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

描述(由申请人提供)：由于疾病或事故而失去的组织无法进行功能性修复，这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性支架，目的是促进骨骼肌再生。然而，这些支架通常由人造聚合物或脱细胞细胞外基质(ECM)组成，模仿成人组织及其 成功受到了东道国有限合并的阻碍。再生生物学家通常没有考虑到的是，在发育、修复和再生过程中，ECM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，通过利用大自然在体内组装肌肉骨骼系统所使用的基本元素，替换结构与宿主的功能整合将显著增强。这项建议的目的是绘制肌肉骨骼发育过程中关键细胞外基质的分布图，并开发一种强大的原位成像方法来阐明细胞外基质的组成是如何调节细胞行为的。我将通过成功完成以下两个具体目标来实现我的目标：1)利用CRE-LOX技术、多光子显微镜和小鼠胚胎培养，创建一种对发育中的小鼠前肢内单个细胞的行为进行成像的方法。肌肉祖细胞将被标记为Pax3-Cre和Rosa-ZsGreen1杂合的双重突变体。通过对Pax3/ZsGreen1肌祖细胞从体节迁移到发育中肢体的成像，将确定和改进实时成像的参数。2)绘制成肌过程中关键细胞外基质的空间分布图，并量化透明质酸(HA)基因敲除对成肌细胞迁移的影响。在肢体发育的早期阶段，关键的ECM的组织，包括HA，将与Pax3肌源性前体和分化肌肉相关的免疫组织化学特征。HA对肌肉祖细胞迁移的影响将通过对小鼠胚胎培养进行成像来直接测试，在小鼠胚胎培养中，HA已经被药理上(通过4-甲基伞形酮)或遗传(通过将HAS2-Flosed小鼠与表达细胞特异性Cre的小鼠交配)击倒。我预计，HA基因的敲除将显著缩短Pax3细胞从体节迁移的距离，并导致肌源性前体细胞的过早分化，从而影响肌肉发育。我的长期目标是使用这种新的方法来评估肌肉骨骼发育过程中细胞外基质-细胞间相互作用的扰动影响，以确定调节肌肉组装的特定成分。这项拟议的研究具有重要意义，因为它将1)创建一种方法来成像细胞对发育中的前肢自然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中的作用，以及3)导致新的组织工程方法来替代受损的肌肉。

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.