Transcriptional upregulation of the C. elegans Hox gene lin-39 during vulval cell fate specification

Extracellular signaling pathways and transcriptional regulatory networks function during development to specify metazoan cell fates. During Caenorhabditis elegans vulval development, the specification of three vulval precursor cells (VPCs) requires the activity of Wnt, Notch, and Ras signaling pathways, and function of the Hox gene lin-39. LIN-39 protein levels are regulated in the VPCs by both Wnt and Ras signaling. In particular, activation of Ras signaling leads to an increase in LIN-39 protein in P6.p at the time of VPC fate specification. We wish to understand the regulation of lin-39 by these pathways. We first show that LIN-39 is a target for MAP kinase in vitro, suggesting that the Ras-dependent LIN-39 upregulation could be mediated post-translationally. To test this idea, we created transcriptional and translational lin-39::GFP fusions that include the entire lin-39 genomic region, allowing observation of lin-39 expression in live animals. The reporters express GFP in most, if not all, sites of expression previously observed by LIN-39 antibody staining. We used these constructs to show that at the time of vulval induction both lin-39::GFP reporters are upregulated in P6.p, indicating that the accumulation of high levels of LIN-39 protein detected previously corresponds to transcriptional upregulation of lin-39 expression. This transcriptional upregulation of lin-39 is dependent on Ras signaling. We tested the requirement for several transcription factors acting downstream of Ras signaling in the VPCs, and found that P6.p upregulation requires the transcription factors LIN-1 and LIN-25, but appears to be independent of LIN-31, SEM-4, EOR-1 and EOR-2.Finally, we found that when the Wnt pathway is over activated, expression from the transcriptional lin-39::GFP increases, suggesting that the Wnt pathway also regulates lin-39 at the transcriptional level.