The fertilized C. elegans egg is loaded with maternal mRNAs whose controlled temporal and spatial decoding will determine the course of embryogenesis. For example, the mRNA for the Delta-related ligand APX-1 is in every cell in the early embryo, but APX-1 protein must accumulate specifically in the P2 blastomere where it functions to differentiate the fates of the two anterior sister cells at the 4-cell stage. Similarly, the mRNA for the twin Zn-finger protein PIE-1 is everywhere but the PIE-1 protein is localized exclusively in germline blastomeres where it is required to prevent somatic differentiation. How is the expression and accumulation of these and other maternal gene products regulated? We have taken both a genetic and a yeast two hybrid based approach to investigate this question. The emerging story suggests that a complex regulatory network that consists of members of the PIE-1 protein family, PIE-1, POS-1, MEX-1, among others, collaborate with members of another conserved gene family that contains KH-domain and includes ALP-1, MEX-3, GLD-1 and others to control maternal mRNA expression. Members of each protein family can physically interact with each other in a partner-specific manner and exhibit intriguing phenotypic similarities and genetic interactions. For example, POS-1 and GLD-1 interact in the two-hybrid assay, and respective mutants exhibit very similar defects in the specification of the E-cell fate (gut) during early embryogenesis. Interestingly, although ALP-1 does not interact with POS-1 or GLD-1 and instead interacts with PIE-1, the
alp-1(
ne157) mutant or
alp-1(RNAi) can completely suppress the no gut phenotype associated with
pos-1 and
gld-1. This is particularly striking since
alp-1 single mutants produce fully penetrant sterile adults but have perfectly normal intestines. Thus it appears that
alp-1 acquires a novel interfering function that inhibits gut formation when
pos-1 or
gld-1 activity is absent. Russell Hill has shown that
pos-1 mutants lack the expression and activity of the APX-1 protein, and we find that
pos-1;
alp-1 double mutants appear nearly identical in phenotype to
apx-1 single mutants. Similarly, rare alleles of both
pos-1 and
mex-1, with missense mutations at conserved cysteines within the zinc finger motifs, exhibit
apx-1-like mutant phenotypes. These data suggest that these factors may regulate the translation of the
apx-1 mRNA as well as of mRNAs for other factors that either specify gut differentiation or can prevent gut differentiation when missexpressed. We propose that different members of the Zn-finger and KH-domain proteins may either positively or negatively control maternal protein expression based on their heterodimer composition and that at least part of the specificity comes from Zn-finger motifs. We think that the physical and genetic interactions we see are the tip of the iceberg. Sorting this complexity out will require new tools for expressing altered forms of each protein family member and for expressing altered candidate target mRNAs in vivo. For example, we would like to identify lesions that abolish specific in vitro protein-protein interactions and test these for their effects in vivo. Toward this end we have recently found that whole yeast genomic DNA prepared from a strain carrying the
pie-1 YAC can give a remarkably robust rescue of the
pie-1(
zu177) mutant strain (no pulse field gels are needed. We followed injection procedures similar to those outlined by Andrew Davies and Jocelyn Shaw, Worm Breeder's Gazette 15(1): 28, 1997). We are now using recombination in yeast to introduce specific point mutations into PIE-1 (in the YAC). In principal it would seem that there are no longer any real barriers to expressing transgenes in the germline. We hope to build new tools such as conditional transgenes for streamlining structure function studies in the germline. Investing in these tools is a must since it appears that understanding the embryo is not going to be a simple task.