Enhancer Evolution and the Origins of Vertebrate Brain Development

转录增强子控制胚胎发育过程中基因表达的时空特异性 发展据估计，人类基因组包含数十万个增强子， 它们的识别和功能的理解对理解人类发育和疾病很重要。 染色质状态的全基因组分析的进展已经推动了以下能力的重大进展： 确定潜在的顺式调控元件，但阐明生物学功能和确定如何 基因调控的变化导致表型创新仍然具有挑战性， 在基因调控和增强子进化的理解差距。感兴趣的一个具体创新是 脊椎动物的大脑和基因组的变化，导致这种形态的新奇。长期目标是 该项目旨在阐明脊椎动物中枢神经系统顺式调控逻辑的进化起源。 神经系统使用半索动物Saccoglossus kowalevskii，一种无脊椎动物的后口， CNS。这里的总体目标是鉴定在初级脊椎动物中有活性的保守增强子元件 脑信号中心，并确定其在S. kowalevskii和老鼠。初步数据 表明小鼠Sonic hedgehog brain enhancer 1（SBE 1）的功能性直系同源物， 在S. kowalevskii和Shh表达背后的顺式和反式决定因素是 古老的起源这些数据表明，相同的SBE 1增强子用于调节Shh表达， 非同源结构的脊椎动物和半索动物，并显示出识别功能的可行性 在具有不同解剖结构的动物中， 进化中心假设是，尽管存在很强的形态差异，但顺式调节元件在 脊椎动物的前神经脊、丘脑内嵴和峡部组织者存在于 半索藻S. kowalevskii，一种具有弥散神经网络的动物，并且具有保守的调节功能。 这项研究的基本原理是不仅要深入了解脊椎动物中枢神经系统的起源， 增强子，而且基因调控的基本机制及其与形态创新的联系。 本研究将通过以下三个具体目标来验证这一假设：1）确定保守的顺式调控元件 使用ATAC-seq和转座子介导的增强子捕获在初级脊椎动物信号传导中心中有活性 色葡萄kowalevskii BACs在小鼠中的表达; 2）测定半索酸和小鼠BACs的时空活性 转基因S. kowalevskii和小鼠胚胎;和3）阐明每个保守的功能 S. kowalevskii使用CRISRP-Cas9基因组编辑工具。方法是创新的 因为它是第一个确定存在关键保守的功能验证的发育 在图案化不同形态结构中起作用的增强子。所提出的研究是有意义的 因为它有望纵向推进和扩展我们对基因调控及其与 形态创新

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.