Cells in the vulval equivalence group in the C. elegans hermaphrodite can adopt one of three different fates, 1o, 2o or 3o. The fates of these 6 cells, termed collectively the VPCs, are thought to be influenced by at least three different signalling events. A cell in the somatic gonad, the anchor cell induces P6.p to adopt the 1o cell fate. Lateral signalling between adjacent VPCs results in P5.p and P7.p adopting the 2o fate. The remaining cells, P3.p, P4.p and P8.p, adopt the 3o fate, it is thought because they do not receive sufficiently high levels of either the inductive or lateral signals to overcome an inhibitory signal from the neighbouring syncytial hypodermal cell
hyp7. Signalling between the anchor cell and P6.p is mediated by a signal transduction pathway involving
let-60 Ras and
mpk-1 MAP kinase. Previous work has shown that one likely target of this pathway is the ETS domain transcription factor LIN-1. Loss-of-function mutations in
lin-1 cause the Multivulva phenotype: P3.p, P4.p and P8.p are induced to adopt a vulval (1o or 2o) fate. Since the Multivulva phenotype caused by
lin-1(lf) is epistatic to the Vulvaless phenotype caused by loss-of-function mutations in
mpk-1 MAP kinase, and since MPK-1 can directly phosphorylate LIN-1 in vitro, the current model is that the Ras/MAP kinase pathway mediates the induction of the 1o fate in part by phosphorylating and inactivating LIN-1, a negative regulator of this fate. One molecular marker for the 1o fate is an FGF-family ligand EGL-17. GFP under the control of
egl-17 promoter sequences is expressed in P6.p and this expression is dependent upon
let-60 Ras. Activation of
let-60 in other VPCs causes ectopic expression of the reporter. Since
let-60 Ras appears to function in part by inactivating LIN-1, if
lin-1 regulated
egl-17, then a reasonable expectation is that inactivation of
lin-1 might, like activation of
let-60, cause increased expression of the EGL-17::GFP reporter. We have found, however, that
lin-1 null mutations have the converse effect: they abolish
egl-17::gfp expression in P6.p. Thus
lin-1 is required positively for the expression of at least one marker for the 1o fate. We reasoned that one possible explanation for this observation might be that loss of
lin-1 function might cause constitutive activation of the lateral signalling pathway which promotes the 2o fate. Consistent with a role for lateral signalling in negatively regulating
egl-17::gfp expression in P6.p, expression of the marker was consistently higher than in wild type in worms homozygous for
n676n930, a hypomorphic mutation in
lin-12, which encodes the receptor for this pathway. However, the marker was not expressed in
lin-12(
n676n930);
lin-1(0) double mutant hermaphrodites.
lin-1(0) also suppressed the ectopic expression of
egl-17::gfp caused by constitutive activation of
let-60 Ras. Together, these observations suggest that the role for LIN-1 in the induction of vulval fates might be more complicated than previously thought. By comparing the sequence of the C. elegans
egl-17 promoter fragment with the analogous sequence from C. briggsae, we have identified 3 elements that show strong sequence conservation between the two species. We are presently assaying these and other fragments for their ability to direct expression of gfp in P6.p, and for their ability to be bound and regulated by LIN-1. We are also examining the effect of
lin-1 mutations on other markers for the 1o fate such as downregulation of LIN-12::GFP, and downregulation of
lip-1::gfp.