Although the Ras signaling pathway is complex, genetic analyses suggest that the major function of the Ras signaling pathway during vulval development is to negatively regulate
lin-1, the C. elegans homolog of the vertebrate oncogene,
elk-1. Loss-of-function mutations in
lin-1 cause a Muv phenotype, indicating that
lin-1 functions to inhibit vulval cell fates. Furthermore, loss of
lin-1 activity bypasses the requirement for
let-60 Ras,
mek-2 MEK, and
mpk-1 MAPK, suggesting that the Ras pathway negatively regulates
lin-1 in P6.p and thereby promotes a vulval cell fate. Gain-of-function mutants of
lin-1 constitutively inhibit Ras-mediated vulval induction resulting in a vulvaless phenotype.
lin-1 encodes a 441 amino acid protein with an 80 amino acid ETS domain in the N-terminus that has recently been shown to exhibit DNA-binding activity. Although
lin-1 has been well studied, little is known about how it functions to affect the downstream gene expression changes necessary to transmit the Ras-mediated signal. In order to identify direct and indirect targets of LIN-1, we have utilized microarray analysis to compare the gene expression profiles in wild type,
lin-1(g.f.), and
lin-1(l.f.) animals. RNA samples from synchronized populations of L3 and L4 animals from N2,
lin-1(
n383), and
lin-1(
n1761) strains were subjected to microarray analysis using the commercial Affymetrix C. elegans genomic arrays. Triplicate samples were analyzed independently and expression results were combined. In our most stringent analysis, genes were required to exhibit two-fold expression level differences when comparing each mutant sample with the wild-type expression level. Surprisingly, only two genes, C32H11.9 and C12C8.1, met these criteria. Both genes showed a significant increase in expression compared to wild-type in the
lin-1(l.f.) animals and a significant decrease in expression in
lin-1(g.f.) animals. C32H11.9 and C32H11.10 have been desingated
dod-21, a largely uncharacterized gene acting downstream of DAF-16. C12C8.1 encodes a member of the
hsp70 protein family which has been previously implicated in the mediation of Ras signaling at the level of Raf/KSR. Hsp-70 possesses a canonical ETS-domain DNA binding element upstream of the coding region, however such elements are not rare. We are attempting immunohistochemical, biochemical, and further genetic studies to determine if
hsp-70 is a direct or indirect target of LIN-1.