The Biomechanics of morphogenesis in the frog

描述(申请人提供)：这项建议的目标是应用多尺度分析会聚伸展的机制，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，建立被动组织属性，如僵硬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中协调。我们将使用已建立的工具包，该工具包由三个元素组成：1)用于直接调节蛋白质功能和基因表达的水生非洲爪蛙；2)高分辨率共聚焦显微镜，用于可视化细胞行为、细胞骨架动力学和组织结构；以及3)用于施加应变、测量组织硬度和力产生的生物物理方法。这项提案中概述的研究将回答：1)胚胎细胞如何使用肌动球蛋白在会聚延伸过程中物理产生力量、改变形状和指导运动？为了了解运动是如何被物理控制的，我们将对中胚层细胞皮质中的F-肌动蛋白进行“自下而上”的分析，因为这些细胞启动了细胞形状的变化，并采取了中间外侧的插入行为。2)整体组织僵硬和组织伸长力的细胞和分子机制是什么？我们对原肠发育和轴伸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部组织汇聚和伸展时的组织僵硬和力产生中的作用。3)在收敛伸展过程中，细胞嵌入和刚性协调的物理机制是什么？我们假设原肠形成依赖于来自延长的背轴的力量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交互作用，并测试我们工作模型的定性预测。这些模型既能证明简单的机械反馈机制的合理性，又能预测实验操作的结果。这项工作将通过为新的假说和生物工程工具提供基本的生物物理原理来检验它们，来补充正在进行的识别形态发生的分子调节因子的努力。我们工作的意义不仅仅是定义将内侧细胞嵌入转化为大规模收敛延伸的力学条件和力，以便更全面地了解组织力学对出生缺陷的贡献，了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。 与公共健康相关：这项建议的目标是了解肌动蛋白动力学驱动细胞形状变化、产生牵引力、建立被动组织属性(如僵硬)、通过汇聚和伸展产生主动力，以及被动力学和主动力如何塑造脊椎动物胚胎的物理机制。我们的工作的意义不仅仅是定义力学条件及其在早期发育中的作用，为未来的组织工程师提供基本的物理原理，允许更全面地了解组织力学对出生缺陷的贡献，以及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.