Our laboratory is interested in the mechanisms controlling cell fate specification by the Vulval Precursor Cells (VPCs) during vulval induction. Established work showed that RTK/Ras and Notch signaling pathways act during this process, and previous work from our laboratory and others has indicated that a Wnt signaling pathway is also required. The first evidence of this was the observation that mutations in
bar-1 , which encodes one of three C.elegans beta-catenin homologs, cause VPC fate specification defects that lead to Pvl and Egl phenotypes. One target of this pathway is the Hox gene
lin-39 , which is coordinately regulated at the transcriptional level by both Wnt and Ras pathways (see Wagmaister et al. abstract). Subsequently, we have shown that other canonical Wnt pathway components such as APC (APR-1), Axin (PRY-1) and TCF/LEF (POP-1), also function during VPC fate specification. However to date, we have not identified the Wnt signal used to activate this pathway, the source of this Wnt signal, or the Frizzled receptor used to transduce this signal in the VPCs. We predict that loss of the Wnt or frizzled gene activity would lead to a vulval phenotype resembling that of
bar-1(
ga80) or
mig-14(
ga62) . There are five Wnt genes in the worm (
lin-44,
egl-20,
mom-2,
cwn-1 and
cwn-2 ), and we took two approaches to determine which of these is acting on the VPCs. First, analysis of existing viable mutants showed that none of them had Pvl/Egl phenotypes like
bar-1(
ga80) , however
egl-20 mutants had a Fused fate phenotype at P3.p and P4.p like that observed in
bar-1(
ga80) . This phenotype was not enhanced in a
lin-44;
egl-20 double mutant. To examine whether redundant Wnts might be used in VPC fate specification, we performed RNAi on
cwn-1,
cwn-2 or
mom-2 in a
lin-44;
egl-20 background, and found that 21 - 36% of
lin-44;
egl-20;
cwn-1(RNAi) animals had an Underinduced phenotype like
bar-1(
ga80), in which fewer than three VPCs adopted induced fates. Subsequently, we analyzed a
cwn-1(
ok546);
egl-20(
n585) double mutant strain (generous gift of Rik Korswagen) and found that 84% of these animals had Underinduced or Vulvaless phenotypes (versus 6% for
cwn-1(
ok546) and 3% for
egl-20(
n585) ). These results suggest that the Wnt pathway in the VPCs is activated by two genetically redundant Wnt ligands, EGL-20 and CWN-1. Second, we created transcriptional GFP reporter constructs for all five Wnt genes to determine which, if any, were expressed in or around the VPCs at a time when they could influence fate specification. Analysis of the expression of these constructs showed that both
cwn-1 and
cwn-2 are expressed in ventral cord neurons during the L2 and L3 stages, and could therefore be affecting the VPCs. Although
egl-20 is expressed in the vulval region, we have not seen expression at a time early enough to corroborate our genetic evidence. This could be due to low level or transient expression, or to the lack of necessary genomic elements in our reporter. Data on the expression of the Wnt reporters, additional Wnt gene RNAi data, and similar experiments to identify the frizzled gene or genes (
lin-17,
mig-1,
mom-5, or
cfz-2 ) acting in the VPCs will be presented.