Establishment of individual ray identities is a complex process involving Hox genes
egl-5 and
mab-5. The regulation of Hox gene expression in cell lineages, however, is not well understood. For example, in the lineage leading to the rays, MAB-5 turns on
egl-5 transcription in V6.ppp, but for reasons unknown, not in V6, V6.p, or V6.pap, where MAB-5 is also present. To understand this regulation, we are now doing promoter dissections of
egl-5, trying to find the regulators of
egl-5 by identifying the regulatory motifs in the promoter. We have been concentrating on the sequences in the
egl-5 promoter that are conserved between C. elegans and C. briggsae, on the assumption that the conservation in DNA sequence implies functional significance. Our approach is to introduce deletions of conserved sequences into
egl-5::gfp reporters, and try to interpret the changes in expression pattern. So far we were able to localize a B lineage enhancer to a highly conserved region of about 200 bp, and are currently searching for regulatory motifs that can account for specific expression of
egl-5 in seam cells and the ray lineages. Two other candidate genes that might affect the expression pattern of Hox genes are
ceh-20, which is a Drosophila Exd/human Pbx homolog, and
ceh-25 (
unc-62), which is a Drosophila Hth/human Meis homolog. EXD/PBX amd HTH/MEIS are noncanonical homeobox proteins that physically interact with Hox proteins to enhance their DNA binding specificity. If
ceh-20 and
ceh-25 act in C. elegans as they do in Drosophila and humans, they should be implicated in the processes of self and cross regulation among Hox genes. Weak alleles of
ceh-20 and
ceh-25 produce males with abnormal ray morphology. RNAi experiments also result in male tail defects, suggesting that
ceh-20 and
ceh-25 do fuction during male tail development. We are now making reporters and constructing dominant-negative forms of
ceh-20 and
ceh-25 to analyze their roles in detail.