To find genes required for differentiation of non-neuronal ectodermal cells, we have looked for mutations displaying similar defects as mutations in
lin-26(
n156), a gene known to specify and/or maintain the fates of hypodermal and glial-like cells. Both
che-14(
e1960) (Perkins et al, Dev Biol 56 : 110-156 1986) and
lin-26(
n156) (Labouesse et al, Development 122 : 2579-2588 1996) lead to ultrastructural defects in glial-like cells. Using a dye-filling test as an assay, we isolated one new
che-14 mutation,
mc16, in a non-complementation screen against
e1960 after examining 10000 genomes. We found by electron microscopy that
mc16, as
e1960, affects chemosensory organs by closing the amphid and phasmid channels. Both alleles induce a weakly penetrant embryonic lethality reminiscent of
lin-26(
mc2) and a partial rod-like larval lethality probably due to hypodermal defects. We have cloned
che-14 by complementation rescue of the dye filling defect due to
e1960.
che-14 encodes a protein, with 10 to 12 transmembrane domains, which displays 45% similarity/20% identity with PATCHED, the HEDGEHOG receptor. The two
che-14 mutations are G-A transitions that affect splice donor sites located towards the 3' end of the gene. To look at the expression pattern of
che-14 we inserted the gfp in frame at the 3' end of
che-14. The fusion protein is expressed in all epithelial ectodermal cells, and is found mostly at the apical membrane but also in the cytoplasm. To find CHE-14 partners, we screened for mutations displaying both the r od-like larval lethality and the dy e-filling defect (Rdy phenotype). After TMP/UV mutagenesis of 13000 genomes, we kept 10 mutants, one of which is a new
che-14 allele. The
mc35 mutation is an insertion combined with a deletion which could produce a CHE-14 protein half its normal size. Together our data suggest that
che-14 is required to maintain the polarity of epithelial cells in the ectoderm. We are currently examining the specific function of the different potential functional domains found in CHE-14 by deletion analysis in the
che-14::gfp fusion. Injection in
che-14 mutants will allow us to follow both rescue and protein localisation.