Regulation of germline stem cells in Drosophila through intrinsic and extrinsic factors: Insulin signaling, microRNA dependent regulation of Cyclin dependent kinase, and the TGF-beta pathway

This work examines the roles of known intrinsic and extrinsic factors regulating the function of Drosophila germline stem cells: the TGF-beta pathway, the insulin pathway, and the microRNA pathway. We have revealed hitherto unsuspected convolvement of these three pathways.;The recently discovered microRNA pathway post transcriptionally regulates the expression of genes by guiding silencing complexes containing Argonaute proteins to messenger RNA. The TGF-beta pathway is a cell to cell signaling pathway informing intracellular functions including growth and tissue identity. The insulin pathway serves to provide information on the nutrition available to cells within an organism.;We show that the insulin pathway regulates Drosophila GSCs by via microRNAs and regulates the G1 to S transition by way of the cyclin dependent kinase inhibitor Dacapo/p21/p27. We also identify two microRNAs active in the regulation of Drosophila GSCs. We further show that in Drosophila cells, the plant viral protein p19 is able to suppress the function of microRNAs, opening the possible use of this ectopic protein to study the function of microRNA in animals.;We then demonstrate a stage specific effect shared by the microRNA pathway and by a component of the the TGF-beta cell/cell signaling transcriptional factor, Mad by demonstrating that loss of Mad or Dicer-1, genes, essential for the TGF-beta or microRNA pathways, respectively, is tolerated by GSCs which are maintained in their niche if and only if either gene is lost in pre-adults. We also demonstrate a genetic interaction between Dicer-1 and Mad. We then show that the phosphorylated and transcriptionally active form of the Mad protein, pMad perdures in GSCs for many cell cycles after loss of the Mad gene.;Finally, we report on our use of plant viral protein, p19, as a tool for the analysis of the microRNA pathway in animals. We show that the expression of this viral gene derepresses a luciferase reporter that is repressed by microRNA and demonstrate the expression of a p19 transgene in the germline of Drosophila, offering the potential of isolating microRNA populations from specific cell types in heterogeneous tissues by immunoprecipitation of p19.