Regulation of Extracellular Matrix Assembly at the Myotendinous Junction

描述(由申请人提供)：肌腱连接(MTJ)的形成是功能性肌肉骨骼系统的基本组成部分。肌肉与肌腱在其附着点相互作用，导致细胞外基质(ECM)重组为结缔组织结构，可以承受肌肉收缩施加的巨大压力。转录因子硬化轴(SCX)指定早期的肌腱前体细胞并调节ECM的产生，我们最近在斑马鱼中发现了一种称为血栓反应蛋白-4b(Thrombospondin-4b)的ECM蛋白，它受SCX的调控，是肌肉附着所必需的。肌腱前体如何到达肌肉附着部位并影响MTJ处的ECM组装和肌肉粘连仍不清楚。目前的提案利用斑马鱼在活体成像和遗传操作方面的优势来解决这些问题。这项拟议研究的长期目标是了解MTJ细胞外基质组装和整合素(ITG)信号的空间动力学。两个基本的假设指导着研究：1)SCX控制着肌腱细胞的规格和ECM的产生，在MTJ重组ITG信号成分；2)Thbs4是ECM中的一个关键的ITG配体，在MTJ的形成过程中充当其他基质蛋白的支架。目的1是显示活斑马鱼胚胎中的肌腱前体，并研究SCX在MTJ细胞外基质产生和重组中的作用。斑马鱼的光学清晰度使它们特别适合于这项研究。目的2通过改变Thbs4b的表达，分析ECM蛋白和ITG信号的空间动态变化，分析Thbs4b在MTJ形成中的作用。每个目标都结合了新的遗传操作、荧光动力学成像和肌肉生理刺激的定量方法，以获得在MTJ组装ECM的机制。

DESCRIPTION (provided by applicant): The formation of myotendinous junctions (MTJs) is a fundamental component of a functional musculoskeletal system. Muscles interact with tendons at their attachment sites, causing a reorganization of the extracellular matrix (ECM) into connective tissue structures that can bear the large forces exerted by muscle contraction. The transcription factor Scleraxis (Scx) specifies early tendon progenitors and regulates ECM production, and we recently discovered an ECM protein called Thrombospondin-4b (Thbs4b) in zebrafish that is regulated by Scx and required for muscle attachment. How tendon progenitors reach sites of muscle attachment and influence ECM assembly and muscle adhesion at the MTJ remains unclear. The current proposal addresses these issues using the advantages of the zebrafish for in vivo imaging and genetic manipulation. The long-term goal of the proposed research is to understand the spatial dynamics of ECM assembly and Integrin (Itg) signaling at MTJs. Two primary hypotheses guide the research: 1) Scx controls tenocyte specification and ECM production that reorganize Itg signaling components at MTJs, and 2) Thbs4 is a critical Itg ligand in the ECM that acts as a scaffold for other matrix proteins during the formation of MTJs. Aim 1 is to visualize tendon precursors in living zebrafish embryos and study roles for Scx in ECM production and reorganization at MTJs. The optical clarity of zebrafish makes them uniquely suited for this study. Aim 2 analyzes Thbs4b functions in MTJ formation, by altering its expression and analyzing the spatial dynamics of ECM proteins and Itg signaling. Each aim combines novel genetic manipulation, fluorescence dynamics imaging and quantitative methods for physiological stimulation of muscles to get at the mechanisms of ECM assembly at the MTJ.