We are studying how extracellular signaling regulates cell fate specification during vulval development. The vulva arises from a group of six Vulval Precursor Cells (VPCs) located in the ventral midline of the body. The Hox gene
lin-39 is expressed in the VPCs and is required for their cell fate specification. At the L3 stage, activation of a conserved RTK/Ras signaling pathway induces vulval formation and the Kenyon lab has shown that LIN-39 levels are upregulated by activation of the Ras pathway. A Wnt signaling pathway also acts during VPC fate determination.
bar-1 encodes a b -catenin homologue, and in
bar-1 mutants the level of LIN-39 is very low in some VPCs and these VPCs adopt abnormal cell fates. Therefore,
lin-39 is regulated by Wnt and Ras pathways. Our goal is to understand how these signaling pathways converge in the regulation of the Hox gene
lin-39 to specify VPC fates. Specifically, we want to know if
lin-39 regulation by these pathways is at the transcriptional or post transcriptional level. MAP kinase acts downstream in the Ras pathway by phosphorylating transcription factors such as LIN-1 and LIN-31. The LIN-39 protein sequence has a single phosphorylation site and a single docking site for MAP kinase, suggesting that the Ras pathway may regulate
lin-39 activity at the post-transcriptional level by phosphorylation. Preliminary results show that 6HIS-LIN-39 purified from E. coli was weakly phosphorylated by vertebrate MAP kinase in an in vitro phosphorylation assay. The addition of CEH-20, a protein known to interact with LIN-39, enhanced LIN-39 phosphorylation under specific binding conditions, either alone or in the presence of a DNA fragment containing putative LIN-39 binding sites. Experiments are underway to determine if CEH-20, DNA or both enhance LIN-39 phosphorylation. We are also mutating the LIN-39 phosphorylation and docking sites to test if phosphorylation requires these sequences. Our goal is to understand the significance of LIN-39 phosphorylation. We wish to determine if phosphorylation of LIN-39 affects 1) the interaction of LIN-39 with DNA, 2) the interaction of LIN-39 with CEH-20, and 3) the stability of LIN-39 in vivo . The
lin-39 genomic sequence contains recognition sites for TCF transcription factors, which are known to mediate Wnt signaling, suggesting a transcriptional mode of regulation of
lin-39 . We are cloning
lin-39 genomic regions containing TCF recognition sites into a
pes10::GFP reporter to determine if these TFC sites are required for
lin-39 expression. Preliminary results using a fragment containing six TCF sites located 5.5 kb upstream of the LIN-39 start codon showed GFP expression in different tissues but only a weak expression in the VPCs of one animal. Finally, we are making two
lin-39 ::GFP reporter constructs that will help us to differentiate between a transcriptional and a post-transcriptional regulation of
lin-39 activity.