FRET-based tension-sensors for studying zebrafish development

描述(申请人提供)：基于FRET的张力传感器用于研究斑马鱼发育力和其他机械变量，在动物发育中发挥重要作用，因为它们塑造组织形态，是信号转导途径的一部分，并推动细胞分化。用于测量发育中胚胎内的力和其他生物物理特性的体内定量工具对于进一步了解这些过程及其在疾病中的放松调控是关键，但这些工具在很大程度上是缺乏的。我们建议开发基因编码的、基于FRET的张力传感器，利用荧光信号来报告力。由于斑马鱼胚胎的透明性和快速发育，是进行这些研究的理想模式生物。我们将构建和筛选基于本地斑马鱼基因的多个张力传感器，在更简单和已建立的细胞培养环境中表征这些传感器的功能，并在更具挑战性的体内环境中测试和充分表征最有希望的传感器结构。我们的初步数据表明，我们的成像和数据分析模式对于拟议的斑马鱼体内测量具有足够的敏感性。我们已经基于天然斑马鱼机械蛋白(Ezrin，EpCAM)构建了多个张力传感器，这些蛋白正确定位于MDCK细胞和斑马鱼。此外，我们已经建立了化学和机械方法来表征这些传感器的体外和体内。该项目的主要目标是将已建立的体外细胞培养测量结果转化为斑马鱼的体内环境，并扩大我们的斑马鱼原生力报告构建库。该项目的风险适合于FOA，并将通过筛选大量探测器来缓解。我们的主要目标是构建和验证一个或多个报告体内张力的传感器，并在一个与发育相关的背景下成功演示至少一种亚细胞分辨率的力测量。根据我们的进展，我们希望使用我们的传感器(S)来显著提高我们对亚细胞或细胞间张力如何驱动外胚层等形态发生过程的理解。我们的团队拥有所有必要的专业知识和积极的合作，到目前为止已经产生了一份联合出版物和初步结果，可以预测拟议项目的成功。因此，所有资源和技术都在手头，通过开发强大、有效的工具来测量机械特性，如细胞间张力，在发育过程中完整、活的动物体内具有亚细胞分辨率，从而为发育生物学研究带来革命性的变化。我们预计，使发育生物界能够在生物体内“看到力量”将对机械生物学领域产生很大影响，就像通过GFP标记“看到基因表达”和通过钙成像“看到神经元活动”分别在发育生物学和神经生物学中打开了巨大的机会一样。

DESCRIPTION (provided by applicant): FRET-based tension sensors to study zebrafish development forces and other mechanical variables play a significant role in animal development, as they shape tissue morphology, are part of signal transduction pathways, and drive cellular differentiation. Quantitative in-vivo tools for measuring forces and other biophysicl properties inside developing embryos are key for further understanding of these processes and their deregulation in disease, but these tools are largely lacking. We propose to develop genetically encoded, FRET-based tension sensors that harness a fluorescent signal to report force. Zebrafish is an ideal model organism for these studies due to the transparency and fast development of the embryo. We will build and screen multiple tension sensors based on native zebrafish genes, characterize the functionality of these sensors in the simpler and established cell-culture context, and test and fully characterize the most promising sensor constructs in the more challenging in-vivo context. Our preliminary data demonstrate that our imaging and data analysis modalities are sensitive enough for the proposed in-vivo measurements in zebrafish. We have already built multiple tension sensors based on native zebrafish mechanoproteins (ezrin, EpCAM) that properly localize in MDCK cells and in zebrafish. Further, we have established chemical and mechanical methods to characterize these sensors in-vitro and in- vivo. The main goal of this project is to translate the established in-vitro cell culture measurements into the in- vivo context of the zebrafish and to expand our repertoire of zebrafish-native force reporter constructs. The risk of this project is appropriate to the FOA and will be mitigated by screening a large number of probes. Our major intended deliverable is the construction and validation of one or more sensors that report in- vivo tension, with the successful demonstration of at least one force measurement at subcellular resolution within one developmentally relevant context. Depending on our progress, we hope to use our sensor(s) to significantly advance our understanding of how subcellular or inter-cellular tension drives a morphogenic process such as epiboly. Our team has all of the necessary expertise and an active collaboration that to date has generated a joint publication and the preliminary results tha predict the success of the proposed project. All resources and technologies are therefore at hand to revolutionize developmental biology research by developing robust, validated tools for measuring mechanical properties such as intercellular tension with subcellular resolution inside intact, living animals during development. We anticipate that empowering the developmental biology community to "see forces" inside living organisms will impact the field of mechano-biology much as "seeing gene expression" via GFP-tagging and "seeing neuronal activity" via Ca2+ imaging opened tremendous opportunities in developmental biology and neurobiology, respectively.