Enhancer Evolution and the Origins of Vertebrate Brain Development

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Transcriptional enhancers control the spatiotemporal specificity of gene expression during embryonic development. It is estimated that the human genome contains hundreds of thousands of enhancers, making their identification and understanding of function important to understanding human development and disease. Advances in whole genome analysis of chromatin states have driven significant progress in the ability to identify potential cis-regulatory elements, but the ability to elucidate biological function and determine how changes in gene regulation lead to phenotypic innovation remains challenging and represents a fundamental gap in the understanding of gene regulation and enhancer evolution. One specific innovation of interest is the vertebrate brain and the genomic changes that led to this morphological novelty. The long-term goal of this project is to elucidate the evolutionary origins of the cis-regulatory logic underlying the vertebrate central nervous system using the hemichordate Saccoglossus kowalevskii, an invertebrate deuterostome that lacks a CNS. The overall objective here is to identify the conserved enhancer elements active in the primary vertebrate brain signaling centers and determine their regulatory function in S. kowalevskii and mouse. Preliminary data indicate a functional ortholog of mouse Sonic hedgehog brain enhancer 1 (SBE1) active in the zona limitans intrathalamica exists in S. kowalevskii and the cis and trans determinants underlying Shh expression are of ancient origin. These data suggest the same SBE1 enhancer is used to regulate Shh expression in diverse, nonhomologous structures in vertebrates and hemichordates, and show the feasibility of identifying functional conserved enhancers in animals with disparate anatomies separated by hundreds of millions of years of evolution. The central hypothesis is despite strong morphological disparity the cis-regulatory elements active in the vertebrate anterior neural ridge, zona limitans intrathalamica, and isthmic organizer are present in the hemichordate S. kowalevskii, an animal with a diffuse nerve net, and have a conserved regulatory function. The rationale for this proposed research is to gain insight not only into the origins of the vertebrate CNS enhancers, but also the fundamental mechanisms of gene regulation and its link to morphological innovation. This hypothesis will be tested by pursuing three specific aims: 1) Identify the conserved cis-regulatory elements active in the primary vertebrate signaling centers using ATAC-seq and transposon-mediated enhancer trapping of S. kowalevskii BACs in mouse; 2) Determine the spatiotemporal activity of hemichordate and mouse enhancers in transgenic S. kowalevskii and mouse embryos; and 3) Elucidate the function of each conserved cis-regulatory element in S. kowalevskii using CRISRP-Cas9 genome editing tools. The approach is innovative because it is one of the first to identify the presence of key conserved functionally validated developmental enhancers with roles in patterning disparate morphological structures. The proposed research is significant because it is expected to vertically advance and expand our understanding of gene regulation and its link to morphological innovation.