In C. elegans, ventral ectoblasts called P cells divide postembryonically to produce ventral cord neurons and hypodermal cells. Initially, the two most posterior P ectoblasts have similar potential to undergo two patterns of development known as the P11 and P12 cell fates. In normal development, the WNT and EGF signaling pathways and the HOX gene
egl-5 direct only the most posterior cell to adopt the P12 cell fate. Through epistasis analysis, we have found that
egl-5 acts downstream of the WNT pathway, which combined with other studies, indicates EGL-5 is a functional target of both signaling pathways. Given the central role of
egl-5 in P12 cell fate specification, we have sought to better understand the molecular mechanisms that regulate
egl-5 expression. Previously, another group identified a 1.3 kb
egl-5 enhancer fragment (present in pLG7) that can activate GFP expression in P12.pa, a cell type unique to the P12 cell fate (Teng et al., 2004, Dev Biol 276:476-492). We have found that pLG7 does not express in the P12 cell lineage in
egl-5(-) and Wnt pathway mutants that lack P12 cell fates. Notably, we found that ectopic expression of EGL-5 protein can activate GFP expression from pLG7, even when the Wnt pathway is inactive. This result suggests that pLG7 contains an EGL-5 response element. By producing deletion variants of pLG7, we have identified a minimal 364bp region (pXL323) sufficient for expression in P12.pa. This minimal region contains a consensus sequence for a HOX binding site and this site is conserved in two other Caenorhabditis species. We have shown that mutating this HOX site in pLG7 or pXL323 causes a loss of GFP expression in P12.pa (but not in other cells). These results are consistent with the model that the pLG7 enhancer contains an EGL-5 autoregulatory site that maintains EGL-5 expression in P12.pa. To test whether the pLG7 enhancer is sufficient to activate expression in the P12 cell lineage, we used this enhancer to express EGL-5 protein in an
egl-5 null mutant and found that this did not restore P12 cell fates. This result suggests that some of the cis regulatory sequences that normally activate
egl-5 expression lay outside of pLG7. We propose that functionally separable regulatory elements initiate and maintain
egl-5 expression in the P12 cell lineage. We are continuing experiments to characterize both types of elements.