Discovery and functional analysis of novel candidate genes and variants underlying craniofacial diversification in Cyprinodon pupfishes

项目概要： 颅面畸形是人类出生缺陷最常见的形式，但其分子 基础仍然知之甚少。高度保守的颅面发育通路 在不同的脊椎动物物种中共享的基因已经被适应性进化所塑造， 适应性颅面表型的巨大多样性基本调查 这些表型的遗传基础将导致更好的诊断，预防和治疗 人类出生缺陷事实上，关于许多遗传基础的补充或新信息， 模型系统中的人类病理学通常可从 表现出相似的不同表型。这些自然系统现在是可行的基因组 和转基因方法，并提供了一个机会，'进化'向前遗传学。 在这里，我建议利用我实验室的丰富经验开发一种新的脊椎动物， 系统从地面向上：在加勒比海pupfishes高度分歧颅面形态。 我们初步的全基因组差异扫描和关联映射已经确定了这两个 已知的颅面候选基因和10个新的与颌骨相关的候选基因 长度变化不同的基因组区域通常仅限于一个基因， 或在上游调控区或内含子区中的一些候选变体。我们的初步 数量性状基因座作图也表明，这些区域中的一些可以解释高达15%的 在实验室饲养的F2交种中的颚长变化。因此，我假设， 这些物种控制已知和新的颅面神经的时空表达 基因潜在的高度分歧的颅面特征在pupfishes。我建议 研究新的适应性表型的遗传基础，在这个非模型系统中使用一个 群体基因组学、从头基因组组装、数量遗传学、 转录组学、原位杂交和CRISPR-Cas9基因组编辑。 我们在加勒比海的初步成功证明了我们的方法的力量， 扩展的潜力。Pupfish展示了新的颅面特征;持续的基因流和强大的 选择提供了一个理想的自然“实验”， 有这些特征。我们还开创了原位杂交和CRISPR-Cas9方法， pupfishes。通过在自然系统中整合候选基因和变异发现， 多样的颅面特征与强大的功能测定，拟议的研究将 展示了新的非模型系统的可行性和力量，以获得新的见解 人类疾病的发育遗传学 1

Project Summary: Craniofacial abnormalities are the most common form of human birth defects, but their molecular basis remains poorly understood. Highly conserved craniofacial developmental pathways shared across diverse vertebrate species have been shaped by adaptive evolution to produce a tremendous diversity of adaptive craniofacial phenotypes. Fundamental investigation of the genetic basis of these phenotypes will lead to better diagnosis, prevention, and treatment of human birth defects. Indeed, complementary or new information on the genetic basis of many human pathologies in model systems is often obtainable from naturally occurring systems that display analogous divergent phenotypes. These natural systems are now feasible for genomic and transgenic approaches and provide an opportunity for ‘evolutionary’ forward genetics. Here I propose to leverage my lab’s extensive experience developing a new vertebrate system from the ground up: highly divergent craniofacial morphology in Caribbean pupfishes. Our preliminary genome-wide divergence scans and association mapping have identified both well-known craniofacial candidate genes and ten new candidate genes associated with jaw length variation. Divergent genomic regions are often restricted to a single gene with only one or a few candidate variants in upstream regulatory or intronic regions. Our preliminary quantitative trait locus mapping also indicates that some of these regions explain up to 15% of jaw length variation in lab-reared F2 intercrosses. Thus, I hypothesize that fixed mutations in these species control spatiotemporal expression of both known and novel craniofacial genes underlying highly divergent craniofacial features in pupfishes. I propose to investigate the genetic basis of novel adaptive phenotypes in this non-model system using a combination of population genomics, de novo genome assembly, quantitative genetics, transcriptomics, in situ hybridization, and CRISPR-Cas9 genome editing. Our initial success in Caribbean pupfishes demonstrates the power of our approach and potential for expansion. Pupfish exhibit novel craniofacial traits; ongoing gene flow and strong selection provide an ideal natural ‘experiment’ for fine-mapping candidate variants associated with these traits. We are also pioneering in situ hybridization and CRISPR-Cas9 approaches in pupfishes. By integrating candidate gene and variant discovery in a natural system exhibiting diverse craniofacial features with powerful functional assays, the proposed research will demonstrate the feasibility and power of new non-model systems to gain novel insights into the developmental genetics of human diseases. 1