum-Nos) complexes. Repression of Pum-Nos activity by Bam-Bgcn complexes permits CB differentiation, presumably through the derepression of CB-promoting mRNAs. In this study, I investigated the transcription- and translation-dependent mechanisms controlling the GSC to CB transition. Through genome-wide expression profiling experiments, I showed that there are only minimal transcriptional differences between the GSC and CB. This supports previous studies that highlight the importance of translational repression in maintaining a GSC state. However, some of the transcriptional differences between the undifferentiated GSC and differentiated germ cells were uncovered by expression profiling experiments of germ cells lacking Stonewall (Stwl), a protein required for GSC maintenance through epigenetic regulation of CB-promoting genes. Data from gene profiling experiments of stwl bam and bam mutant germ cells suggest that a suite of genes is normally repressed by Stwl to maintain a GSC fate. In addition, I examined whether putative "stemness" genes identified from mammalian systems also affected Drosophila GSC fate using a pilot screen in a hypomorphic bam background. Components from the COP9 signalosome complex (CSN) and the SCF E3 ubiquitin ligase complex were identified as dominant Suppressors of bam, Su(bam). Since both the CSN and SCF complexes are involved in protein degradation, the suppression of the bam phenotype suggests that they may be involved in stabilizing Bam function through abrogation of Bam turnover. Indeed, though ubiquitinated Bam isoforms were not identified, the abundance of Bam protein was increased in Su(bam) heterozygous animals. In other studies, I examined the requirement for the microRNA pathway in GSC maintenance through the examination of the double-stranded RNA-binding protein Loquacious (Loqs). Loqs enhances Dicer-1's ability to process pre-miRNA hairpin moieties to mature miRNA duplexes. Loqs is required for viability and germline mosaic analysis of loqs GSCs indicates an intrinsic, cell-autonomous requirement for the miRNA pathway in GSC maintenance. Loqs localizes to putative RNP complexes and a specialized region of the oocyte cytoplasm, termed the pole plasm. These data suggest that Loqs is a component of protein-RNA complexes that may be involved in mRNA translational inhibition. In summary, my studies revealed that GSC maintenance is achieved through the repression of CB-promoting factors; epigenetically through the actions of Stwl and other histone-associated proteins and translationally through the actions of the miRNA pathway via Loqs and Dicer-1. My studies provide insights into the understanding of the CB-promoting factors that are held inactive in the GSC and suggest that other stem cell systems may similarly employ multiple layers of repressive mechanisms to maintain a stem cell state.