Enhancer Co-option During Vertebrate Evolution

Abstract

Phenotypic diversity across species is often driven by changes in gene regulation rather than the gene content itself. Cis-regulatory elements, including enhancers, form the cornerstone of regulating any developmental program. While enhancer sequences typically diverge across multiple species, a certain subset of these enhancers have their sequences conserved between zebrafish and mice. This project aims to investigate whether enhancers that remain conserved despite the water-to-land transition of vertebrates have been co-opted to carry out novel functions. The lateral line is a great system to study enhancer co-option because it was lost in terrestrial vertebrates, and despite this loss, some lateral line enhancer sequences are still present in the mouse. This gives us a unique set of enhancers that are conserved in a species that no longer possesses the organ itself. This raises compelling questions about what these enhancers are doing in the mouse. We hypothesize that these sequences have been evolutionarily conserved because they acquired new developmental functions associated with the emergence of novel phenotypes. To test this hypothesis, we conducted analyses of ATAC-seq data combined with sequence conservation, to identify a set of 72 putative lateral line enhancers whose sequences were conserved in both zebrafish and mice. To assess enhancer activity, we carried out enhancer-reporter assays, which revealed 12 enhancer candidates that could drive reporter expression. Comparative analysis of the activity of zebrafish and mouse enhancer orthologs in zebrafish revealed that while many enhancer orthologs exhibited a great overlap in their spatial activity, notable differences in tissue and cell-type-specific expression were observed. Additionally, comparison of the orthologs of the same enhancer in their native species, as well, revealed differences in their spatial activity, while some overlap in their regulatory outputs was still retained. Together, these findings suggest that conserved enhancers may have been repurposed across vertebrate species during evolution, while function in similar sensory structures was still retained. This work provides insights into how enhancer elements associated with lost organs may have been co-opted to contribute to novel developmental programs during vertebrate evolution.