lin-31, a transcriptional regulator of vulval development, is expressed in vulval precursor cells and is downregulated by the anchor cell signalling pathway. Patrick Tan and Stuart K. Kim, Dept of Developmental Biology, Stanford University Medical Center, Stanford, CA94305 During vulval development, a signal from the anchor cell causes nearby Pn.p cells (P5.p, P6.p and P7.p) to adopt induced vulval cell fates. Pn.p cells far from the anchor cell (P3.p, P4.p and P8.p) do not receive the anchor cell signal and consequently remain uninduced. The
lin-31 gene encodes a member of the HNF-3/fkh class of transcription factors. Iin-31 is involved in the proper establishment of both the induced and uninduced vulval cell fates since
lin-31 null mutants exhibit both an incompletely penetrant multivulva (Muv) and vulvaless (Vul) phenotype. Analysis of
lin-31 mosaic animals indicates that
lin-31 acts in the Pn.p cells (Miller et al, WBG 13.1
p72). Genetically, the Muv phenotype of
lin-31 is epistatic to the Vul phenotype of all of the signalling genes tested, including the MAP Kinase homolog mpk-l/suJ-l (M. Lackner, personal comrnunication). Since LIN-31 acts in the vulval precursor cells downstream of MPK-1 /SUR-1, it is a good candidate to be a transcriptional response element in the anchor cell signaling pathway. Iin-31 is apparently expressed in the Pn.p cells since a
lin-31-GFP reporter gene shows strong expression in P3.p-P8.p beginning in L2 larvae. This expression pattern persists until vulval induction begins. This result suggests that LIN-31 is expressed at the right time and in the right place to respond to the anchor cell signalling pathway. We found that
lin-31 expression was regulated by the process of vulval induction itself. We observed that even before P3.p-P8.p had begun to divide,
lin-31-GFP expression disappeared in cells adopting induced fates (P5.p, P6.p and P7.p and their descendants), but persisted in cells adopting the uninduced fates (P3.p, P4.p, and P8.p and their progeny cells). We next observed the expression pattern of
lin-31-GFP in mutants where all the Pn.p cells adopt either uninduced (
lin-7) or induced cell fates (
lin-15,
let-60(gf). In
lin-7 animals, GFP expression was maintained in all the Pn.p cells and their descendants, indicating that activation of the anchor cell signaling pathway is necessary for downregulation of
lin-31 expression. Conversely, in
lin-15 or
let-60(gf) mutants, GFP expression was repressed in all the Pn.p cells after vulval induction had begun, indicating that activation of the anchor cell signaling pathway is sufficient to repress
lin-31 expression. Previous work on other HNF-3/fkh family members suggested that these transcription factors can regulate their own expression. To investigate
lin-31 autoregulation, we studied the expression pattern of
lin-31-GFP in
lin-31 mutants and observed cases in which P6.p (despite being induced) nevertheless still expressed GFP. These results suggest that the
lin-31 gene product can act, directly or indirectly, to negatively regulate its own expression. In order to confirm these (and the above mentioned) results, we plan to extend these observations using LIN-31 antibodies or RNA in-situ hybridization. We are currently studying whether repression of
lin-31 expression is functionally important for vulval development. We will determine whether persistent
lin-31 expression from a heat-shock promoter is able to perturb vulval development. These results should provide a better insight into how cell signalling controls cell fates during vulval development.