Basement membranes are specialized extracellular matrices that provide structural support, tissue separation, and have essential signaling roles during development. Vertebrate type XVIII collagen is a homotrimeric basement membrane molecule whose functions are largely unknown. Its carboxyl terminal fragment, endostatin (ES), inhibits endothelial cell proliferation and migration, and angiogenesis. The C. elegans homologue,
cle-1, is being characterised in our lab. An internal deletion allele,
cg120, that removes the ES/NC1 domain, was shown to cause multiple cell migration and axon guidance defects. Rescue of some of these defects are obtained by expressing transgenically, in
cg120, the shorter
cle-1 isoform, containing just the NC1 domain. In
cg120 mutants truncated CLE-1 is localized in a nearly wild-type pattern, but at reduced level. A second deletion allele,
cg122 (see below), which causes embryonic and larval arrest, severely reduces
cle-1 mRNA and protein. RNAi against
cle-1 by injection of double-stranded RNA results in some embryonic and larval lethality. These results suggested that a
cle-1 null mutant might exhibit some degree of lethality. Most forms of
cle-1 are mainly expressed in neurons, which are refractory to RNAi. We tried to overcome this problem by transgenically producing double-stranded RNA against
cle-1 in neurons using a heat shock vector. However, the effect using this method was not more severe than using microinjections. We examined the persistence of CLE-1C::GFP derived fluorescence after heat shock treatment, and found no detectable reduction in fluorescence. The allele
cg122, removes part of the NC1 domain plus 350 bp of 3' UTR, including the putative polyadenylation signal. Most
cg122 mutants arrest as embryos or L1s, 15% become small, Muv, sterile adults. Deletion of the 3'UTR might destabilize the
cle-1 mRNA and these phenotypes could represent the near null state for
cle-1. However, the deletion also appears to remove 160 bp of the 3'UTR of the adjacent gene, F39H11.3, a putative CDK8 protein kinase. The F39H11 cosmid transgenically rescues the
cg122 lethality. To determine whether one or both of these genes are responsible for the phenotype we have tested subclones containing only
cle-1 or F39H11.3. Unfortunately, for our purposes, the F39H11.3 subclone completely rescues the lethality, while the
cle-1 subclone does not. So the
cg122 lethality appears to result from affects on the adjacent kinase. The
cle-1 gene produces at least three isoforms,
cle-1A-C, expressed from different promoters, and with different temporal and spatial expression. These isoforms are being tagged with a c-myc epitope tag to further investigate the localization to function of the expressed proteins.