EDGE CT: NSF-BSF: Functional Genotype-Phenotype Mapping in the Mexican Blind Cavefish, Astyanax mexicanus.

生物学中的一个中心问题是，生态和环境如何塑造有机体的发育、生理和行为。盲目的墨西哥洞穴鱼已经成为解决这个问题的有力典范。该物种有两种形式：一种是有眼的祖先形式，另一种是至少29种独立进化的穴居形式。这一物种在解决进化生物学问题的模型系统中是独一无二的，但该模型中遗传工具的缺乏限制了它在识别复杂特征进化的遗传和分子机制方面的使用。这个项目中的实验将建立一个广泛的遗传工具包，在盲目的墨西哥洞穴鱼和表面共生物种中。这项提议中产生的工具将允许操纵精确组织中的基因，并在祖先表层鱼和进化的洞穴动物之间交换基因。该项目将建立在一个既定的推广计划的基础上，将研究带给更广泛的公众。该项目将建立一套K-12培训计划，利用穴居鱼向中小学生传授各种进化主题。其次，该项目将建立一个名为《研究日记》的播客。《研究日记》最近完成了第一季，第二季将专注于非模型系统。第三，为了将生成的工具带给更广泛的公众，并为尽可能多的非模型系统提供便利，该项目将为非模型系统生成年度研讨会，研究人员可以来到PI的实验室并与开发的试剂一起工作。模型系统中的遗传工具使生物学发生了革命性的变化，并提供了对生物学原理的无与伦比的洞察。然而，可转基因生物的数量相对较少，对生物过程的多样性和复杂性提供了有限的视角。该项目将为进化生物学中的一种新兴系统--盲目墨西哥穴居鱼--墨西哥文昌鱼开发转基因和基因靶向方法。墨西哥文昌鱼有两种不同的形式：一种是祖先的眼睛形式，另一种是至少29种进化而来的穴居鱼类。该项目将首先产生组织特异性Gal4线，以及相应的UAS效应线，用于在精确组织中操纵基因功能。该项目将专注于与洞穴鱼项目相关的几个组织，包括神经元、眼睛和肝脏组织。该项目还将生产他莫昔芬诱导型Gal4线。接下来，该项目将生成具有特定于attP位点的线，并且该项目将使用这些线来整合表面和洞穴形式的启动子区域。最后，该项目将在这些鱼类中开发CRISPR/Cas9技术，并将制定在表层鱼类和洞穴鱼之间交换基因的策略。综上所述，这些实验不仅将在这个非模型系统中建立遗传工具，还将建立确定基因-表型相互作用和复杂试验的分子基础的方法。该奖项由EDGE计划和环境生物学部门整合组织系统部门的行为系统集群和进化过程集群共同资助。该奖项反映了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。

A central question in biology is how does ecology and environment shape development, physiology and behavior of an organism. The blind Mexican cavefish has emerged as a powerful model to address this question. The species comes in two forms: an eyed, ancestral form, and at least 29 independently evolved cave-dwelling forms. This species is unique among model systems for addressing questions in evolutionary biology, but the lack of genetic tools in this model has limited its use in identifying genetic and molecular mechanisms underlying the evolution of complex traits. The experiments in this project will establish a broad genetic toolkit in the blind Mexican cavefish and surface conspecifics. The tools generated in this proposal will allow for the manipulation of genes in precise tissues, and a swapping of genes between ancestral surface fish and evolved cave-dwelling animals. The project will build on an established outreach program to bring the research to the broader public. The project will establish a set of K-12 training plans utilizing the cavefish to teach various topics in evolution to primary and secondary school children. Second, the project will establish a podcast entitled, the Research Diaries. The Research Diaries recently completed its first season, and season two will focus on non-model systems. Third, in order to bring the tools generated to the broader public, and in order to facilitate as many non-model systems as possible, the project will generate an annual workshop for non-model systems, were researchers can come to the PI's lab and work with the developed reagents.Genetic tools in model systems have revolutionized biology, and has provided unparalleled insight into biological principles. However, the relatively few numbers of genetically amendable organisms provides a limited view of the diversity and complexity of biological processes. This project will develop transgenic and gene-targeted approaches for an emerging system in evolutionary biology, the blind Mexican cavefish, Astyanax mexicanus. A. mexicanus comes in two distinct forms: an ancestral eyed form, and at least 29 populations of evolved cave-dwelling fish. The project will first generate tissue specific Gal4 lines, and corresponding UAS effector lines for manipulating gene function in precise tissues. The project will focus on several tissues relevant to cavefish programs including neuronal, eye, and liver tissues. The project will also generate tamoxifen inducible Gal4 lines. Next, the project will generate lines with site-specific attP site, and the project will use these lines to integrate promoter regions in surface and cave forms. Finally, the project will develop CRISPR/Cas9 technologies in these fish, and will devise strategies for swapping genes between surface fish and cavefish. Taken together, these experiments will not only establish genetic tools in this non-model system, but will also establish methods for determining genotype-phenotype interactions and the molecular basis for complex traits.This award was co-funded by the EDGE program and the Behavioral Systems Cluster in the Division of Integrative Organismal Systems and the Evolutionary Processes Cluster in the Division of Environmental Biology.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.