Worm Breeder's Gazette 11(3): 15
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.
Experiments in vertebrate systems have identified several genes which can apparently function as switches to regulate myogenesis. In the last gazette, cloning and characterization of a C. elegans homologue of one of these genes, 'MyoD', was reported (MK and HW, WBG 11#2p40). We have been using transformation to analyze the expression and activities of the C. elegans MyoD homologue (the gene is currently designated 'CeMyoD').A restriction fragment starting 3.3 kb upstream of the CeMyoD coding region and extending 2.1 kb into the gene was used to make translational fusions to -galactosidase with 3' ends either from unc-54 or from CeMyoD. When these constructs were injected into wild type C. elegans and the first generation stained with Xgal, strong staining in body wall type muscles was observed. No staining in pharyngeal muscles was seen. In order to analyze embryonic expression we made transformed lines by coinjecting the CeMyoD- Gal-unc54 (3') fusion pPD37.48 with either the unc-22 antisense plasmid pPD10.46 (WBG10#2 p89) or the rol-6(su1006) plasmid pRF4 (Mello et al WBG 11#1 p18). Essentially identical embryonic patterns are seen in several independent lines transformed with each of the two co-selection markers, so that we attribute the pattern to properties of the pPD37.48 fusion construct. No staining is seen earlier than the 28 cell stage. At that stage staining is seen in just four cells that are apparently the four MS derived blastomeres MSaa, MSap, MSpa, and MSpp. One or two cell divisions later, staining begins to appear in posterior cells which are presumably C and D derived myoblasts. At approximately 256 cells distinct staining is seen in many body wall muscle cell precursors, but staining is absent in the region of the pharyngeal precursors. Very strongly staining cells are seen in young larvae in a position consistent with these being the M myoblast and immediate descendents. Several conclusions and speculations derive from the staining patterns. First it is clear from the limitation of staining at the 28 cell stage to the MS granddaughters that cells at this stage are already different in their gene expression. Second, the lack of stain in the MS derived pharyngeal cells suggests that expression of the fusion construct pPD37.48 must be turned off in these cells and the fusion protein lost. An intriguing speculation is that the MS sublineage has some sort of intrinsic muscle identity, with additional muscle cells recruited from other lineages and subtracted from the MS lineage as 'afterthoughts'. Sequences upstream of the myoD coding region are apparently required for the body wall muscle staining pattern. A deletion which removes all but 300 b of flanking DNA 5' to the coding region was co- transformed with rol-6 and produces a pattern of larval gut staining but no staining of muscles or their precursors. Using expression vectors based on the unc-54 (body wall muscle myosin) and myo-2 (pharyngeal myosin genes) we have made fusions to the CeMyoD cDNA which should express intact CeMyoD product in either of the two mature muscle cell types. Constructs designed to express CeMyoD product in pharyngeal muscle lead to arrest of worms as young larvae. Although these animals have a superficially normal pharynx, it is conceivable that expression of CeMyoD in pharyngeal muscle cells interferes with normal function by causing a transformation in fate from pharyngeal to body wall muscle types. The construct designed to overexpress CeMyoD in body wall muscle produces a much more subtle phenotype: animals carrying this construct in an extrachromosomal array are viable but are abnormally pale. Although occasional defects in egg laying or slightly sluggish movement in these strains are seen, most of the animals appear to move and behave normally. A parallel construct with the CeMyoD cDNA in antisense orientation produces no evident phenotype upon injection, consistent with the possibility that CeMyoD normally plays an early role in body wall muscle cell determination or differentiation.