Collaborative Research: EDGE CMT: A four-dimensional genotype-phenotype map of poison frog skin color

颜色在动物中起着重要的生态作用，包括选择配偶、调节体温和避免捕食。然而，对动物如何产生色彩的了解仅基于少数物种，知识上仍存在很大差距。例如，作为脊椎动物的主要群体之一，两栖动物是如何产生美丽的颜色和图案的，人们对此知之甚少。该项目将利用基因组和发育生物学的最新进展来确定控制三种毒蛙颜色和颜色模式的基因，这三种毒蛙代表了一些最五颜六色和最有魅力的脊椎动物。毒蛙作为着色研究的模型系统的发展将确定导致着色的关键生物学机制，并为其他动物的颜色遗传学研究开辟新的途径。这项拟议的研究还将培训本科生、研究生和博士后研究人员使用尖端基因组技术。项目人员将使用活青蛙的视觉展示、用西班牙语编写的儿童书籍以及基于科学实地工作的虚拟实地体验向公众交流研究成果，这些虚拟实地体验将在网上免费提供。这项拟议的研究将使用将颜色和图案与其潜在遗传学联系起来的方法，创建毒蛙皮肤着色的四维基因型到表型图。颜色和图案表型将使用图像计算分析、分光光度分析、代谢组学的组合进行检查，其遗传基础将使用几个属、物种和种群的基因组测序、全基因组关联研究和RNAseq进行检查。结果将使用原位杂交来跟踪候选基因在发育过程中的表达，并使用空间RNA测序来跟踪青蛙特定皮肤区域的基因表达，然后将这两种结果映射到使用透射电子显微镜(TEM)获得的皮肤结构图像上。最后，关键候选图案基因将使用CRISPR进行验证。结果将被整合到一个四维图中，将遗传变异和空间基因表达与颜色、颜色图案和色素组织的表型变化联系起来，并使用透射电子显微镜图像进行可视化。这种整体的方法，集中在斑纹和条纹图案的三个独立起源上，将为脊椎动物颜色变化的一般机制提供关键的见解。此外，毒蛙以拥有庞大而复杂的基因组而闻名，因此这项工作将为未来的研究人员提供如何在困难的系统中将基因与表型联系起来的指南。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Coloration plays important ecological roles in animals, including mate choice, thermoregulation, and avoiding predation. However, the understanding of how animals produce coloration is based on only a few species and large gaps in knowledge remain. For example, very little is known about how amphibians, one of the major groups of vertebrate animals, produce their beautiful array of colors and patterns. This project will leverage recent advances in genomic and developmental biology to identify the genes that control color and color patterning in three species of poison frogs that represent some of the most colorful and charismatic vertebrates. The development of poison frogs as a model system for the study of coloration will identify key biological mechanisms that contribute to coloration, and open new avenues of research into genetics of color in other animals. The proposed research will also train undergraduate students, graduate students, and postdoctoral researchers in cutting-edge genomic techniques. Project personnel will communicate research results to the public using visual displays of live frogs, children’s books written in Spanish, and a virtual field experience based on scientific field work which will be made available freely online. The proposed research will create a four-dimensional genotype-to-phenotype map of poison frog skin coloration using methods that link color and pattern to their underlying genetics. Color and pattern phenotypes will be examined using a combination of computational analysis of images, spectrophotometry, metabolomics and their genetic underpinnings will be examined using genome sequencing of several genera, species and populations, genome wide association studies, and RNAseq. Results will be synthesized using in-situ hybridization to track the expression of candidate genes over the course of development and spatial RNA sequencing to track gene expression in specific skin regions of the frogs, both of which will then be mapped onto images of skin structure obtained using transmission electron microscopy (TEM). Finally, key candidate patterning genes will be validated using CRISPR. Results will be integrated into a four-dimensional map connecting genetic variants and spatial gene expression with phenotypic variation in color, color patterning, and pigment organization, and visualized using TEM images. This holistic approach, focusing on three independent origins of spot and stripe patterning, will provide key insights into general mechanisms underlying color variation in vertebrates. Furthermore, poison frogs are known for having large, complex genomes, so this work will provide a guide to future investigators on how to connect genotype to phenotype in difficult systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.