Extrinsic Regulation of Muscle Stem Cell Quiescence

维持和修复成体组织的干细胞对周围微环境提供的信号有很强的依赖性，通常被称为生态位。在骨骼肌中，干细胞的这种生态位调节特别复杂，并且依赖于以严格调节的时空方式分泌生长因子和有益的细胞外基质（ECM）的多种不同细胞类型。肌肉干细胞（MuSC）的基本特性，如静止、自我更新和分化能力，由小生境的组成决定。 然而，尽管其对MuSC的功能具有重要作用，但小生境产生的结构和指导性信号仍然不完全清楚。值得注意的是，它仍然是特别神秘的细胞和细胞外的线索在生态位控制MuSC静止，重要的休眠维持状态，是必不可少的哺乳动物的终身能力，以维持他们的肌肉和从损伤中恢复。我们提出的NSERC研究计划的一个主要重点将是通过研究静止MuSC的微环境调节来填补我们对生态位的生物学理解中的这一空白。 我们最近的进展和初步实验表明，静止的MuSC采用称为肌营养不良蛋白聚糖（DAG）的受体粘附到小生境ECM上。我们观察到，在他莫昔芬诱导的MuSC特异性DAG敲除小鼠模型（iDAG-KO）中DAG的损失导致打破静止和过早分化。重要的是，我们还发现DAG配体层粘连蛋白α 2（LAMA 2）缺陷的小鼠具有过度活化的过度分化的MuSC。在这里，我们提出了一些目标，以调查通过DAG和其ECM配体LAMA 2维持MuSC静止的分子机制。 具体目标是： (I)研究DAG作为MuSC的ECM受体的作用，并提供其诱导维持静止状态的途径的见解。这一目标将基于iDAG-KO小鼠的分析，并将依赖于允许研究培养物中MuSC静止的基于新型水凝胶的测定。 (II)确定丰富的DAG配体LAMA 2在静止MuSC小生境中的作用。该目标涉及在LAMA 2耗尽的环境中超活化表型MuSC的体内和体外询问。 (III)目的：研究LAMA 2在肌肉组织中的沉积动力学及细胞来源。这一目标是基于LAMA 2的创新报告小鼠模型的生成和分析。 总之，本提案中概述的实验将为静止MuSC的生态位调节提供基本见解，这些MuSC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了解层粘连蛋白的生物学及其对发育，器官结构和功能的重要影响。

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.