The goal of this project is to identify and characterize new germ plasm components that are required for primordial germ cell development in the nematode, Caenorhabditis elegans and, in so doing, gain a better understanding of the molecular mechanisms controlling germ cell development. To this end, two approaches will be used, one biochemical and one genetic, both of which will employ the germ plasm associated protein, PIE-1.
Specific Aim I, will use a biochemical approach to identify mRNAs that associate with PIE-1 in vivo. Endogenous PIE- 1 protein will be immunoprecipitated (IPed) from embryo protein extracts. mRNAs that bind to PIE-1 should co-IP, and can then be isolated by phenol/chloroform extraction.
In Specific Aim II, RNAi, in situ hybridization and immunofluorescent cytochemistry will be used to begin analyzing the function and possible role of the PIE- l associating RNAs in germ cell development.
Specific Aim III, will employ a genetic screen for maternal-effect-sterile mutants, performed in a pie-1/+ background. It is anticipated that this sensitized genetic background will allow the identification of previously undetectable maternal-effect-sterile phenotypes, specifically mutations that affect primordial germ cell development, and/or perhaps additional PIE-1 signaling elements.
