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SCX和MKX以及膜糖蛋白Tenomodrin(Tnmd)指定早期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ECM蛋白和整合素(ITG)配体，称为血栓反应蛋白-4b(Tsp4b)，受SCX调控，需要肌肉附着。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肌腱和韧带中细胞外基质组装和细胞命运规范的空间动力学。本研究有两个基本假设：1)MKX和Tnmd与SCX相互作用，控制MTJ的肌腱细胞形态发生和ECM组装；2)机械负荷作用下的ITG和TGFb信号调节MTJ形成过程中SCX、MKX和Tnmd的表达。目的1是在活的野生型、MKX和Tnmd突变胚胎中显示肌腱祖细胞。目的2研究ITG和TGFb信号在调控SCX、MKX和Tnmd表达中的作用。目的3研究ITG和TGFb信号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、荧光动力学成像和肌肉生理刺激的定量方法，以获得在这些专门的结缔组织结构上组装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 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.