Kinase Signaling Pathway, Encodes Two Different Proteins

The Caenorhabditis elegans locus lin-15 negatively regulates an intercellular signaling process that induces formation of the hermaphrodite vulva. The lin-15 locus controls two separate genetic activities. Mutants that lack both activities have multiple, ectopic pseudo-vulvae resulting from the overproduction of vulval cells, whereas mutants defective in only one lin-15 activity appear wild-type. lin-15 acts noncell-autonomously to prevent the activation of a receptor tyrosine kinase/ras signaling pathway. We report here the molecular characterization of the lin-15 locus. The two lin-15 activities are encoded by contiguous genomic regions and by two distinct, non-overlapping transcripts that may be processed from a single mRNA precursor by trans-splicing. Based on the DNA sequence, the 7 1 4 and 1,440-amino acid lin-15 proteins are not similar to each other or to kn wn proteins. lin-15 multivulva mutants, which are defective in both lin-15 activities, contain deletions and insertions that affect the lin-15 genomic region. T HE vulva of the Caenorhabditis elegans hermaphrodite is formed by the 22 descendants of three ectodermal blast cells, P5.p, P6.p and P7.p (SULSTON and HORVITZ 1977) (Figure 1A). Three other cells, P3.p, P4.p and P8.p, also have the potential to produce vulval cells. Since all six of these cells are able to express any of three alternative cell lineages (referred to as lo, 2" and 3") and are equivalent in their developmental potential, they are considered to define the vulval equivalence group (SULSTON and WHITE 1980; KIMBLE 1981; STERNBERG and HORVITZ 1986). Cells that adopt the 1 O and 2" cell fates generate eight and seven descendants, respectively, that together form the vulva, whereas those that express the 3" fate generate two non-vulval descendants that fuse with the syncytial hypoderm that envelops the animal. Cell interactions determine the fates of the cells of the vulval equivalence group (see HORVITZ and STERNBERG (1991) for review). A signal from the gonadal anchor cell induces the nearest Pn.p cells to express vulval cell lineages: P6.p adopts a 1" cell fate, while P5.p and P7.p adopt 2" cell fates (Figure 1B). The more distant cells P3.p, P4.p and P8.p adopt 3" cell fates. The elimination of the anchor cell causes all six cells to express a nonvulval 3" fate, resulting in a vulvaless (Vul) phenotype (KIMBLE 1981) (Figure 1C). Genetic experiments suggest that an inhibitory signal from the syncytial hypoderm prevents the expression of vulval cell fates (HERMAN and HEDGECOCK 1990). The removal of the hypodermal inhibitory signal allows all six cells to express vulval cell fates, resulting in a rnultivulva (Muv) phenotype (Figure 1D). These results suggest that during wildFrancisco, California 94143. I Current address: Department of Anatomy, University of California, San Genetics 137: 987-997 (August, 1994) type development, the anchor cell signal promotes the expression of vulval cell fates by overcoming the hypodermal inhibitory signal. In addition, interactions among the induced Pn.p cells prevent adjacent cells from both expressing a 1" fate (STERNBERG 1988). Many mutants with altered vulval cell lineages have b en characterized (HORWTZ and SULSTON 1980; FERGUSON and HORVITZ 1985, 1989; BEITEL et al. 1990; I-IAN et al. 1990; KIM and HORVITZ 1990; h o r n and STERNBERG 1991; CLARK et al. 1992; H.m et al. 1993; MILLER et al. 1993). Some mutations cause all six cells P3.p-P8.p to express a 3" cell lineage, and no vulva is formed. As this Vu1 phenotype is identical to that of animals lacking the anchor cell, these mutations define genes that could be involved in the signaling process required for vulval induction. Five such Vu1 genes encode proteins imilar to those involved in intercellular signaling in other organisms: lin-3 encodes a molecule related to TGFa (HILL and STERNBERG 1992), let-23 encodes a receptor tyrosine kinase ( h o r n et al. 1990), sem-5 encodes an adaptor protein with SH2 and SH3 domains ( C m et al. 1992), let-60 encodes a ras protein (W and STERNBERG 1990) and lin-45 encodes a raf serine/threonine kinase (HAN et al. 1993). Recently, mutations in the mpk-l/sur-l gene, which encodes a mitogen-activated protein ( M A P ) kinase, have been identified as suppressors of activated let-60 ras mutations, suggesting that mpk-l/sur-1 also functions in the signaling pathway required for vulval induction (LACKNER et al. 1994; Wu and HAN 1994). Other mutations, including some lin-15 mutations, cause all six cells P3.pP8.p to express 1" and 2" cell lineages, resulting in a multivulva phenotype (FERGUSON et al. 1987). 988 S. G. Clark, X. Lu and H. R. Horvitz A P3.p P4.p P5.p P6.p P7.P P8.D h h & A & h L L T N T T T T N T L L