We are interested in the mechanisms establishing muscle diversity in C. elegans. Analysis of pharyngeal myosin regulation led Pete Okkema to the identification of the first member of the NK2 class of homeodomain proteins,
ceh-22, which appears to mediate aspects of pharyngeal gene expression). NK class homeodomains have been shown to play myogenic roles in several diverse organisms, but have also been implicated in nonmyogenic functions.We have now identified three additional members of this family, initially using degenerate PCR (
ceh-24) and subsequently genome gazing (
ceh-27 and
ceh-28). We are in the process of characterizing both expression and function for these genes, with a focus on regulation and activity in muscle development. To date, our analysis has proceeded furthest with
ceh-24: These results will be presented. We have begun a genetic analysis of
ceh24 using a TC1 insertion derived from a mutator strain (provided by R. Plasterk). The TC1 has inserted in the coding region near the C-terminus. Using this strain we have generated four deletion alleles. All four alleles delete the homeodomain and an acidic patch just downstream. Each is homozygous viable. The largest deletion (ccS39) has been outcrossed extensively and we have begun to characterize this strain. ccS39 worms have functional vulval muscle, mate normally, and appear indistinguishable from wild type worms. We are presently constructing double mutants with other loci involved in myogenesis to further analyze the deletion alleles. Expression studies with
ceh-24::gfp and
ceh-24::1acZfusions indicate that the
ceh-24 promoter is active in muscle
m8 of the pharynx, the vulval muscles, and a subset of the sublateraVsubventral head neurons. The sequences responsible for vulval and
m8 reporter gene expression have been identified. Vulval expression is dependent on two elements, a GAGA sequence and two repeated sequences designated "NdEboxes" (these are divergent E boxes containing the core sequence CATATG). Concatomers of NdEboxes can activate a naive promoter in vulval muscles. Extensive mutational analysis has generated a set of derivatives of the NdEbox which have been characterized for activity in vivo. These are being used in screens to identify transactivators responsible for expression in differentiating vulval muscles. Expression in
m8 is dependent on a 115bp sequence. Three regions of this fragment are necessary for
m8 expression. These contain GAGA sequences, a pair of GATA's, and a CT rich region respectively. 1. P. Okkema and A. Fire, Development 120:2175-2186