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.

Public Health Relevance

Forces and other mechanical variables play a significant role in animal development, but quantitative in-vivo tools for measuring these biophysical properties are lacking. We develop novel genetically encoded, in-vivo FRET-based tension sensor (i.e. a fluorescent signal reports on force), and test their utility studying zebrafish gastrulation (whichis the ideal model organism for this kind of studies given its optical properties and fast development). With this work we aim to develop the tools that allow seeing forces inside a living organism, which will enable 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.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1)
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Mukhopadhyay, Mahua
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Stanford University
Schools of Medicine
United States
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Du, XinXin; O'Brien, Lucy Erin; Riedel-Kruse, Ingmar Hans (2017) A Model for Adult Organ Resizing Demonstrates Stem Cell Scaling through a Tunable Commitment Rate. Biophys J 113:174-184