The slender body shape of C. elegans depends on successful extracellular matrix (ECM) synthesis. Viable mutants for ECM components/modifying enzymes display abnormal body morphologies. Characterisation of morphogenetic genes can shed light on the mechanisms of ECM deposition. Here we report the cloning of a new dpy gene,
dpy-31.
dpy-31(
e2770) mutants display a strong recessive Dpy phenotype and are ts-lethal. The strain is 50% viable at 15C, while at 25C all mutants die as embryos or arrest at L1 with severe body abnormalities. The phenotype is suggestive of a defect in cuticle synthesis. The
e2770 mutation was recovered as a spontaneous event in a
mrt-2(
e2663) mutant background. The complete ts-lethality of the
e2770 mutants was exploited to isolate revertant strains in EMS and ENU screens followed by shift to 25C. Many wild-type revertants and non-ts Dpy revertants were isolated in such screens. The wild-type revertants are intragenic dominant, indicating that in these strains the
dpy-31 function has been restored. The
dpy-31 locus was of particular interest to us for two reasons: first, the general phenotype suggested a fundamental role for this gene in C. elegans morphogenesis. Secondly, we wished to investigate the peculiar genetic properties of the locus. Genetic mapping placed
dpy-31 on the centre of LGIII. Germline transformation experiments showed that gene R151.5 (
toh-2), a secretory zinc-metalloprotease of the BMP-1/TOLLOID family, is sufficient for phenotypic rescue of
dpy-31. The pattern of expression of
toh-2, explored by means of a GFP promoter fusion, was found to be hypodermal at all stages. Sequencing of the
e2770 allele identified an LtoP mutation in
toh-2, causing the disruption of a conserved -strand structure in the CUB domain of the enzyme. The wild-type revertants carry compensatory mutations on the same codon altered by
e2770. As far as the non-ts Dpy revertants are concerned, we found that most carry dominant extragenic suppressors of the lethality. Notably, 4 suppressors are alleles of a known gene,
rol-4.
rol-4 remains uncloned, and could encode a target of
toh-2, or an enzyme involved in cuticle formation. We are trying to clone
rol-4 by cosmid rescue. The properties of TOH-2 suggest that it is an essential Procollagen-C-Proteinase (PCP) involved in cuticular collagen maturation. TOH-2 is the first PCP cloned in C. elegans. The
dpy-31 mutants and revertants may provide a useful model for investigating collagen processing in C. elegans.