EDGE: Developing techniques for linking genotype to phenotype in amphibians

几个世纪以来，两栖动物一直是科学研究中的重要模式系统。青蛙和蝾螈的生理、生命周期和行为很容易理解，可以用来研究生物学中的一般问题。有超过5，000种青蛙，显示出大量的自然多样性，每个物种都以独特的方式适应其环境。这些物种中有许多面临灭绝的危险，主要是由于栖息地退化和致命真菌在世界各地的传播，这种真菌可以杀死整个青蛙种群。该项目的研究人员与两栖动物研究人员网络一起，为青蛙和蝾螈开发基因编辑工具。这个基因工具包的目的是使研究人员能够在原始实验室和泥泞的雨林中测试基因或蛋白质如何影响行为和发育的预测。该项目还涉及通过实践培训课程和开发在线社区资源，对来自世界各地的本科生和研究生以及研究人员进行培训。在斯坦福大学举办了一个培训班，以支持在美国培训研究人员。另一个国际研讨会在南美洲举行，那里拥有世界上大部分的两栖动物生物多样性。此外，该项目还将一个名为“蛙人学校计划”的成功区域项目扩展为全国性资源，为农村和市中心公立学校的K-12教室提供教育材料。两栖动物是一种多样化的脊椎动物，在形态、行为和生理学方面具有独特的进化创新。这些引人注目的表型中的大多数在具有功能基因组学工具的少数两栖动物物种中并不存在，严重阻碍了理解遗传变异如何影响在这个物种群中观察到的令人难以置信的表型变异的进展。南美洲的毒蛙进化出了警戒色、化学防御和复杂的父母行为，这些特征在物种内部和物种之间都有所不同。此外，这套特征在一些非洲青蛙物种中独立进化，允许内置复制来解决基因型和表型之间的联系。这项研究计划的重点是在毒蛙中开发功能基因组学工具，并计划将这些技术扩展和传播到更广泛的两栖动物社区。具体来说，该项目的目标是开发一个通用的两栖动物遗传工具包，以（1）使用CRISPR/Cas9和morpholino技术进行基因破坏，（2）使用表达载体插入表达转基因材料，以及（3）开发电穿孔技术来操纵特定细胞类型或组织中的基因。另一个目标是通过在线分发协议和实践研讨会来快速传播这个工具包，以培训下一代两栖动物生物学家将新开发的尖端技术整合到他们的研究计划中。最后，现有的区域K-12外展计划扩展为全国性的资源，供教育工作者和当地两栖动物研究人员相互联系，并将两栖动物生物学，保护和进化带入公立学校课堂。该项目由生物科学理事会综合有机系统司行为系统组共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Amphibians have been an important model system in scientific research for centuries. Frogs and salamanders have easily accessible physiology, life cycles, and behavior that can be used to study general questions in biology. There are over 5,000 species of frogs that show a great deal of natural diversity, where each species has adapted in unique ways to its environment. Many of these species are at risk of extinction, mainly due to habitat degradation and spread of a lethal fungus throughout the world that can kill entire frog populations. The investigators of this project, together with a network of amphibian researchers, develop gene-editing tools for frogs and salamanders. The purpose of this genetic toolkit is to enable researchers to test predictions of how genes or proteins influence behavior and development, both in the pristine laboratory as well as the muddy rainforest. This project also involves training of undergraduate and graduate students, and researchers from around the world through hands-on training courses and the development of online community resources. One training course is held at Stanford University to support the training of researchers in the United States. Another, international workshop is held in South America, which holds much of the world's amphibian biodiversity. In addition, the project expands a successful regional program called the Frogger School Program to a nationwide resource with education materials for K-12 classrooms in rural and inner-city public schools.Amphibians are a diverse group of vertebrates with unique evolutionary innovations in morphology, behavior, and physiology. Most of these striking phenotypes are not represented in the few amphibian species with functional genomics tools, severely hindering progress on understanding how genetic variation influences the incredible phenotypic variation observed within this speciose group. South American poison frogs have evolved warning coloration, chemical defenses, and complex parental behaviors, where each of these traits varies both within and between species. Moreover, this suite of traits has evolved independently in some African frog species, allowing built-in replication for addressing links between genotype and phenotype. This research proposal focuses on the development of functional genomics tools in poison frogs, with plans to expand and disseminate these technologies to the broader amphibian community. Specifically, the goals of the project are to develop a general amphibian genetic toolkit to (1) enable gene disruption using CRISPR/Cas9 and morpholino technologies, (2) express transgenic materials using expression vector insertions, and (3) develop electroporation techniques to manipulate genes in specific cell types or tissues. Another goal is rapid dissemination of this toolkit through the online distribution of protocols and hands-on workshops to train the next generation of amphibian biologists to integrate newly developed, cutting-edge technologies into their research programs. Finally, an existing regional K-12 outreach program is expanded into a nationwide resource for educators and local amphibian researchers to connect with one another and bring amphibian biology, conservation, and evolution into public school classrooms. This project is co-funded by the Behavioral Systems Cluster in the Division of Integrative Organismal Systems of the Directorate for Biological Sciences.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.