Extrinsic Regulation of Muscle Stem Cell Quiescence

维持和修复成人组织的干细胞强烈依赖于周围微环境提供的信号，微环境通常被称为利基。在骨骼肌中，干细胞的这种生态位调控特别复杂，依赖于多种不同类型的细胞，以严格调控的时空方式分泌生长因子和指导性细胞外基质(ECM)。肌肉干细胞(MuSCs)的基本特性，如静止、自我更新和分化能力，由生态位的组成决定。然而，尽管它在MUSCs的功能中扮演着重要的角色，但它的结构和利基产生的指示信号仍然没有完全被理解。值得注意的是，利基中的细胞和细胞外信号控制着MUSC的静止，这是一种重要的休眠维持状态，对于哺乳动物维持肌肉和从损伤中恢复的终身能力来说是必不可少的，这仍然是一个谜。我们建议的NSERC研究计划的一个主要焦点将是通过研究静止的MUSCs的微环境调节来填补我们对利基生物学理解的这一空白。 我们的最新进展和初步实验表明，静止的MUSCs使用一种名为dystroglan(DAG)的受体来附着在小生境ECM上。我们观察到，在他莫昔芬诱导的MUSC特异性DAG基因敲除小鼠模型(IDAG-KO)中，DAG的丢失会导致断裂、静止和过早分化。重要的是，我们还发现缺乏DAG配体层粘连蛋白α2(LAMA2)的小鼠过度激活了过度分化的MuSCs。在这里，我们提出了一些目标来研究DAG及其ECM配体LAMA2维持MUSC静止的分子机制。 具体目标包括： (I)研究DAG作为MUSCs的ECM受体的作用，并对其诱导维持静止状态的途径提供见解。这一目标将基于对IDAG-KO小鼠的分析，并将依赖于新的水凝胶分析，允许研究MSC在培养中的静止。 (Ii)确定丰富的DAG配体LAMA2在静止的MUSC生态位中的作用。这一目标涉及在LAMA2耗尽的环境中对高激活表型MUSC的体内和体外询问。 (3)研究LAMA2在肌肉组织中的沉积动力学和细胞来源。这一目标是基于对LAMA2创新型报告鼠模型的产生和分析。 综上所述，这项提案中概述的实验将为静止的MSC的利基调控提供基本的见解，这种调节很可能从骨骼肌外推到其他组织。此外，我们的工作将有助于更好地理解层粘连蛋白的生物学及其对发育、器官结构和功能的所有重要影响。

Stem cells maintaining and repairing adult tissues have a strong dependency on signals provided by the surrounding microenvironment, often referred to as the niche. In skeletal muscle this niche regulation of stem cells is particularly complex and depends on a multitude of different cell types secreting growth factors and instructive extracellular matrix (ECM) in a tightly regulated spatio-temporal manner. Fundamental properties of muscle stem cells (MuSCs), such as quiescence, self-renewal and the ability to differentiate, are determined by the composition of the niche. However, in spite of its important role for the function of MuSCs, the architecture and the instructive signals the niche generates remain incompletely understood. Notably, it remains particularly enigmatic which cellular and extracellular cues in the niche control MuSC quiescence, the important dormant maintenance state that is essential to the life-long ability of mammals to maintain their muscles and recover from injuries. A major focus of our proposed NSERC research program will be to fill this gap in our biological understanding of the niche by studying the microenvironmental regulation of quiescent MuSCs. Our recent progress and preliminary experiments have revealed that quiescent MuSCs employ a receptor called dystroglycan (DAG) to adhere to the niche ECM. We observed that loss of DAG in a tamoxifen inducible MuSC specific DAG knockout mouse model (iDAG-KO) leads to a break quiescence and to premature differentiation. Importantly, we also discovered that mice deficient for the DAG ligand Laminin alpha 2 (LAMA2) have hyperactivated excessively differentiating MuSCs. Here we propose a number of objectives to investigate the molecular mechanisms through which DAG and its ECM ligand LAMA2 maintain MuSC quiescence. Specific objectives are: (I) To study the role of DAG as an ECM receptor for MuSCs and provide insights into the pathways it induces to maintain the quiescent state. This objective will be based on the analysis of iDAG-KO mice and will rely on novel hydrogel based assays that allow to study MuSCs quiescence in culture. (II) To determine the role of the abundant DAG ligand LAMA2 in the quiescent MuSC niche. This objective involves the in-vivo and in-vitro interrogation of the hyperactivated phenotype MuSC in a LAMA2 depleted environment. (III) To study the deposition dynamics and cellular sources of LAMA2 in muscle tissue. This objective is based on the generation and analysis of an innovative reporter mouse model for LAMA2. Taken together, the experiments outlined in this proposal will provide fundamental insights into the niche regulation of quiescent MuSCs that may well be extrapolated from skeletal muscle to other tissues. In addition, our work will contribute to a better understanding of the biology of Laminins with all their important implications for development, organ structure and function.