The NK-2 family homeodomain factor CEH-22 is an important regulator of gene expression in pharyngeal muscle, where it binds the enhancer and activates expression of the pharyngeal muscle-specific myosin gene,
myo-2 .
ceh-22 is expressed exclusively in pharyngeal muscle beginning at the bean stage of embryogenesis and is the earliest marker of pharyngeal muscle differentiation. A 1.9 kb promoter fragment is sufficient to direct reporter gene expression in a pattern identical to the endogenous
ceh-22 gene. We are characterizing regulatory sequences in the
ceh-22 promoter region to elucidate early steps in pharyngeal muscle development. We have identified two restriction fragments from the
ceh-22 promoter region that function as transcriptional enhancers and have designated them the distal and proximal enhancers. When assayed in transgenic lines, these enhancer fragments activate reporter gene expression in distinct temporal and spatial patterns. The distal enhancer fragment activates gene expression in both muscle and non-muscle cells in the pharynx, as well as in a subset of non-pharyngeal tissues. The earliest we have observed distal enhancer activity in the pharynx is at the bean stage. In contrast, the proximal enhancer fragment primarily activates gene expression in pharyngeal muscles. However, the earliest we observe proximal enhancer activity is at the 1-1/2 fold stage. We are interested in understanding how activity of both the proximal and distal enhancers is regulated. We have identified a 143 bp region near the 5' end of the proximal enhancer fragment that contributes substantially to pharyngeal muscle enhancer activity. Within this region we have found two distinct segments sufficient to activate reporter gene expression primarily in the same subset of pharyngeal muscles that express
ceh-22 . Interestingly, one of the segments contains a candidate CEH-22 binding site, suggesting the proximal enhancer may be a site of
ceh-22 autoregulation. Similarly, within the distal enhancer fragment we have localized a 119 bp fragment sufficient to enhance reporter gene expression specifically in pharyngeal muscle and marginal cells. We are analyzing both
ceh-22 enhancers and plan to use these sequences to identify factors regulating
ceh-22 expression.