The secondary vulval lineages display specific orientations, visible in the spatial arrangement of adherent and nonadherent cells, in the direction of cell migration, and in the shapes of individual cells during vulval morphogenesis. Cell ablation and molecular genetic experiments indicate that a signal from the gonad is required for correct vulval cell orientation, and that excess activity of the vulval inductive pathway overcomes the need for orienting signal from the gonad. We are examining the role of
lin-18 in specifying correct vulval cell orientation. Mutations in
lin-18 cause some vulval cells to be misoriented. Because we had seen that excess vulval inductive activity promotes correct vulval cell orientation, we tested whether reduced inductive activity enhanced the penetrance of the
lin-18 orientation defect. We used viable reduction-of-function mutations in raf to reduce activity of the vulval induction pathway. We found that the fraction of animals with misoriented vulval cells was increased in the double mutants: a greater fraction of raf;
lin-18 double mutants displayed misoriented vulval cells than did
lin-18 single mutants. We were surprised to find also that
lin-18 mutations increased the penetrance of the raf vulval induction defect.
lin-18 mutations alone did not affect the extent of vulval differentiation, but raf;
lin-18 double mutants showed reduced vulval differentiation compared to raf single mutants. We have performed these experiments using two different alleles of each gene, to control for the possibility that enhancement was due to a cryptic mutation in one of the strains. The genetic interaction we have observed suggests that LIN-18 might exert a novel influence on the RAS/RAF-mediated vulval inductive pathway. We have cloned
lin-18 by germline transformation experiments. The minimal rescuing fragment contains the predicted gene C16B8.1. This gene encodes a predicted receptor tyrosine kinase. The predicted protein shares 56-60% similarity and 33-39% identity with amino acid sequences of the Ryk subfamily of predicted receptor tyrosine kinases. The role of the mammalian homologs is not known. We are currently sequencing C16B8.1 from
lin-18 mutants; one allele contains a 382 base pair internal deletion within C16B8.1, supporting the conclusion that this gene is
lin-18. Analysis of
lin-18 expression is in progress.