In invertebrates gap junction channel proteins are encoded by innexin genes. In C. elegans there are 25 innexins present in the genomic sequence. We are investigating the roles that gap junctions may play in embryogenesis and have been analyzing
inx-3 , which is expressed strongly in embryos. Of the remaining 24 innexins,
inx-13 is the most similar to
inx-3 (42% identity, 60% similarity over 371 amino acids), and so we examined the pattern of
inx-13 expression to determine whether it might also be expressed during embryogenesis, possibly in association with
inx-3 . Antibodies raised against a peptide representing the carboxyl terminus of INX-13 were used to examine INX-13 expression. INX-13 is expressed in embryos and is detected at the plasma membranes of contacts between hypodermal cells, in a pattern similar to that observed for the zonula adherens protein JAM-1, except that INX-13 outlines hypodermal cells discontinuously with large puncta. This expression pattern overlaps with that of
inx-3 . Postembryonically INX-13 continues to be expressed in seam cells. Additionally, INX-13 is strongly expressed in the excretory cell throughout all larval and adult stages, and is expressed in the developing vulva and spermatheca. An
inx-13 deletion mutant was isolated by the Gene Knockout Consortium (Univ. Oklahoma).
inx-13(
ok236) has a 1.6-kb deletion that removes 2 exons, including the predicted fourth transmembrane domain of INX-13. Homozygous
inx-13(
ok236) mutants all arrest at the L1 stage. There is no embryonic lethality associated with this mutation. It appears that the hypodermis and/or body cavity of an L1 mutant animal fills with fluid, and the animal arrests as a dead rod. This phenotype is similar to the description of animals after ablations of the excretory pore, duct, or excretory cell (Nelson and Riddle. 1984. J. Exp Zool. 231:45-56.), components of the excretory/secretory system that functions in osmoregulation. Other cell types, such as pharyngeal, intestinal, and neuronal cells, do not appear to be affected. The RNAi phenotype for
inx-13 is similar, though not as severe -- RNAi animals often survive to later larval stages before they assume a dead rod phenotype. Interestingly, the RNAi phenotype for
inx-12, which lies only 2 kb from
inx-13 , is similar, raising the possibility that these two innexins function together. [
inx-12 has only 39% amino acid identity with
inx-13 and so we do not think that RNAi directed against
inx-12 also affects
inx-13 .] Extensive gap junctions between the excretory canals and the hypodermis have been described (Buechner et al. 1999. Dev. Biol. 214: 227-241). We therefore hypothesize that INX-13, and possibly INX-12, contribute to the formation of the gap junctions formed between the canals and hypodermis and play an essential role in osmoregulation. Because homozygous
ok236 embryos show no apparent hypodermal defects, INX-13 likely plays a redundant role during embryogenesis. We know of at least three other innexins expressed in the hypodermis at this time, including
inx-3 , and it is possible that any of them may compensate for loss of INX-13. We have not yet addressed the potential role of INX-13 during vulval and spermathecal development.