The receptor-tyrosine kinase/Ras pathway for vulval induction is negatively regulated by two functionally redundant pathways consisting of the Class A and Class B synthetic multivulva (synMuv) genes (1). Animals are multivulva (Muv) only if they are mutant in both Class A and B genes and have a wild-type vulval phenotype if they carry one or more mutations of a single class. To understand the molecular mechanisms by which the synMuv genes regulate the Ras signaling pathway, we have cloned the Class B genes
lin-35 and
lin-53.
lin-35 maps on LGI between
unc-40 and
stP124. We rescued the Muv phenotype of the strain
lin-35(
n2977);
lin-15(
n433) with a 9 kb DNA fragment from that region that contains a single predicted gene similar to genes of the pocket protein family, which includes
p130,
p107 and the tumor suppressor gene Rb. Six of the eight
lin-35 alleles contain nonsense mutations; the other two have identical mutations at a splice-acceptor site. Using anti-LIN-35 peptide antibodies, we observed staining in most if not all nuclei in embryos and newly hatched L1s. In older larvae, staining was visible in the nuclei of the P(3-8).p cells and their descendants, which give rise to the vulva, and in cells in the head and tail regions.
lin-53 encodes a 7 WD-repeat protein similar to mammalian
p48 and
p46, two closely related proteins each of which binds Rb. The
lin-53(
n833) allele appears to be dominant-negative in its effect. A rescuing GFP::LIN-53 fusion transgene is expressed in the nuclei of the P(3-8).p cells and their descendants during vulval development. Localization of LIN-35 and LIN-53 in the P(3-8).p cells as detected by antibodies and a GFP transgene, respectively, is consistent with the hypothesis that these proteins act cell-autonomously. We are attempting mosaic analysis to establish the site of action of
lin-35. In mammals,
p130,
p107 and Rb act as negative regulators of cell proliferation and have overlapping functions with one another. Hypophosphorylated Rb can bind to E2F transcription factors (which consist of DP/E2F heterodimers) and recruit histone deacetylase, possibly via
p48, to repress transcription from target promoters (2). Interestingly, C. elegans DP and E2F genes act in the class B synMuv pathway to regulate vulval induction (see abstract by Ceol and Horvitz). We propose that
lin-35 and
lin-53 antagonize the Ras pathway by forming a complex with DNA-bound E2F, thereby repressing transcription. In this model, the class B gene
lin-37, which we previously found to encode a novel protein (3), could act in
hyp7 (4) to regulate a signal that leads to LIN-35-mediated transcriptional repression. The identification of
lin-35 target genes should help reveal how the class B synMuv pathway interface with the Ras pathway. 1. Ferguson and Horvitz, Genetics 123:109-121, 1989 2. Luo, Postigo and Dean, Cell 92: 463-473, 1998 3. Lu, Thomas and Horvitz, 1997C. elegans meeting abstract, p. 389 4. Hedgecock and Herman, Genetics 141:989-1006, 1995