During vulval development the 'vulval precursor' cells P3.p-P8.p adopt invariant cellular fates - P3.p, P4.p and P8.p adopt 'uninduced' fates (3?), while P5.p, P6.p and P7.p adopt 'induced' fates (1?, 2?, 1? respectively). In
lin-31 mutants, P3.p-P8.p appear to become deregulated, such that a Pn.p cell is able to adopt any vulval cell fate. The
lin-31 gene encodes a putative transcription factor with high homology to the HNF-3/fkh class of transcription factors. Genetic mosaic experiments indicate that
lin-31 acts in the Pn.p cells, and epistasis experiments suggest that
lin-31 acts downstream of genes in the anchor cell signalling pathway, most notably the MAP Kinase homolog
mpk-1. These results suggest that LIN-31 function may be regulated by MPK-1. We have tested this hypothesis in a number of ways. Firstly, we constructed a
lin-31-GFP reporter gene and observed strong GFP expression in P3.p-P8.p before vulval induction began. This result suggests that LIN-31 is expressed at the right time in the right place to act as an effector molecule in response to activation by MPK-1. Secondly, we have mutagenized the single strong MAP Kinase consensus phosphorylation site in
lin-31. Following transformation into
lin-31 mutants, this putative 'phosphorylation-defective'
lin-31 gene can rescue lineage defects in Pn.p cells far from the anchor cell (P3.p, P4.p and P8.p), but not in cells near to the anchor cell (P5.p, P6.p and P7.p). This result is consistent with the idea that the
lin-31 protein is normally unphosphorylated in Pn.p cells far from the anchor cell (in these cells, the 'phosphorylation defective'
lin-31 protein is able to provide basal
lin-31 function), and is phosphorylated in cells near to the anchor cell (in these cells, the 'phosphorylation defective'
lin-31 protein would not be expected to provide activated
lin-31 function). In additional experiments, we have found that
lin-31 expression is down-regulated by the process of vulval induction. We observed that
lin-31-GFP expression disappeared in induced cells (P5.p, P6.p and P7.p) shortly before the first vulval cell division, but persisted in uninduced cells (P3.p, P4.p, and P8.p) throughout vulval development. Furthermore, in a
lin-7 vulvaless mutant,
lin-31-GFP expression persisted in all 6 Pn.p cells, while in a in-15 multivulva mutant,
lin-31-GFP expression disappeared in all 6 Pn.p cells. These results suggest that
lin-31-GFP expression may be down-regulated by the anchor cell signaling pathway. Interestingly, in
lin-31 mutants,
lin-31-GFP expression was not down-regulated, suggesting that
lin-31 protein in the auto-regulates its own promoter. These results suggest that both transcriptional control of in-31 gene expression, as well as active regulation of
lin-31 protein activity may be necessary to achieve proper vulval cell fate choices.