Satellite Cell Development and Potential

说明(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，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基本功能已被认识多年，但关于其胚胎起源、发育潜力和更新机制的基本问题仍未解决。目前的建议使用独特的转基因小鼠品系和基于Cre/loxP重组的谱系追踪方法来研究体内卫星细胞生物学的这些关键方面。为了研究卫星细胞来自胎儿成肌细胞和血管细胞(如内皮前体细胞)的相互竞争的假设，将通过两个小鼠品系的杂交，将相关细胞特异性地永久标记为LacZ，即在细胞类型特异的启动子/增强子控制下表达Cre重组酶基因的转基因小鼠(成肌细胞为MyoD-cre，血管衍生细胞为Tie2-cre和flk1-cre)，以及LacZ表达依赖于CRE的报告鼠株。这些细胞是否代表卫星细胞前体，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，以及通过单纤维制剂的标准和共聚焦显微镜来确定。对受损肌肉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卫星细胞在肌肉损伤和单纤维培养中的命运来确定。野生型小鼠的实验将与MyoD或Myf-5突变的小鼠的平行实验进行比较，以测试这些肌肉调控基因在卫星细胞生肌承诺中的功能。对胚胎中MyoD和Myf-5功能的分析将确定在缺乏这些调控基因的情况下胚胎成肌前体所能选择的全部细胞命运，并将为胚胎和出生后肌肉发生的调控提供新的比较见解。总而言之，这些研究将极大地提高对卫星细胞生物学的现有知识，并将为未来研究调控卫星细胞及其前体细胞命运选择的信号和基因转录途径提供基础。

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.