Pharyngeal muscle gene expression is activated by a combination of cell type- and organ-specific signals (1). In the myo-2 enhancer these distinct developmental pathways cooperate to activate transcription through discrete pharyngeal muscle-specific and organ-specific sub-elements which we refer to as B and C, respectively. The gene ceh-22 appears to be a key component of the pharyngeal muscle-specific pathway for activating gene expression (1). CEH-22 protein binds a site essential for B sub-element function and ectopic ceh-22 expression can activate expression of the endogenous myo-2 gene (1,2). We have previously described the mutant ceh-22(cc8266) that results in a partially penetrant L1 arrest phenotype (2). Although these mutants have pharyngeal muscle defects, expression of both myo-2 and an antigen recognized by MAb 3NB12 appear normal. We do not know if ceh-22(cc8266) is a null allele. The pha-1 gene is also required for normal differentiation of pharyngeal muscle, as well as differentiation of all other pharyngeal cell types (3). This organ-specific phenotype suggests pha-1 could function in parallel to ceh-22 as a direct activator of gene expression via the C sub-element. To explore the relationship between pha-1 and ceh-22 we have: 1) examined ceh-22 expression in a pha-1 mutant; 2) characterized pharyngeal muscle differentiation in a pha-1; ceh-22 double mutant; and 3) examined transcriptional activity of the C sub-element in a pha-1 mutant. The results of these experiments suggest that ceh-22 and pha-1 affect related processes required for pharyngeal muscle differentiation, but it is unlikely that pha-1 functions directly through C. ceh-22 is expressed normally in pha-1 mutant embryos: pha-1(e2123ts) animals raised at the non-permissive temperature (25C) were stained with anti-CEH-22 antibodies. In these mutants, both the temporal and spatial pattern of CEH-22 expression appeared normal indicating that pha-1 is not required upstream to activate ceh-22 expression. Synthetic interactions between ceh-22 and pha-1 mutations: The pharyngeal muscles of pha-1 null mutants and pha-1(e2123ts) mutants grown at 25! stain very well with the antibody 3NB123. Likewise, ceh-22(cc8266) mutants stain normally with this antibody. In contrast, pha-1(e2123ts); ceh-22(cc8266) double mutants raised at 25C show almost no 3NB12 staining. myo-2 expression also appears to be reduced in pha-1(e2123ts); ceh-22(cc8266) at 25C, however this reduction has been more difficult to characterize because myo-2 expression is already partially inhibited in the pha-1 single mutant. A similar synthetic interaction is also observed at the permissive temperature where pha-1(e2123ts) enhances the lethal phenotype of ceh-22(cc8266). This synergism between mutations in pha-1 and ceh-22 indicates the functions of these genes are closely related. pha-1 is not required for C sub-element function: To test whether the C sub-element requires wild-type pha-1, we assayed function of an enhancer consisting of four copies of C in wild type animals and in pha-1(e2123ts) grown at the non-permissive temperature. This enhancer activates expression of a linked lacZ reporter in pharyngeal muscle and non-muscle cells in both wild-type and pha-1 mutants. Thus, C sub-element function does not depend on wild-type pha-1. 1 Okkema, P.G. and A. Fire (1994) Development 120, 2175-2186. 2 Okkema et al., (1994) WBG 13 #4, pg. 90. 3 Schnabel, H. and R. Schnabel (1990) Science 250, 686-688.

Affiliation:
- Department of Biological Sciences, University of Illinois at Chicago Department of Embryology, Carnegie Institution of Washington