C. elegans pharyngeal muscle development involves
ceh-22 , an NK-2 family homeobox gene structurally and functionally related to genes controlling heart development in other species.
ceh-22 is the first gene known to be expressed as cells commit to a pharyngeal muscle fate. We are studying how
ceh-22 expression is regulated to understand the early steps in pharyngeal muscle differentiation.
ceh-22 is expressed in a subset of pharyngeal muscle cells, beginning at the bean stage of embryogenesis. Its expression is regulated at the level of transcription, and within the
ceh-22 promoter we have identified two transcriptional enhancer regions, which we call the distal and proximal enhancers. The proximal enhancer is a pharyngeal muscle specific autoregulatory element that becomes active during embryogenesis shortly after the endogenous
ceh-22 gene, and we suggest it maintains
ceh-22 expression. The distal enhancer has a broader cell type specificity, but it becomes active in the pharynx at the bean stage of embryogenesis, coincident with expression of
ceh-22 . We believe that the distal enhancer is involved in initiating
ceh-22 expression. At least three separable subelements we call DE1 , DE2 , and DE3 , contribute to distal enhancer activity. DE3 appears to be most active and is sufficient to enhance abundant gene expression specifically in the
ceh-22 expressing muscles. We have identified two short segments of DE3 , which we call
de199 and
de209 , that together are sufficient to reproduce the activity of DE3 .
de199 appears to be a more general regulatory site that alone activates transcription in pharyngeal muscles, epithelial cells, marginal cells, as well as body wall muscle. In contrast,
de209 is a more specific regulatory site that activates transcription predominantly in the pharyngeal muscles.
de209 contains a consensus GATA factor binding site and a candidate binding site for PHA-4, a winged helix transcription factor that is expressed in all pharyngeal cell types and is required for
ceh-22 expression. Disruption of the PHA-4 site reduces
de209 activity in the pharynx, while ectopic expression of PHA-4 results in expression of a
de209 regulated reporter outside the pharynx. These results suggest that PHA-4 directly regulates
ceh-22 through
de209 . A mutation outside the PHA-4 and GATA sites also disrupts the activity of
de209 . We hypothesize this mutation affects a site for a factor that works with PHA-4 to activate
ceh-22 expression specifically in the pharyngeal muscles. We are currently performing a one-hybrid screen in yeast to identify this factor.