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

Michalis Barkoulas,Amhed M Vargas Velazquez,Alexandre E Peluffo,Marie-Anne Félix,Susan E Mango

doi:10.1371/journal.pgen.1006278

Abstract

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.

Highlights

Developmental systems operate in the presence of stochastic, environmental and genetic perturbations
We find that the lin-3 expression level is remarkably conserved, with 20–25 messenger RNAs per anchor cell, in species that are molecularly as distant as fish and mammals
We previously showed by modulating lin-3 expression via single-copy transgenesis that the genomic level of lin-3 expression is limited within a four-fold range for the vulva to develop normally in the C. elegans N2 background [2]

Summary

Introduction

Developmental systems operate in the presence of stochastic, environmental and genetic perturbations. To reach a quantitative understanding of developmental systems, a key approach is to measure the sensitivity of the developmental system output to induced variation in an intermediate developmental phenotype. Whether and how this intermediate developmental phenotype varies within and among species becomes a relevant evolutionary question [1]. Cis-regulatory sites containing binding sites for transcription factors often occur upstream of the coding region or in introns These binding sites are often organized in modules, the designation as cis-regulatory modules (CRMs), acting in concert to enhance or repress gene expression in a given tissue at a given time. Lin-48 expression was gained in the excretory duct cell of C. elegans due to the acquisition of upstream binding sites for the transcription factor CES-2 [18,19]

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Discussion

Conclusion