We are interested in dissecting the network of genes that control pharynx development in C. elegans. Many of these genes encode transcription factors that function combinatorially to regulate the expression of target genes within the developing pharynx. One consequence of this combinatorial control is that there is significant redundancy within the pharyngeal gene network. One example of redundancy in the regulation of pharyngeal gene expression is the activity of two regulatory elements, Early-1 and Early-2, in the promoters of pharyngeally-expressed genes. Mutation of either element alone in the context of a pharyngeal promoter (K07C11.4) has only mild effects on expression, while simultaneous mutation of both sites abolishes expression (Gaudet et al. 2004). Our current goal is to identify the trans-acting factors that bind to the Early-1 and Early-2 elements. Here we focus on Early-2, which closely resembles a nuclear hormone receptor (NHR) half-site. C. elegans has ~270 NHR-encoding genes, some of which are expressed in the pharynx. Unfortunately, RNAi of candidate NHR genes has failed to identify an Early-2 factor and yeast one-hybrid screens did not yield promising candidates. We therefore employed a genetic screen to identify mutations affecting Early-2 activity. Our screen uses a transgenic strain in which CFP expression is dependent on Early-1 and YFP expression is dependent on Early-2. By screening for mutants that affect expression of YFP or CFP (but not both) we are able to rule out mutations that generally affect pharyngeal expression or transgene expression. Candidate mutants represent possible mutations of transcription factors that act through the Early-1 or Early-2 elements. We have so far identified 5 mutants that specifically affect Early-2-dependent YFP and are continuing to screen for mutations affecting Early-1-dependent CFP expression. The most promising mutant, mut
(iv1), completely abolishes YFP expression. Interestingly, these mutants possess a morphologically normal pharynx but are slow growing. This mild phenotype is consistent with the mutation affecting a gene that is redundantly involved in regulating pharynx gene expression. Our preliminary mapping places
iv1 in a region of LG V that has an abundance of NHR genes. We are currently mapping
iv1 to a smaller interval in an effort to clone the gene that affects Early-2 activity.