Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis

Patterning of C . elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C . elegans wild isolates and only a low level of change—less than 30%—in the Caenorhabditis genus and in Oscheius tipulae . In C . elegans , lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR) binding site. Mutation of any of these three elements in C . elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C . angaria , while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C . angaria bearing a single E-box is sufficient for normal expression in C . elegans . Even a short 58 bp cis -regulatory fragment from C . angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C . elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C . elegans . This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis -regulatory site requirements and highlights the evolution of new cis -regulatory elements required for cell-specific gene expression.