Worm Breeder's Gazette 14(3): 51 (June 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

ceh-22 and pha-1 mutations have synergistic effects on pharyngeal muscle gene expression.

Peter Okkema1, David Moons1, Andrew Fire2

1 Department of Biological Sciences, University of Illinois at Chicago
2 Department of Embryology, Carnegie Institution of Washington

        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.