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. © 2016 barkoulas et al.