The C. elegans pharynx is a neuromuscular organ consisting of five distinct cell types. We have been characterizing how gene expression is regulated in one of these cell types, the pharyngeal muscles, by examining the pharyngeal muscle-specific myosin gene,
myo-2 .
myo-2 expression is regulated by a combination of cell type- and organ-specific signals. The organ-specific signals target a site in the
myo-2 enhancer, termed the C subelement. We and others have shown that the C subelement contains binding sites for multiple factors, including the Forkhead family transcription factor PHA-4 and the Smad protein DAF-3. We have identified another DNA-binding protein, PEB-1, that binds the C subelement and is strongly expressed in the pharynx (see abstract for this meeting by Thatcher, etal.). PEB-1 is a novel factor that does not share apparent similarity with any known DNA-binding protein. To learn more about PEB-1 function during pharyngeal development, we are following three experimental approaches. First, we are producing antibodies against PEB-1 to determine its tissue distribution and sub-cellular localization. Two PEB-1 fusion proteins have been used to immunize mice and rabbits. Polyclonal ascites fluid has been isolated from the mice and antibodies recognizing both PEB-1 fusion proteins are detectable on Western blots. Second, we are using a PCR-based screen to identify a
peb-1 deletion mutant (following the protocols of G. Moulder and R. Barstead).
peb-1 maps on the left arm of the X chromosome in a region containing no pharyngeal defective mutants, and RNAi has not produced an informative phenotype. We hope to use a deletion mutant to understand
peb-1 function in the pharynx. Third, we are examining the effect of expressing
peb-1 ectopically in C. elegans . Strains expressing the
peb-1 cDNA under control of the hsp 16-2 , hsp 16-41 ,
unc-54 , or
mec-7 promoters have been generated. Thus far we have not seen ectopic expression of pharyngeal-specific markers in these strains. However, the hsp::
peb-1 strains exhibit a heat-inducible lethal phenotype, and the
unc-54::
peb-1 strain displays an adult onset Unc phenotype. These phenotypes suggest we are expressing ectopic
peb-1 in these strains, and we will verify this using antibodies against PEB-1.