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 genes
pha-1 and
pha-4 are also required for normal differentiation of pharyngeal muscle, as well as differentiation of all other cell types in the pharynx (3,4). This phenotype suggests
pha-1 and
pha-4 might play role in organ-specific activation of pharyngeal muscle genes.
pha-4 is likely to play an upstream role, because
ceh-22 is not expressed in
pha-4 mutants (4). In contrast,
pha-1 mutations inhibit only later stages of pharyngeal muscle differentiation, suggesting this gene could play a more direct role in the organ-specific activation of gene expression. To more fully understand the relationship between
ceh-22 and
pha-1 we have 1) examined
ceh-22 expression in
pha-1 mutants, and 2) characterized pharyngeal muscle differentiation in a
pha-1;
ceh-22 double mutant.
ceh-22 is expressed normally in
pha-1 mutant embryos: To determine whether
pha-1 functions upstream to activate
ceh-22 expression,
pha-1(
e2123ts) animals raised at the non-permissive temperature (25) were stained with anti-CEH-22 antibodies. In these mutants, both the temporal and spatial pattern of CEH-22 expression appeared normal. This result is consistent with a model that
pha-1 functions in parallel to
ceh-22 to induce pharyngeal muscle differentiation. Interactions between
ceh-22 and
pha-1 mutations: If
pha-1 and
ceh-22 promote pharyngeal muscle differentiation via parallel pathways, we expect the double mutant would have a more severe defect than either single mutant. To test this hypothesis, pharyngeal muscle differentiation was examined in a
pha-1(
e2123ts);
ceh-22(
cc8266) strain.
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 25. This reduction has been more difficult to characterize, because
myo-2 expression is already partially inhibited in
pha-1. A similar synergistic interaction can also be observed at the permissive temperature where
pha-1(
e2123ts) enhances the lethal phenotype of
ceh-22(
cc8266). Does
pha-1 promote pharyngeal muscle differentiation through the C sub-element? Our results are consistent with model in which
pha-1 promotes pharyngeal muscle differentiation in a pathway which is distinct from that requiring
ceh-22. An attractive hypothesis is that
pha-1 is directly involved in organ-specific activation of pharyngeal muscle genes. We are currently constructing strains to test this hypothesis by directly assaying C sub-element function in a
pha-1 mutant background. 1 Okkema, P.G. and A. Fire (1994) Development 120, 2175-2186. 2 Okkema, P.G. and A. Fire. 1995 C. elegans meeting abstracts 3 Schnabel, H. and R. Schnabel (1990) Science 250, 686-688. 4 Mango, S.E., E.J. Lambie, and J. Kimble (1994) Development 120, 3019-3031.