Regulation of Germ Cell Fate During Embryogenesis

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DESCRIPTION (provided by applicant): The germ line is essential for reproduction and the perpetuation of species; yet little is known about the molecular mechanisms that first distinguish germ cells from all other embryonic cells (somatic cells). The long-term goal of this proposal is to define these mechanisms using the genetic model system Caenorhabditis elegans. In this transparent worm, the germ cells arise from asymmetrically dividing precursors (germline blastomeres) during the first 2 hours of development. This proposal focuses on three evolutionarily conserved mechanisms essential for the establishment of the germline. The first mechanism involves asymmetric partitioning of maternal proteins to the nascent germline. In the previous funding period, we showed that CCCH finger proteins (putative RNA binding proteins) are targeted for degradation in somatic blastomeres by a novel E3 ubiquitin ligase, which recognizes CCCH fingers. CCCH proteins become asymmetrically distributed in germ line blastomeres before each asymmetric division, and we hypothesize that this asymmetry also results from localized protein degradation. We will test this hypothesis using a combination of transgenic studies and time-lapse analyses in live embryos. The second mechanism involves global inhibition of mRNA transcription in germline blastomeres by the CCCH finger protein PIE-1. Our previous work suggests that PIE-1 inhibits a kinase that phosphorylates the carboxy-terminal domain of RNA polymerase II. We will test this hypothesis by determining the activity in vivo of PIE-1 mutants and PIE-1 interacting proteins. The third mechanism involves translational activation of nanos RNA in primordial germ cells. Nanos is essential for primordial germ cell development, and our initial studies indicate that CCCH proteins regulate nanos expression by controlling both RNA stability and translational status. We will test this hypothesis using structure/function studies in vivo to define the functional and physical interactions connecting the CCCH proteins to the nanos 3'UTR (3' untranslated region). These studies will provide insights into basic developmental processes, including asymmetric partitioning of proteins and RNAs, transcriptional repression, translational regulation, and the control of germ cell fate. As our previous studies indicate, the many conserved characteristics between C. elegans and vertebrate germ cells make likely that principles gathered in this simple model will be applicable to other animals, including humans.