Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells
Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells
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DOI：
10.1006/dbio.1997.8721
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发表时间：
1997-11-15
影响因子：
2.7
通讯作者：
Wold, BJ
中科院分区：
文献类型：
--
作者：
Cornelison, DDW;Wold, BJ
成年骨骼肌的修复和再生由卫星细胞介导。在健康肌肉中，这些罕见的单核肌肉前体细胞处于有丝分裂静止状态。当肌肉受伤或退化时，这个自我更新群体的成员被激活增殖，然后分化。在此，我们在单个卫星细胞中分析了一组调节基因的表达，这些基因是卫星细胞激活、成熟和分化中因果作用的候选基因。单个细胞被鉴定为卫星细胞，并根据它们与单个外植肌纤维的物理关联或它们在未受干扰的肌肉组织中基膜下方的位置进行选择分析。利用多重单细胞逆转录聚合酶链反应分析，我们同时监测了所有四种肌肉决定和分化的MyoD家族调节因子（MRPs）以及卫星细胞身份的两个候选标记物c - met和m - cadherin的表达。通过在卫星细胞激活的时间进程中对大量单个细胞进行这些测量，我们能够确定哪些表达状态（六种基因的可能组合）存在，并明确每种状态的呈现如何随时间变化。被激活的卫星细胞在MRFs中首先开始表达MyoD或myf5；大多数细胞随后同时表达myf - 5和MyoD；肌生成素在同时表达MyoD和myf5的细胞中稍后出现；并且许多细胞最终同时表达所有四种MRFs。来自主要是快肌或慢肌的与纤维相关的卫星细胞的结果彼此无法区分。还监测了c - met受体酪氨酸激酶，因为它是介导静止卫星细胞激活的候选因子（Alien等人，1995年），并且它也可能是卫星细胞的候选分子标记。研究小鼠卫星细胞的一个重大困难是缺乏能够在MRFs或结蛋白表达之前的静止状态下识别它们并将它们与成纤维细胞区分开来的分子标记。我们在此表明，c - met受体存在于完整肌肉中假定的卫星细胞的基膜下方，并且从外植时起，经过激活、增殖和分化过程，所有与肌纤维相关的卫星细胞都表达c - met mRNA和蛋白质。在肌肉来源的成纤维细胞或来自健康肌肉外植体的其他单核细胞中未检测到c - met。当与先前被提议作为静止卫星细胞标记物的m - cadherin直接比较时，m - cadherin mRNA仅在肌纤维外植后的早期在一小部分卫星细胞中被检测到。然而，在激活后的后期（在这个纤维培养系统中为96小时），c - met和m - cadherin均匀共表达。根据观察到的单个卫星细胞表达类型，生成了一个静止状态以及激活时间进程中的卫星细胞群体模型。（C）1997年学术出版社。
Repair and regeneration of adult skeletal muscle are mediated by satellite cells. In healthy muscle these rare mononucleate muscle precursor cells are mitotically quiescent. Upon muscle injury or degeneration, members of this self-renewing pool are activated to proliferate and then differentiate. Here we analyzed in single satellite cells the expression of a set of regulatory genes that are candidates for causal roles in satellite cell activation, maturation, and differentiation. Individual cells were identified as satellite cells and selected for analysis based on their physical association with single explanted myofibers or their position beneath the basal lamina in unperturbed muscle tissue. Using a multiplex single-cell RT-PCR assay we simultaneously monitored expression of all four MyoD family regulators of muscle determination and differentiation (MRPs) together with two candidate markers of satellite cell identity, c-met and m-cadherin. By making these measurements on large numbers of individual cells during the time course of satellite cell activation, we were able to define which expression states (possible combinations of the six genes) were represented and to specify how the representation of each state changed with time. Activated satellite cells began to express either MyoD or myf5 first among the MRFs; most cells then expressed both myf-5 and MyoD simultaneously; myogenin came on later in cells expressing both MyoD and myf5; and many cells ultimately expressed all four MRFs simultaneously. The results for fiber-associated satellite cells from either predominantly fast or slow muscles were indistinguishable from each other. The c-met receptor tyrosine kinase was also monitored because it is a candidate for mediating activation of quiescent satellite cells (Alien et al., 1995) and because it might also be a candidate molecular marker for satellite cells. A significant difficulty in studying mouse satellite cells has been the absence of molecular markers that could identify them in the quiescent state before expression of MRFs or desmin and distinguish them from fibroblasts. We show here that c-met receptor is present beneath the basal lamina on presumptive satellite cells in intact muscle and that c-met mRNA and protein are expressed by all myofiber-associated satellite cells from the time of explant through the course of activation, proliferation, and differentiation. c-met was not detected in muscle-derived fibroblasts or in other mononucleate cells from healthy muscle explants. When compared directly with m-cadherin, which has previously been suggested as a marker for quiescent satellite cells, m-cadherin mRNA was detected only in a small subset of satellite cells at early times after myofiber explant. However, at late times following activation (by 96 hr in this fiber culture system), c-met and m-cadherin were uniformly coexpressed. From the individual satellite cell expression types observed, a model of the satellite cell population at rest and during the time course of activation was generated. (C) 1997 Academic Press.