Project Summary Adult stem cells must meet the dual demands of lifelong persistence and continued production of derivatives, as needed. Highly proliferative stem cells that support continuously renewing epithelia, as in human skin and intestines, are especially important medical targets of investigation to understand how mutations initiate pre-cancerous amplification, and how stem cells might be used for regenerative therapies. Recent investigations of Follicle Stem Cells (FSCs) in the Drosophila ovary have illuminated a detailed picture of their behavior, including direct differentiation to two different cell types, locally graded proliferation, independent regulation of division and differentiation, and remarkable similarities to the organization of mammalian intestinal stem cells. FSCs now provide an exceptional opportunity to exploit Drosophila genetics, accessibility to comprehensive high-resolution fixed and live imaging, and a relatively simple system to examine the organization, dynamics and regulation of highly proliferative adult stem cells in detail. FSCs function as a community in which individual stem cells behave stochastically and compete for niche space. FSCs directly differentiate to a quiescent cell type at the anterior face of the FSC domain and to proliferative Follicle Cells (FCs) at the posterior face. FSCs also divide more rapidly in posterior regions and dynamically exchange locations. FSC behaviors are substantially guided by the strength of two graded signaling pathways (Wnt and JAK-STAT) of opposite polarity. Potential effectors of these signals will be sought, in part, through single-cell expression profiling. Their function in regulating FSC proliferation and differentiation will be tested using established lineage analyses of genetic variants that yield quantitative measures of location, cell division and differentiation responses. These will be supplemented by new methods that examine cell cycle reporters in fixed and live samples, and by developing “GRASP” reporters of surface interactions between marked FSCs and neighboring FCs, ECM and germline cysts to explore the mechanism of FSC conversion to FCs. Adult stem cell function depends on prior development of appropriate numbers and organization of stem cells and supporting niche cells. Recent insights show that FSCs and niche cells share common precursors, with fates acquired gradually through progressive refinement of cell locations. Potential signals and mediators that guide specification of the first FCs will be investigated to provide complementary insights to those gained from studying FC formation in adults, where the morphological changes and signaling environment differ significantly. Genetic manipulations affecting division rates, differentiation and signaling profiles of the whole FSC community will be used to expose community regulative mechanisms that can modify FSC behavior, responses or the signaling environment in order to maintain stem cell numb...