Defining the mechanical link that unites the musculoskeletal system during limb development

项目摘要 功能性肌肉骨骼系统的形成依赖于收缩肌的整合 到承重肌腱到骨头这些组织最初是相互独立的，但随后 随着开发的进行而无缝集成。研究人员调查背后的机制 肌肉骨骼组装主要集中在调节细胞行为的信号通路上。 目前的研究范式是，肌肉、肌腱和骨之间的界面是通过细胞 - 细胞相互作用和通过分泌信号在分离的组织之间的相互信号传导。然而，在这方面， 我的实验室的初步研究表明，细胞外基质（ECM）为基础的模板，沉积前， 这些界面的形成也可以指导肌肉、肌腱和骨骼之间的适当组合。 ECM在早期肌肉骨骼发育中的作用在很大程度上被忽视， 缺乏工具。为了应对这些挑战，我的实验室一直在开发新的方法来绘制 在肌肉骨骼发育过程中，ECM的组成和组织。最近我们 证明小鼠胚胎可以用非典型氨基酸（ncAA）标记，这些非典型氨基酸是 强大的生物化学工具，使可视化，富集和识别新合成的 复杂混合物中的蛋白质。此外，我的实验室还开创了光学清除方法， 发育和成人肌肉骨骼组织深处生物标志物的ECM结构。 本项目的目标是通过以下方式确定企业内容管理模板的组成和机制： 模板变得对齐并适应生长。建立了敲除模型， 肌肉骨骼模式被破坏将被用于测试基于ECM的模板， 在肌腱和肌肉分化之前指定，是对齐和正确整合所必需的。 脊椎动物前肢发育过程中的承重肌肉和肌腱。调节的下游目标 模板形成和重塑将通过质谱结合ncAA标记来鉴定。的 候选分子的时空表达将相对于模板可视化， 利用我实验室开发的透明化技术，最强的候选人将 以组织特异性方式被敲除，表型的严重程度将使用3D 形态测量模板的延展性将通过胚胎的延时成像来测量 离体培养并量化网络变形。 成功完成拟议的研究，将确定 ECM为肌肉骨骼结构提供了基础，揭示了肌肉和肌腱是如何 对齐并提供对体内ECM力学的洞察。这些结果将共同提供设计参数 用于再生支架，可以成功地恢复受损肌肉骨骼组织的功能。

PROJECT SUMMARY Formation of a functional musculoskeletal system is dependent on the integration of contractile muscle to load-bearing tendon to bone. These tissues are initially specified independently of each other, but then seamlessly integrate as development progresses. Researchers investigating the mechanisms behind musculoskeletal assembly have primarily focused on the signaling pathways that regulate cellular behavior. The current paradigm is that the interfaces between muscle and tendon and bone are established through cell – cell interactions and reciprocal signaling between the separate tissues via secreted signals. However, preliminary studies by my lab indicate that an extracellular matrix (ECM)-based template, deposited before the formation of these interfaces, may also direct the proper combinations between muscle and tendon and bone. The role of the ECM during early musculoskeletal development has been largely overlooked due to a lack of tools. To address these challenges, my laboratory has been developing novel methods to map the composition and organization of the ECM over the course of musculoskeletal development. Recently, we demonstrated that the murine embryo could be labeled with non-canonical amino acids (ncAAs), which are powerful biochemical tools that enable the visualization, enrichment and identification of newly synthesized proteins within complex mixtures. In addition, my lab has pioneered optical clearing methods to image the 3D ECM architecture of biomarkers deep within developing and adult musculoskeletal tissues. The goal of this project is to identify the composition of the ECM template and the mechanisms by which the template becomes aligned and adapts to growth. Established knockout models in which musculoskeletal patterning is disrupted will be used to test the hypothesis that an ECM-based template, specified before tendon and muscle differentiation, is necessary for the alignment and correct integration of load-bearing muscles and tendons during vertebrate forelimb development. Downstream target(s) that regulate template formation and remodeling will be identified via mass spectrometry combined with ncAA labeling. The spatio-temporal expression of candidate molecules will be visualized with respect to the template and connective tissue cells using clearing techniques developed within my laboratory. The strongest candidates will be knocked down in a tissue specific manner and severity of phenotype will be quantified using 3D morphological measurements. Template extensibility will be measured by time-lapse imaging of embryos cultured ex vivo and quantifying network deformation. Successful completion of the proposed studies will identify the composition and spatial distribution of the ECM that provide the foundation for musculoskeletal architecture, reveal how muscles and tendons are aligned and provide insight into ECM mechanics in vivo. Together, these results will provide design parameters for regenerative scaffolds that can successfully restore functionality to damaged musculoskeletal tissues.