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.