lin-39, one of the six C. elegans Hox genes, encodes a transcription factor expressed in cells of the mid-body region, including the vulval precursor cells (VPCs), the Q cell daughters, and ventral cord neurons. We are interested in the role of
lin-39 in vulval development.
lin-39 is required twice for vulval development. First,
lin-39 is required in the L1 stage for the generation of the VPCs. In
lin-39 null mutants, the VPCs fuse with
hyp7 in the L1, like the other Pn.p cells that do not express
lin-39. Second,
lin-39 is required in the L3 stage for proper vulval cell fate specification, and loss of
lin-39 activity at this time leads to defects in vulval induction.
lin-39 acts downstream of both RTK/Ras and Wnt signaling pathways at this time. We hypothesize that LIN-39 regulates multiple target genes that prevent fusion of the VPCs and promote VPC fate specification.Some LIN-39 targets are known. LIN-39 was shown by gel shift assays to interact with its co-factor, CEH-20, an Extradentical homologue, and bind sites in the
egl-18 and
hlh-8 genes. Other potential targets, such as,
egl-17 and
eff-1, show differential expression with the loss of
lin-39; however, no direct binding has been shown. The goal of my project is to identify unknown targets of
lin-39 through microarray analysis. We will compare gene expression patterns between an inducible
lin-39 strain, containing muIs23 (Hslin-39), and a control strain. Preliminary RT-PCR analysis of the muIs23 strain shows an increase in
lin-39 and
egl-18 transcripts after induction. Time course experiments will tell us when after induction to collect RNA for microarray analysis.We are taking two approaches to identify unknown
lin-39 targets: a global approach and a cell-specific approach. The global approach will identify
lin-39 targets though out the worm by using total mRNA as a template. Adapting the cell-specific method of Roy et al. (Nature, 2002) we will use a VPC-specific promoter driving FLAG::PAB-1 to enrich for mRNA expressed in the VPCs or VCNs only (see B. Jackson abstract).Once we have identified genes differentially regulated upon LIN-39 expression we will use qRT-PCR, chromatin immunoprecipitation, GFP reporters and RNAi to validate candidate target genes and examine their role in vulval development.