Satellite Cell Development and Potential

描述（由申请人提供）：肌肉损伤刺激正常静止的肌肉卫星细胞重新进入细胞周期并执行生肌程序，导致肌肉结构和功能的恢复。虽然卫星细胞在肌肉修复中的重要功能已被认识多年，但有关其胚胎起源，发育潜力和更新机制的基本问题仍未解决。本提案使用独特的转基因小鼠品系和谱系追踪方法的基础上Cre/loxP重组，调查这些关键方面的卫星细胞生物学在体内。探讨卫星细胞来源于胎儿成肌细胞和血管细胞的竞争假说在细胞类型特异性启动子/增强子（例如内皮前体）的控制下，相关细胞类型将通过使两种小鼠品系（在细胞类型特异性启动子/增强子的控制下表达Cre重组酶基因的转基因小鼠）杂交而用lacZ特异性地和永久地标记。（MyoD-cre用于成肌细胞，Tie 2-cre和Flk 1-cre用于血管来源的细胞）和报道小鼠品系，其中lacZ表达是Cre依赖性的。这些细胞是否代表卫星细胞祖细胞将通过使用透射电子显微镜评估卫星细胞的β-半乳糖苷酶标记以及通过单纤维制备物的标准和共聚焦显微镜来确定。在受损肌肉中进行的类似研究将确定卫星细胞库是通过自我更新还是通过骨骼肌内外的干细胞来源来维持的。卫星细胞的发育潜力将通过跟踪肌肉损伤后和单纤维培养物中卫星细胞的命运来确定。将野生型小鼠中的实验与MyoD或Myf-5突变的小鼠中的平行实验进行比较，以测试这些肌肉调节基因在卫星细胞肌原性定型中的功能. MyoD和Myf-5在胚胎中的功能分析将定义在没有这些调控基因的情况下胚胎肌原性前体细胞命运选择的完整库，并将为胚胎和出生后肌发生的调控提供新的比较见解。总的来说，这些研究将显着推进目前的卫星细胞生物学知识，并将提供一个基础，为未来的调查信号和基因转录途径，调节卫星细胞及其祖细胞的细胞命运的选择。

DESCRIPTION (provided by applicant): Muscle injury stimulates normally quiescent muscle satellite cells to re-enter the cell cycle and execute the myogenic program, resulting in the restoration of muscle structure and function. While the essential function of satellite cells in muscle repair has been recognized for many years, fundamental questions concerning their embryological origin, developmental potential, and mechanism of renewal remain unresolved. The present proposal uses unique transgenic mouse lines and lineage tracing approaches based on Cre/loxP recombination to investigate these key aspects of satellite cell biology in vivo. To investigate the competing hypotheses that satellite cells are derived from fetal myoblasts and cells of the vasculature (e.g. endothelial precursors), relevant cell types will be specifically and permanently labeled with lacZ by intercrossing two mouse strains, transgenic mice that express the gene for Cre recombinase under the control of a cell type- specific promoter/enhancer (MyoD-cre for myoblasts, Tie2-cre and Flk1-cre for vascular-derived cells) and a reporter mouse strain in which lacZ expression is Cre-dependent. Whether these cells represent satellite cell progenitors will be determined by assessing beta-galactosidase labeling of satellite cells using transmission electron microscopy, and by standard and confocal microscopy of single-fiber preparations. Similar studies in injured muscle will determine whether the satellite cell pool is maintained by self-renewal, or by a stem cell source within or outside of skeletal muscle. The developmental potential of satellite cells will be determined by tracing the fates of satellite cells following muscle injury and in single-fiber cultures. Experiments in wild- type mice will be compared to parallel experiments in mice that are mutant for either MyoD or Myf-5 to test the function of these muscle regulatory genes in satellite cell myogenic commitment. Analysis of MyoD and Myf-5 functions in the embryo will define the full repertoire of cell fate choices available to embryonic myogenic precursors in the absence of these regulatory genes and will provide new comparative insights into the regulation of embryonic and postnatal myogenesis. Collectively, these studies will significantly advance current knowledge of satellite cell biology and will provide a foundation for future investigations into the signaling and gene transcriptional pathways that regulate cell fate choices of satellite cells and their progenitors.