Regulation of Morphogenesis and 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部位相互作用，导致细胞外基质（ECM）重组为结缔组织结构，可以承受肌肉收缩产生的巨大力量。转录因子Scleraxis（Scx）和Mohawk（Mkx）以及膜糖蛋白Tenomodulin（Tnmd）指定早期肌腱祖细胞并调节ECM的产生，我们最近在斑马鱼中发现了一种ECM蛋白和整合素（Itg）配体，称为血小板反应蛋白-4b（Tsp 4 b），由Scx调节并为肌肉附着所需。肌腱祖细胞如何到达肌肉附着部位并影响MTJ处的ECM组装和肌肉粘附仍不清楚。目前的提议利用斑马鱼在体内成像和遗传操作方面的优势来解决这些问题。拟议研究的长期目标是了解肌腱和韧带中ECM组装和细胞命运规范的空间动力学。两个主要假设指导研究：1）Mkx和Tnmd与Scx相互作用以控制肌腱细胞形态发生和在MTJ处的ECM组装，以及2）响应于机械负荷的Itg和Tgfb信号传导在MTJ形成期间调节Scx、Mkx和Tnmd表达。目的1是可视化活的野生型，Mkx和Tnmd突变体胚胎中的肌腱祖细胞。目的二是研究Itg和Tgfb信号通路在Scx、Mkx和Tnmd表达调控中的作用。目的3研究Itg和Tgfb信号通路在机械负荷反应中的作用。每个目标都结合了新的遗传操作，荧光动力学成像和定量方法，用于肌肉的生理刺激，以获得ECM组装在这些专门的结缔组织结构中的机制。

 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 factors Scleraxis (Scx) and Mohawk (Mkx), as well as the membrane glycoprotein Tenomodulin (Tnmd), specify early tendon progenitors and regulate ECM production, and we recently discovered an ECM protein and Integrin (Itg) ligand called Thrombospondin-4b (Tsp4b) 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 cell fate specification in tendons and ligaments. Two primary hypotheses guide the research: 1) Mkx and Tnmd interact with Scx to control tenocyte morphogenesis and ECM assembly at MTJs, and 2) Itg and Tgfb signaling in response to mechanical load regulates Scx, Mkx and Tnmd expression during the formation of MTJs. Aim 1 is to visualize tendon progenitors in living wild-type, Mkx and Tnmd mutant embryos. Aim 2 is to study roles for Itg and Tgfb signaling in regulation of Scx, Mkx and Tnmd expression. Aim 3 is to study the roles of Itg and Tgfb signaling in response to mechanical load. 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 these specialized connective tissue structures.