The Biomechanics of morphogenesis in the frog

描述（由申请人提供）：本提案的目标是应用聚合伸展力学的多尺度分析，确定调节细胞形状和驱动中外侧细胞行为的生物力学机制，建立被动组织特性，如刚度和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中协调。我们将使用由三个要素组成的既定工具包：1)水蛙爪蟾用于直接调节蛋白质功能和基因表达；2)用高分辨率共聚焦显微镜观察细胞行为、细胞骨架动力学和组织结构；3)应用应变、测量组织刚度和力产生的生物物理方法。本提案中概述的研究将回答：1)胚胎细胞如何利用肌动球蛋白在会聚伸展过程中物理地产生力、改变形状和直接运动？为了理解运动是如何被物理控制的，我们将对中胚层细胞皮层中的f -肌动蛋白进行“自下而上”的分析，因为这些细胞启动细胞形状变化并采取中外侧嵌入行为。2)趋同伸展过程中组织体刚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期间胚胎组织刚度的表征揭示了随着胚胎年龄的增长，刚度的广泛调节以及从一个胚层到下一个胚层对刚度的精确控制。我们建议测试f -肌动蛋白细胞骨架的物理状态在调节背侧组织收敛和伸展时组织刚度和力量产生中的作用。3)收敛伸展过程中协调单元插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延长的背轴和周围组织阻力的适当平衡。为了验证这一点，我们建议构建基于有限元的模型来研究这些相互作用，并测试我们工作模型的定性预测。这些模型既可以证明简单机械反馈机制的合理性，也可以预测实验操作的结果。这项工作将通过为新的假设提供潜在的生物物理原理和生物工程工具来测试它们，从而补充正在进行的识别形态发生分子调节因子的工作。我们工作的意义超越了定义将中侧细胞嵌入转化为大规模会聚延伸的机械条件和力，从而更全面地理解组织力学对出生缺陷的贡献，理解组织力学在肿瘤发生中的作用，并为未来的组织工程师提供基本的物理原理。

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.