RNA-binding protein regulation of germline development in zebrafish

Abstract

The developmental stages of oocyte maturation, egg fertilization, and early embryonic development take place while the genome is transcriptionally silent. During this period, posttranscriptional mechanisms regulate these vital developmental processes. The most prominent regulators are RNA-binding proteins (RNAbps) that assemble their targets into ribonucleoprotein granules where they are stored and regulated for activation at the appropriate time and place. The germ plasm is a specialized portion of cytoplasm in the oocyte that is required for germ cell specification and germline development in the embryo, and most known components of the zebrafish germ plasm include RNAbps. In this dissertation, I have characterized the functions of two RNAbps, Vasa and Rbpms2, in the development of the zebrafish germline.;The RNA-helicase Vasa is universally expressed in germ cells of all organisms, and is essential for various aspects of germline development. I have shown that the zebrafish vasa mutant germline develops normally until the juvenile stage, likely due to residual Vasa function from maternally deposited gene products. At the juvenile stage, vasa mutants have stem cell deficits resulting in reduced germ cell cluster sizes and germ cell marker loss. Germ cell loss occurs upon sexual maturation due to meiotic arrest, and zebrafish vasa mutants eventually become sterile males through a p53-independent process. Thus, as in other animals, zebrafish Vasa is required for germ cell survival and fertility.;Rbpms2 localizes to the germ plasm of oocytes where it interacts with RNAs and proteins with essential roles in reproduction and egg patterning; however, rbpms2 functions in the germline have not been rigorously studied. I have mutated the duplicated and redundant zebrafish rbpms2 genes, rbpms2a and rbpms2b. Zebrafish rbpms2a;2b (rbpms2) double mutants develop exclusively as fertile males. Zebrafish will differentiate as males if germ cell numbers are diminished or oogenesis fails. In rbpms2 mutant embryos primordial germ cell numbers are comparable to wild type with no discernable defects. Analysis of juvenile stages indicates comparable germ cell morphology in rbpms2 mutants until the onset of sexual differentiation, during which rbpms2 mutants fail to make mature oocytes. These findings reveal a novel and essential role for rbpms2 in oogenesis.