Morphogenesis is the process by which an embryo is transformed from a ball of cells into its mature worm shape. During morphogenetic elongation of the C. elegans embryo, cortical actin in the epidermis reorganizes into an ordered circumferential microfilament array, perpendicular to the anterior-posterior axis (Priess & Hirsch, Dev Bio 117:156). We are interested in how this actin reorganization occurs and its role in C. elegans morphogenesis. We have isolated embryos defective in morphogenesis that carry mutations in the
sma-1 gene. In
sma-1 mutant embryos elongation initiates 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. Phalloidin/actin staining (protocol kindly provided by Mike Costa) of
sma-1 embryos reveals abnormal microfilament organization in the epidermis 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. Injection of antisense RNA derived from the 11 kb DNA fragment produces
sma-1 phenocopies. 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 H isoform of spectrin, an actin binding protein. Drosophila H-spectrin expression is developmentally regulated and restricted to musculature and ectodermally-derived epithelia. In general, H-spectrin protein localizes to the apicolateral portions of cells, but in cells undergoing morphogenesis, an apical cap of H-spectrin is observed in invaginating cells (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 -spectrins, contains an actin binding domain, and is the C. elegans homolog of H-spectrin. We are also testing whether Drosophila H-spectrin antibodies recognize C. elegans SMA-1 protein. 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.