Caenorhabditis elegans pharyngeal muscle development involves
ceh-22, an NK-2 family homeobox gene related to genes controlling heart development in other species.
ceh-22 is the earliest known gene expressed in the pharyngeal muscles and is likely regulated directly by factors specifying pharyngeal muscle fate. We have previously implicated the
ceh-22 distal enhancer in initiating
ceh-22 expression. Here we analyze the distal enhancer using functional and comparative assays. The distal enhancer contains three subelements contributing additively to its activity, and functionally important regulatory sequences are highly conserved in Caenorhabditis briggsae. One subelement, termed DE3, is strongly active in the pharyngeal muscles, and we identified two short oligonucleotides (
de199 and
de209) contributing to DE3 activity. Multimerized
de209 enhances transcription similarly to DE3 specifically in the pharyngeal muscles, suggesting it may be an essential site regulating
ceh-22.
de209 binds the pan-pharyngeal Forkhead factor PHA-4 in vitro and responds to ectopic
pha-4 expression in vivo, suggesting that PHA-4 directly initiates
ceh-22 expression through
de209. Because
de209 enhancer activity is primarily limited to the pharyngeal muscles, we hypothesize that
de209 also binds factors functioning with PHA-4 to specifically activate
ceh-22 expression in pharyngeal