The long-term objective of the proposed research is to elucidate cellular and molecular controls of growth and differentiation in the animal germline. The proposed experiments address three fundamental problems of reproductive biology. First, how are germ cells controlled to continue mitosis or enter meiosis? Second, how are germ cells controlled to differentiate as sperm or oocyte in response to somatic cues? Third, how are the transitions from germline stem cell to transit-amplifying cell and then to meiotic entry controlled by the stem cell niche? Our proposed experiments focus on the C. elegans nematode germline, which is superbly poised to address these fundamental problems with molecular clarity. The power of this model derives from its experimental tractability and the rich foundation already laid. A single cell, the distal tip cell (DTC), creates the stem cell niche and germ cell fates are controlled by an emerging network of conserved molecular regulators, including GLP-1/Notch signaling, FBF/PUF and FOG-1/CPEB RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase and MPK- 1/MAP kinase, among others. Indeed, where known, the conserved regulators retain conserved regulatory relationships and biological functions. For example, PUF proteins control germline stem cells in nematodes, flies and probably humans, and PUF proteins control MAP kinase expression in both nematodes and humans. Our specific aims will identify FBF target mRNAs and investigate conserved PUF controls of proliferation and differentiation; analyze the molecular mechanism by which FOG-1/CPEB controls both proliferation and differentiation in a dose-dependent manner; elucidate fog-3 regulation and investigate how FOG-3/Tob controls both proliferation and the sperm fate; investigate GLD-2 and its combinatorial control of germline fates; and investigate the Niche Region, the Transit Amplifying Compartment and their molecular regulation. The unifying theme is that the aims together will delineate how these conserved proteins work within a regulatory network to orchestrate germline growth and differentiation. Moreover, our studies promise to reveal general mechanisms that operate broadly in animal development, because of our focus on conserved regulators and their control of fundamental fate decisions. The elucidation of cellular and molecular controls of germline fate decisions will likely shed light on the mechanistic basis of germline cancers and infertility, two major health problems. Our studies may also impact use of germ cells as a potential source of stem cells for regenerative medicine. PUBLIC HEALTH RELEVANCE: This proposal investigates the molecular regulation of germline stem cells, germline transit-amplifying cells and the sperm/oocyte decision. Regulators of these germline fates, when defective, cause cancer or infertility, two major health problems. Moreover, germ cells may well be the ultimate source of stem cells for regenerative medicine to replace tissues in diseased or injured individuals.