Worm Breeder's Gazette 15(1): 64 (October 1, 1997)

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.

Isolation of Non-Conditional emb-30 Alleles

Bryan Koch, Roy Auty, Tokiko Furuta, Kim Rose, David Greenstein

Department of Cell Biology, Vanderbilt University School of Medicine Nashville, TN 37232 USA

Germ cells differ from somatic cells in undergoing both mitosis and meiosis. We would like to understand how these two types of cell cycles are regulated and coordinated with gametogenesis. In C. elegans, oocytes complete both meiotic divisions after fertilization (Albertson, Dev. Biol. 101: 61-72, 1984). In a genetic screen for oocyte-defective mutants, we isolated a strong allele of the emb-30 locus, tn377ts III, that blocks meiotic divisions of oocytes (Furuta et al., 1997 Meeting Abstracts). Ovulated oocytes from emb-30(tn377ts) are fertilized but produce no polar body and never form pronuclei. The oocytes are able to set up a meiotic spindle, but apparently cannot progress to anaphase I. This meiotic cell cycle block results from a defect in the oocyte. Surprisingly, if emb-30(tn377) mutant embryos are shifted to the non-permissive temperature, they grow into sterile adults that display a strong Glp phenotype, producing only a few germ cells and no gametes, in both hermaphrodites and males. This strong Glp phenotype is maternally rescued by a wild-type allele. emb-30 Glp adults are extremely healthy, and no defect has been observed in the soma.

We sought additional emb-30 alleles in an F1 non-complementation screen in order to: 1) determine the null phenotype; 2) dissect the multiple emb-30 germline functions; and 3) evaluate whether the emb-30 mutant defects are specific to the germline. N2 males were mutagenized with EMS and crossed to emb-30(tn377ts) ced-7(n1892) unc-69(e587) hermaphrodites at the permissive temperature (16C). F1 cross progeny were singled and allowed to lay approximately half of the brood at 16C. The parent was then removed and cultured at 16C, while the F2 eggs were cultured at the non-permissive temperature (25C). We screened for plates in which all the F2 progeny were Glp or laid fertilized oocytes that did not complete meiosis (Mel). In a screen of 10,871 mutagenized haploid genomes we isolated 13 independent mutations that failed to complement emb-30(tn377)for either the Glp or Mel phenotypes. All these mutations map to chromosome III and three mutations were three-factor mapped and found to map just left of ced-7. Based on these results, we have assigned these new mutations as emb-30 alleles. These new mutations are non-conditional and can be classified into two groups based on their homozygous mutant phenotypes (4 alleles have yet to be classified).

Class I mutations (7 alleles) are homozygous sterile and have an everted vulva or are vulvaless. The class I alleles are recessive and fail to complement tn377 for both the Glp and Mel phenotypes. Hermaphrodites that are homozygous for class I alleles produce reduced numbers of germ cells and no gametes. These animals also exhibit somatic gonadal defects. Since this mutant phenotype seems similar to that described for several evl mutants (Seydoux et al., Dev. Biol. 157: 423-436, 1993 ) we performed complementation tests between emb-30(tn377), emb-30(tn471) [a class I allele] and the mutations evl-8(ar102)III and evl-19(ar98)III. Each of these emb-30 alleles complemented these evl mutations and thus they are likely to represent distinct loci.

By contrast, class II mutations (2 alleles) are lethal. Homozygous mutant hermaphrodites die as embryos or young larvae. The class II alleles are weakly dominant and can be further subdivided based on complementation tests with emb-30(tn377ts): tn475 fails to complement tn377 for both the Glp and Mel phenotypes, whereas tn474 complements tn377for the Glp phenotype, but not the Mel phenotype. Thus the germline-specific functions of emb-30 are separably mutable. Based on these results, we propose that emb-30 has germline-specific as well as general functions and emb-30(tn377) appears to disrupt the germline functions specifically.