FRET-based tension-sensors for studying zebrafish development

描述（由申请人提供）：用于研究斑马鱼发育力和其他机械变量的基于FRET的张力传感器在动物发育中起着重要作用，因为它们塑造组织形态，是信号转导途径的一部分，并驱动细胞分化。用于测量发育中的胚胎内的力和其他生物学特性的定量体内工具是进一步理解这些过程及其在疾病中的失调的关键，但这些工具在很大程度上缺乏。 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。斑马鱼是这些研究的理想模式生物，因为胚胎的透明性和快速发育。我们将建立和筛选基于原生斑马鱼基因的多个张力传感器，在更简单和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前途的传感器结构。 我们的初步数据表明，我们的成像和数据分析模式是足够敏感的建议在斑马鱼体内测量。我们已经建立了多个张力传感器的基础上，本地斑马鱼机械蛋白（ezrin，EpCAM），适当地定位在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在体外和体内。该项目的主要目标是将建立的体外细胞培养测量转化为斑马鱼的体内环境，并扩展我们的斑马鱼-天然力报告基因构建体的库。本项目的风险适用于FOA，并将通过筛选大量探针来缓解。 我们的主要预期交付成果是构建和验证一个或多个报告体内张力的传感器，并成功证明在一个发育相关背景下以亚细胞分辨率进行至少一次力测量。根据我们的进展，我们希望使用我们的传感器来显着推进我们对亚细胞或细胞间张力如何驱动形态发生过程（如外延）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，迄今为止已经产生了一个联合出版物和初步结果，预测拟议项目的成功。因此，所有的资源和技术都可以通过开发强大的、经过验证的工具来彻底改变发育生物学研究，这些工具用于测量机械特性，例如在发育过程中完整的活动物体内具有亚细胞分辨率的细胞间张力。 我们预计，使发育生物学社区能够“看到生物体内部的力量”将影响机械生物学领域，就像通过GFP标记“看到基因表达”和通过Ca 2+成像“看到神经元活动”分别在发育生物学和神经生物学中开辟了巨大的机会一样。

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.