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，包括配偶选择，体温调节和避免捕食。然而，对动物如何产生颜色的理解仅基于少数物种，并且在知识上存在很大的差距。例如，我们对两栖动物（脊椎动物的主要类群之一）如何产生它们美丽的颜色和图案知之甚少。该项目将利用基因组学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，这些毒蛙代表了一些最丰富多彩和最具魅力的脊椎动物。将毒蛙作为着色研究的模型系统的发展将确定有助于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。拟议的研究还将培训本科生，研究生和博士后研究人员在尖端基因组技术。项目人员将利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传播研究成果，这些信息将在网上免费提供。这项拟议中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，创建一个毒蛙皮肤颜色的四维基因型-表型图。颜色和图案表型将使用图像的计算分析、分光光度法、代谢组学的组合进行检查，并且其遗传基础将使用几个属、种和种群的基因组测序、全基因组关联研究和RNAseq进行检查。将使用原位杂交来合成结果，以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，然后将两者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，将使用CRISPR验证关键候选模式化基因。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、颜色模式和色素组织的表型变异联系起来，并使用TEM图像进行可视化。这种整体的方法，专注于三个独立的起源点和条纹图案，将提供关键的见解，在脊椎动物的颜色变化的一般机制。此外，众所周知，毒蛙具有庞大而复杂的基因组，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型与表型联系起来的指导。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值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.