Imaging the role of hyaluronic acid in skeletal muscle assembly during murine for

描述（由申请人提供）：无法功能性修复因疾病或事故而丧失的组织仍然是再生医学的一个重大挑战。组织工程师已经开发了各种类型的替代支架，目的是促进骨骼肌再生。然而，这些支架通常由人工聚合物或脱细胞细胞的细胞外基质（ECM）组成，其模拟成人组织及其功能。 由于东道国公司有限，阻碍了成功。再生生物学家通常不考虑的是，ECM在发育、修复和再生过程中经历了戏剧性的重塑，创造了一个在生物力学上不同于稳态成人的环境。我推测，通过利用自然界用于在体内组装肌肉骨骼系统的基本元素，将显著增强替换结构与宿主的功能整合。这项建议的目的是映射在肌肉骨骼发育过程中的关键ECM的分布，并开发一个强大的原位成像方法，以阐明细胞行为是如何调节ECM组合物。我将通过以下两个具体目标的成功完成来实现我的目标：1）使用Cre-lox技术，多光子显微镜和小鼠胚胎培养物创建一种方法来成像发育中小鼠前肢内单个细胞的行为。将在Pax 3-Cre和ROSA-ZsGreen 1杂合的双突变体中标记肌肉祖细胞。通过对Pax 3 +/ZsGreen 1+肌肉祖细胞从体节向发育肢体的迁移进行成像，将确定并细化用于实时成像的参数。2)绘制肌生成过程中关键ECM的空间分布图，并量化透明质酸（HA）敲低对成肌细胞迁移的影响。关键ECM的组织，包括HA，将在肢体发育的早期阶段与Pax 3+肌源性前体和分化肌肉相关的化学表征。HA对肌肉祖细胞迁移的影响将通过对小鼠胚胎培养物进行成像来直接测试，在所述小鼠胚胎培养物中HA已被敲除（通过4-甲基伞形酮）或遗传敲除（通过使HAS 2-floxed小鼠与细胞特异性Cre表达小鼠交配）。我预计HA的敲除将通过显著减少Pax 3+细胞从体节迁移的距离并导致肌源性前体的过早分化来影响肌肉发育。我的长期目标是使用这种新方法来评估肌肉骨骼发育过程中扰动对ECM细胞相互作用的影响，以确定调节肌肉组装的特定组件。这项研究意义重大，因为它将1）创建一种方法来成像细胞对发育前肢的天然3D环境中广泛的生化和分子干预的反应，2）表征透明质酸在骨骼肌发育过程中的作用，3）导致新的组织工程方法用于替换受损肌肉。

DESCRIPTION (provided by applicant): The inability to functionally repair tissues lost as a consequence of disease or accident remains a significant challenge for regenerative medicine. Tissue engineers have developed various types of replacement scaffolding with the aim of enhancing skeletal muscle regrowth. However, these scaffolds are typically comprised of artificial polymers or decellularized extracellular matrix (ECM) that mimic adult tissues and their success has been hindered by limited host incorporation. What is not commonly taken into consideration by regenerative biologists is that the ECM undergoes dramatic remodeling during development, repair and regeneration, creating an environment that is biomechanically distinct from the homeostatic adult. I hypothesize that by harnessing the basic elements nature uses to assemble the musculoskeletal system in vivo, the functional integration of replacement constructs into the host will be significantly enhanced. The objectives of this proposal are to map the distribution of key ECM during musculoskeletal development and to develop a robust in situ imaging method to elucidate how cell behavior is regulated by ECM composition. I will achieve my objective through the successful completion of the following two specific aims: 1) Create a method to image the behavior of individual cells within the developing mouse forelimb using Cre-lox technology, multiphoton microscopy and murine embryo cultures. Muscle progenitors will be labeled in double mutants that are heterozygous for Pax3-Cre and ROSA-ZsGreen1. By imaging the migration of Pax3+/ZsGreen1+ muscle progenitors from the somites into the developing limb, the parameters for live imaging will be determined and refined. 2) Map the spatial distribution of key ECM during myogenesis and quantify the effect of hyaluronic acid (HA) knockdown on myoblast migration. The organization of key ECM, including HA, will be characterized immunohistochemically in relation to Pax3+ myogenic precursors and differentiating muscle during the early stages of limb development. The influence of HA on muscle progenitor migration will be directly tested by imaging murine embryo cultures in which HA has been pharmacologically (via 4-methylumbelliferone) or genetically (by mating HAS2-floxed mice with cell-specific Cre-expressing mice) knocked down. I expect that the knockdown of HA will affect muscle development by significantly decreasing the distance Pax3+ cells migrate from the somites and resulting in premature differentiation of myogenic precursors. My long-term goal is to use this novel method to assess the effect of perturbations on ECM-cell interactions during musculoskeletal development to identify specific components that regulate muscle assembly. The proposed research is significant because it will 1) create a method to image the response of cells to a wide range of biochemical and molecular interventions in the native 3D environment of the developing forelimb, 2) characterize the role of hyaluronic acid during skeletal muscle development and 3) lead to new tissue engineering approaches for the replacement of damaged muscle.