Worm Breeder's Gazette 13(4): 90 (October 1, 1994)
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.
| 1 | Carnegie Institution of Washington, Baltimore, MD. |
| 2 | Netherlands Cancer Institute, Amsterdam. Andy Fire Carnegie Institution of Washington, Baltimore, MD. |
We have been characterizing regulation of the pharyngeal myosin gene, myo-2 ,to identify factors controlling pharyngeal muscle development. One candidate trans-acting factor we have identified, CEH-22 ,is an NK-2 class homeoprotein that binds an element in the myo-2 enhancer'. CEH-22 is expressed specifically in pharyngeal muscle prior to myogenic differentiation. Given it's binding specificity and expression pattern, we believe CEH-22 is an important regulator of myo-2 expression. Isolation of ceh-22 mutants To determine the role CEH-22 plays in pharyngeal muscle development, we needed to identify a mutation in the gene. ceh-22 maps near daf-11 on LGV. None of the point mutations or deficiencies in the region affect ceh-22 .We therefore generated a mutation by insertion and imprecise excision of Tc1 (see ref. 2). ceh-22 ( cc8266 )contains a 1248 bp deletion that removes ceh-22 exon 2 and much of exon 3. Animals homozygous for this allele have pharyngeal defects (see figure) and do not express CEH-22 detectable with our antibodies. However, cc8266 does not remove the homeobox and we do not know if it is null. (see figure) ceh-22 ( cc8266 )animals have pharyngeal defects, but they express myo-2 ceh-22 ( cc8266 )results in a partially penetrant, recessive larval lethal phenotype. Approximately 1/4 of cc8266 homozygotes arrest as L1 -L2larvae. The remainder grow slowly to adulthood but appear thin and starved. Many of these adults produce small broods or are sterile; we have not characterized this fertility defect extensively. The pharynxes of ceh-22 ( cc8266 )mutants have morphological defects: the isthmus is thicker than in wild type, and the metacorpus is variably misshapen. In addition, the pharyngeal grinder does not come to a full forward position. This latter phenotype is similar to that of phm-2 (3). Pharyngeal muscle contractions are severely defective in those animals that arrest as young larvae, we believe these animals arrest because they are unable to feed. Pharyngeal contractions appear less defective in animals that reach adulthood, but in all cc8266 animals the grinder does not invert as completely as in wild type. Both the pharyngeal and fertility defects are rescued by transformation with an 11 kb genomic DNA fragment containing ceh-22 . As judged by staining with an antibody against myo-2 protein (4), cc8266 animals express the endogenous myo-2 gene similarly to wild type. This is consistent with our analysis of cis-acting sequences regulating myo-2 (1,5). myo-2 contains multiple regulatory elements in addition to the CEH-22 binding site that appear to be sufficient to induce myo-2 expression in the absence of CEH-22 .We had previously identified 3 elements that contribute to activity of the myo-2 enhancer (1). CEH-22 binds a site necessary for activity of one of these elements, which we call B. As a more specific test of CEH-22 function, we assayed activity of an enhancer consisting of two copies of the B element. Although this duplication of B functions as a strong pharyngeal muscle-specific enhancer in wild type animals, it is inactive in cc8266 .Thus, CEH-22 is required for transcriptional activity of the myo-2 B element. Ectopic expression of CEH-22 in body wall muscle activates myo-2 expression We have also asked whether CEH-22 is sufficient to activate myo-2 expression in body wall muscle. A construct expressing CEH-22 protein under control of the unc-54 promoter and enhancer induces CEH-22 protein accumulation in body wall muscle nuclei. This expression activates the endogenous myo-2 gene in these cells. We are currently testing whether CEH-22 is capable of inducing other pharyngeal muscle markers in body wall muscle, and whether it can induce myo-2 expression in non-muscle cell types. Our working model is that CEH-22 is one of several factors regulating myo-2 .In the absence of CEH-22 ,these other factors are sufficient to activate myo-2 expression. The ceh-22 mutant phenotype might result either from subtle defects in myo-2 expression or from defects in expression of other genes regulated by CEH-22 . (1) Okkema and Fire (1994) Development 120, 2175. (2) Zwaal et al.(1993). PNAS 90, 7431. (3) Avery (1993) Genetics 133, 897. (4) Miller et al. (1986). PNAS 83, 2305-2309. (5) 0kkema et al. (1993) Genetics 135, 385-404.