Worm Breeder's Gazette 15(2): 42 (February 1, 1998)
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 Zoology, University of British Columbia, Vancouver, Canada
An L1 lethal mutant ra409 was isolated from a screen designed to isolate embryonic lethals with defects in muscle development. ra409 animals have a unique phenotype in that mutant animals do not complete morphogenesis and do not elongate entirely. Although all tissues types examined have differentiated in ra409 homozygotes, many do not appear wild type. In particular, the body wall muscle and the overlying basement membrane are 2-fold wider than in wild type animals. In addition, muscle filaments in ra409 animals are oriented at a steeper angle to the longitudinal axis of the embryo than what is seen in wild type. This may be a consequence of either the lack of complete elongation or the expansion of muscle cells or basement membrane. We have also observed that the pharynx is much shorter in these mutant animals and labors during contraction. The actin filament organization and the antigen recognized by MH4 (intermediate filament like antigen) in the hypodermis both appear normal, with the exception that the MH4 staining pattern is two fold wider corresponding to the wider muscle quadrants in this mutant. Recent characterization and cloning of sma-1 (McKeown, C., Praitis, V. And Austin, J. 1997 Worm Meeting) provided a clue to aid in the identification of the ra409 gene. sma-1 is a viable mutant that has defects during morphogenesis similar to ra409 mutants. At hatching sma-1 mutants are half the length of wild type, a phenotype that is identical to ra409 homozygotes. Transformation rescue of sma-1 animals revealed that sma-1 encodes a beta-spectrin (McKeown, C., Praitis, V. And Austin, J. 1997 Worm Meeting). We have genetically mapped ra409 to a region on the X chromosome containing an alpha-spectrin (C. elegans genome sequencing consortium). Spectrins are a major components of the membrane cytoskeleton and are important for maintaining cell shape and polarity. Since beta-spectrin forms heterodimers with alpha-spectrin, we felt this alpha-spectrin might be a likely candidate gene for ra409. To determine if the ra409 lesion is in the alpha-spectrin gene we initially took an antisense RNA approach using a 2.8 kb partial cDNA obtained from Y. Kohara. Antisense as well as RNA injection into the gonad of wild type worms has been shown to phenocopy the mutant phenotype of several genes in the progeny of injected worms (Driver, S.E., Ali, M., Mello, C.C. 1997 Worm Meeting). The 2.8 kb cDNA to the 3! end of the alpha spectrin gene was used to generate RNA and this RNA was injected into wild type animals and the progeny were screened for the presence of the ra409 phenotype. Several L1 larvae from the injected wild type hermaphrodite resembled ra409 homozygotes, but unlike ra409 animals these were viable. Next, in an attempt to rescue ra409 animals we transformed animals with a purified cosmid clone, M01F12, that contains the entire alpha-spectrin gene (supplied from the Sanger Center). We obtained transformation rescue of the ra409 mutants thus demonstrating that ra409 is an allele of the alpha-spectrin gene. This alpha-spectrin, asp-1 (alpha spectrin), is 58% identical to the vertebrate non-erythroid alpha-spectrin and 63% identical to the Drosophila alpha-spectrin. The non-erythroid alpha-spectrins and ASP-1 contain an SH3 domain, a calmodulin binding domain and a calcium binding region. Now that the gene is identified the goal will be to determine how it functions during development. One area we will investigate is what role spectrin plays in the patterning of the muscle cytoarchitecture.