Germline Stem Cell Establishment and the Role of Dazl in Gametogenesis

PROJECT SUMMARY In all animals, including humans, establishment and maintenance of the germline stem cells (GSCs) is critical for life-long reproductive health and fitness. Yet, how GSCs are specified in vertebrates remains largely unknown. GSCs arise from primordial germ cells (PGCs) and serve to establish, renew, and expand the germ cell population. Differentiation of PGCs to GSCs is conserved and critical for establishment of the germline, yet a major gap in our knowledge of reproductive development and fertility remains understanding what factors influence this process. To examine early GSC development, I am using zebrafish – a genetically tractable model with high fecundity and rapid external development. Recently published data from our laboratory showed that, in zebrafish, PGCs lacking the conserved germline-specific RNA-binding protein Dazl (Deleted in Azoospermia- like) fail to differentiate into GSCs and are lost shortly thereafter. Dazl is a translational regulator which was previously implicated in meiosis; however, our finding that dazl mutant PGCs fail to differentiate raises interesting questions as to how GSCs arise from PGCs, and the specific role(s) of Dazl during this process. I hypothesize that Dazl translationally regulates its associated factors during PGC differentiation to maintain PGC germline fate and promote GSC establishment. However, Dazl has been characterized as both an activator and repressor of translation of thousands of germline targets in various contexts, making the specific mechanisms by which it promotes GSC establishment unclear. Herein, I propose to functionally define Dazl’s role in GSC establishment using a combination of genetic and molecular approaches. Specifically, my proposed research strategy will (1) trace dazl mutant germ cell fates to determine whether Dazl maintains PGC identity, (2) screen for GSC-specific markers to investigate the mechanism of GSC fate establishment, and (3) test the functions of conserved Dazl- associated mRNAs which I hypothesize are translationally regulated by Dazl during PGC differentiation. Together, these approaches will delineate Dazl’s role in PGC differentiation and GSC specification. More broadly, these studies may shed light on mechanisms to maintain, renew, and prevent premature loss of the GSC population, which has important implications for reproductive health. Completion of these aims will provide rigorous training in all aspects of genetics-based functional analysis, including generation, characterization, and validation of mutant and transgenic zebrafish lines, Cre-based lineage tracing, and genomic cloning, which will foster my development as a successful independent investigator committed to using genetic models to study reproductive development.