Worm Breeder's Gazette 14(2): 83 (February 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.
Department of Molecular Genetics and Cell Biology University of Chicago 920 East 58th St. Chicago, IL 60637
Morphogenesis is the process by which an embryo is transformed from a ball of cells into its mature form. At the start of C. elegans morphogenesis, the actin cytoskeleton in hypodermal cells reorganizes into a highly ordered structure which, if disrupted, prevents embryonic elongation (Priess & Hirsch, Dev. Bio. 117:156-173). We are interested in how this cytoskeletal reorganization occurs, what cytoskeletal components are required for morphogenesis, and how the process of elongation is regulated. We have isolated embryos defective in morphogenesis that carry mutations in the sma-1 gene. In sma-1 mutant embryos elongation is initiated but ceases prematurely, resulting in short, fat larvae with round heads. The length of newly hatched sma-1 larvae ranges from 50% of wild type for severe alleles to 75% of wild type for weaker alleles. The reduction in length occurs along the entire animal, with head, body and tail lengths decreased proportionately. Interestingly, the pharynx of sma-1 mutants is also short and fat. Additional sma-1 phenotypes include a defect in extension of the excretory canal (see Buechner, Hall, and Hedgecock, WBG 13(5):75), and a twisted cuticle which produces a weak roller phenotype in adults. Preliminary results from phalloidin staining experiments indicate sma-1 embryos have abnormalities in the actin cytoskeleton during morphogenesis. We have not yet determined whether any of the sma-1 alleles we have isolated is a null, and are using complementation screens to isolate additional alleles including sma-1 deletions. The sma-1 mutant phenotype is rescued by injection of either the entire cosmid C10C2 or an 11 kb subclone. Partial sequence from the rescuing subclone contains homology to a number of interesting cytoskeletal protein domains, including an SH3 domain, multiple spectrin repeats, and a pleckstrin homology domain. The order and sequences of these domains show strong homology to the Drosophila ßH isoform of spectrin. Drosophila ßH-spectrin expression is developmentally regulated and restricted to musculature and ectodermally-derived epithelia. In general, ßH-spectrin protein localizes to the apicolateral portions of cells, but in cells undergoing morphogenesis, an apical cap of ßH-spectrin is observed (Thomas & Kiehart, Dev. 120: 2039-50). We are continuing to sequence the 11 kb rescuing fragment and flanking regions to determine if this molecule, like ß-spectrins, contains an actin binding domain, and is the C. elegans homolog of ßH-spectrin. We have performed in situ hybridizations in C. elegans embryos using a 2 kb subclone of the 11 kb rescuing fragment. RNA staining first appears at the lima bean stage of early morphogenesis in the dorsal hypodermis. By comma stage, we observe decreased RNA staining intensity in dorsal hypodermis and new RNA staining in ventral hypodermis and gut. RNA staining in the hypodermis is nearly gone by the 1.5-fold stage, while the gut still contains RNA staining. We observe no RNA staining in two-fold and later stage embryos. We are continuing to examine the RNA expression pattern of the gene encoded by the rescuing fragment, both by Northern blot and in situ analysis, in larval and adult stages. We are also planning to generate antibodies to examine the expression pattern and intracellular localization of the sma-1 protein in C. elegans.