The Biomechanics of morphogenesis in the frog

描述（由申请人提供）：本提案的目标是应用会聚伸展力学的多尺度分析，确定调节细胞形状和驱动内外侧细胞行为的生物力学机制，建立被动组织特性（如刚度）以及产生伸展力的主动过程，以及被动力学和主动力产生过程如何在青蛙胚胎内协调。我们将使用由三个要素组成的成熟工具包：1）用于直接调节蛋白质功能和基因表达的水生青蛙非洲爪蟾; 2）高分辨率共聚焦显微镜，以可视化细胞行为，细胞骨架动力学和组织结构;和3）用于施加应变，测量组织刚度和力产生的生物物理方法。在这个建议中概述的研究将回答：1）胚胎细胞如何使用肌动球蛋白在物理上产生力，改变形状，并在会聚延伸过程中指导运动？为了了解运动是如何物理控制的，我们将采取“自下而上”的中胚层细胞皮层中的F-肌动蛋白的分析，因为这些细胞启动细胞形状的变化，并采取mediolateral嵌入行为。2)在会聚伸展过程中，大体积组织硬度和组织伸长力的细胞和分子机制是什么？我们对原肠胚形成和轴延伸过程中胚胎组织硬度的表征揭示了随着胚胎年龄的增长，硬度的广泛调节以及从一个胚层到下一个胚层对硬度的精确控制。我们建议测试的物理状态的F-肌动蛋白细胞骨架的作用，在调节组织刚度和力生产的背部组织收敛和扩展。3)在收敛伸展过程中，协调细胞嵌入和刚度的物理机制是什么？我们假设原肠胚形成依赖于来自伸长的背轴的力和来自周围组织的阻力的适当平衡。为了测试这一点，我们建议构建基于有限元的模型来研究这些相互作用，并测试我们的工作模型的定性预测。这些模型将用于演示简单的机械反馈机制的可扩展性以及预测实验操作的结果。这项工作将补充正在进行的努力，以确定形态发生的分子调节器，提供潜在的生物物理学原理的新的假设和生物工程工具来测试它们。我们的工作的意义超出了定义的机械条件和力量，转换成大规模的收敛性延伸细胞插入，使组织力学的贡献，出生缺陷，了解组织力学在肿瘤发生中的作用，并提供基本的物理原则，为未来的组织工程师。 公共卫生相关性：该提案的目标是了解肌动球蛋白动力学驱动细胞形状变化的物理机制，产生牵引力，建立被动组织特性，如刚度，通过会聚和延伸产生的主动力，以及被动力学和主动力如何塑造脊椎动物胚胎。我们的工作的意义超出了定义的机械条件及其在早期发展中的作用，为未来的组织工程师提供基本的物理原理，允许更完整地了解组织力学对出生缺陷的贡献，并了解组织力学在肿瘤发生中的作用。

DESCRIPTION (provided by applicant): The goal of this proposal is to apply a multi-scale analysis of the mechanics of convergent extension, identifying biomechanical mechanisms that regulate cell shape and drive mediolateral cell behaviors, establish passive tissue properties such as stiffness as well as active processes that generate forces of extension, and how passive mechanics and active force generating processes are coordinated within the frog embryo. We will use an established toolkit consisting of three elements: 1) the aquatic frog Xenopus laevis for direct modulation of protein function and gene expression; 2) high resolution confocal microscopy to visualize cell behaviors, cytoskeletal dynamics, and tissue architecture; and 3) biophysical methods for applying strains, measuring tissue stiffness and force production. Studies outlined in this proposal will answer: 1) How do embryonic cells use actomyosin to physically generate force, change shape, and direct movement during convergent extension? To understand how movements are physically controlled we will take a "bottom-up" analysis of F-actin in the cortex of mesodermal cells as these cells initiate cell shape changes and adopt mediolateral intercalation behaviors. 2) What are the cell and molecular mechanisms underlying bulk tissue stiffness and tissue elongation forces during convergent extension? Our characterization of stiffness of embryonic tissues during gastrulation and axis extension has revealed both broad regulation of stiffness as the embryo ages as well as precise control over stiffness from one germ layer to the next. We propose to test the role of the physical state of the F-actin cytoskeleton in regulating of tissue stiffness and force-production as dorsal tissues converge and extend. 3) What are the physical mechanisms coordinating cell intercalation and stiffness during convergent extension? We hypothesize that gastrulation relies on a proper balance of forces from the elongating dorsal axis and resistance from surrounding tissues. To test this we propose to construct finite element based models to investigate these interactions and test qualitative predictions of our working models. These models will serve to both demonstrate the plausibility of simple mechanical feed-back mechanisms as well as predict the outcome of experimental manipulations. This work will complement ongoing efforts to identify the molecular regulators of morphogenesis by providing underlying biophysical principles for new hypotheses and bioengineering tools to test them. The significance of our work extends beyond defining the mechanical conditions and forces that convert mediolateral cell intercalation into large-scale convergent extension to allow a more complete understanding of the contribution of tissue mechanics to birth defects, to understand the role of tissue mechanics in oncogenesis, and to provide fundamental physical principles for future tissue engineers. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand the physical mechanisms by which actomyosin dynamics drive cell shape changes, generate traction forces, establish passive tissue properties such as stiffness, active force production by convergence and extension, and how passive mechanics and active forces shape a vertebrate embryo. The significance of our work extends beyond defining the mechanical conditions and their role in early development to provide fundamental physical principles for future tissue engineers, allow a more complete understanding of the contribution of tissue mechanics to birth defects, and to understand the role of tissue mechanics in oncogenesis.