The germ plasm is a specialized region of cytoplasm in oocytes and early embryos that is both necessary and sufficient to direct cells to enter germline development, and thus, is believed to harbor "determinants" that singly or collectively specify germ cell fate (Ikenishi, 1998; Wylie, 1999). The identity of these determinants, however, remains elusive. With few exceptions, the factors required to specify germ cell fate are still unknown. Among the germ plasm components identified in C. elegans thus far, PIE-1 and
nos-2 are the only ones with a known function in primordial germ cells (PGCs). PIE-1 is a putative RNA binding protein characterized by two CCCH fingers (Mello et al, 1996). CCCH fingers in other proteins, such as mammalian TIS11, have been shown to bind to mRNAs and have been implicated in mRNA stability (Lai et al, 2000). Work in our lab has shown that missense mutations in the second CCCH finger (ZF2) of PIE-1 result in a very specific maternal-effect sterile phenotype (Tenenhaus et al, 2001). Embryos derived from mothers that express the ZF2 mutant version of PIE-1 make abnormal PGCs, which are not associated with the somatic gonad, and often remain on the surface of the embryo. As a result, these embryos develop into sterile animals with empty gonads. These observations suggest that PIE-1 regulates PGC development by binding to and regulating specific mRNAs. Consistent with this hypothesis, PIE-1 has been shown to be required for the efficient translation of the germ plasm mRNA,
nos-2.
nos-2 is a maternal RNA that segregates with the germ plasm and is enriched on P granules. The NOS-2 protein is translated specifically in primordial germ cells and is required for these cells to associate efficiently with the somatic gonad (Subramaniam and Seydoux, 1999). NOS-2 protein is significantly decreased in PIE-1 ZF2 mutant embryos, suggesting that
nos-2 RNA is not translated efficiently in these mutant embryos (Tenenhaus et al, 2001). Interestingly, the PIE-1 ZF2 mutant phenotype is more severe than the
nos-2 mutant phenotype (in the latter, the primordial germ cells manage to gastrulate, but have trouble joining up with the somatic gonad). These data strongly suggest that PIE-1 regulates other germ plasm mRNAs in addition to
nos-2 . We are attempting to identify new germ plasm components required for primordial germ cell development, using the essential germline protein, PIE-1. A biochemical method using immunoprecipitation, developed for C. elegans by Lee and Schedl, will be used to identify RNAs and proteins that are complexed with PIE-1 in vivo (Lee and Schedl, manuscript in preparation). In preliminary experiments, we have shown that PIE-1 can be efficiently immunoprecipitated from embryonic cell extracts, using an anti-PIE-1 monoclonal antibody. We have also detected several bands, using SDS-PAGE, that are unique to PIE-1 IP samples. Further progress will be reported at the meeting.