Worm Breeder's Gazette 10(3): 104
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.
We looked for mutants in which the pattern of body wall muscles is disrupted and muscle cells are inappropriately attached (i.e. in more than one quadrant). Screen: 600 F1 of mutagenized daf-2 (e1368) were shifted to 25 C and screened for the presence of larval lethals which do not move and contain bends and kinks with a constant position in the animal (a phenotype which Ed Hedgecock named 'mup'). Candidates were screened by polarized light microscopy for the presence of muscles which attach across quadrants. The candidates picked had a normal pharynx and a smooth, non-blistered cuticle. Three mutants were identified: - e2346 is a ts larval lethal on the left arm of X close to and probably to the left of dpy-7. It complements dpy-(e14), & ev400), unc-18(e81) and dpy-8(e130). It has not yet been complemented to unc-97 and unc-98.- e2347 is a non-ts larval lethal and is on the left arm of II. It is not balanced by mnC1 and is not under mnDf30.- e2348 is a larval lethal allele of unc-15 (paramyosin). Phenotypes of e2346 and e2347: e2346 and e2347 embryos hatch as curled up L1's with many muscles detached and some attached across quadrants. Most die as L1 or L2. e2347 larvae are dpy. Antibody staining with NE8/4C6.3 (a muscle specific antibody from the LMB) indicates that at the 1.25-1.5 fold stage of embryogenesis, when in wild type myoblasts are already arranged in quadrants, some muscles extend processes across quadrants and some cells are displaced from the quadrants of muscle cells. In further elongated embryos more inappropriately attached and possibly detached muscles are observed. These cells extend processes containing muscle filaments in many directions. These ectopic processes contain contracting muscle filaments which have nice muscle attachments as seen on E.M. sections of larvae (courtesy N. Thompson) . In three fold stage embryos the arrangement of body wall muscles becomes very disorganized. In e2346 the arrangement of overlying hypodermal cells is normal as seen by the anti-desmosome antibody MH27 (courtesy of Francis & Waterston) and an anti-seam antibody (courtesy of H. & R. Schnabel). At 15 C e2346 is viable but most hermaphrodites are sterile or produce only a few eggs (males are quite fertile). e2346 embryos shifted from 15 C to 25 C before the 2.5 fold stage invariably develop into mup L1's. Later embryos have not yet been shifted. This together with the fact that ectopic muscle processes are already observed at the 1.5 fold stage indicates that e2346 may be required throughout morphogenesis. A more detailed analysis of the temperature sensitive period of e2346 is in progress. L2's shifted to 25 C invariably develop into sterile (non unc) hermaphrodites. At the junction of the spermatheca and oviduct the spermatocytes and oocytes are extruded into the pseudocoelomic cavity. The vulva, uterus and oviduct appear normal. These sterile hermaphrodites produce only a few oocytes. e2347 is suppressed by dpy 2, dpy-10 and dpy-11:e2347/mnC1(dpy-10 unc-52) produced some dpy's (F1) which gave offspring consisting mainly of mup's, partially suppressed but fertile dpy's with one or two kinks in the body and some dpy's (F2) which have normal body wall muscles. These F2 dpy's were e2347/e2347 (determined by outcrossing to wt males and checking for segregation of mnC1). The suppressed dpy F2 daughters gave only mup offspring (F3). e2347 dpy-10 / e2347 dpy- 10 are strong dpy's or partially suppressed mup's. The dominant suppression by mnC1 therefore is likely to be due to the maternal product of the dpy-10 mutation on mnC1. We are now determining the genotype of F1 dpy's (e2347/e2347 or e2347 mnC1/ e2347 ) to find out whether the F2's are suppressed by dpy-10 product from the parent or grandparent. Dpy-11(e224) also suppresses e2347 to give a viable strain. About 10% of the offspring of e2347;e224 are fertile piggy dpy's but most are mup's suppressed to different extents and die as later stage larvae than e2347/e2347. The (partially) suppressed dpy e2347 have muscle filaments crossing quadrants and muscle cells attaching nicely in both quadrants. Some muscles have ragged edges. We do not know whether there is a dominant or maternal component to the suppression of dpy-11. e2347 dpy-2 (e8) / e2347 dpy-2 are (like e2347 dpy-10) strong dpy's or partially suppressed mup's but dpy-2 rescues a higher proportion of mup's than dpy-10. Dpy-5(e61) does not suppress e2347, indicating that not all dpy's suppress e2347. Summary: We have isolated two mutants e2346 and e2347 required for appropriate body wall muscle development. The mutations appear not to affect the pharynx nor the ultrastructure of the muscle filaments ( from EM sections, courtesy N. Thomson). The observation of inappropriately placed myoblasts and myoblasts sending processes in several directions in early embryos (1.25-1.5 fold) of e2346 and e2347 suggests an early function of these genes in the development (possibly attachment) of body wall muscles. The phenotypes of myo-3 (Bob Waterston, WBG, 9(2), 30) and e2348, a lethal allele of unc-15 indicate that the inappropriate attachment or detachment of body wall muscles could also be due to a failure to lay down appropriate (or appropriately patterned) muscle filaments in the body wall muscles. The suppression of e2347 by several dpy's which are mutations of genes of the collagen family are known to suppress various dpy's, bli's, rol's, or sqt's which are also collagen genes (Schuyler et al. WBG 10( 1), 23; Cox. et al., Genetics, 95:317-339), may indicate a basement membrane/cuticle defect in e2347. A more detailed phenotypic analysis and further mapping of e2346 and e2347 are in progress.