Single-Cell Analysis of Regulatory Gene Expression in Quiescent and Activated Mouse Skeletal Muscle Satellite Cells

• Published in: Developmental biology 1997-11, Vol.191 (2), p.270-283
• Main Authors: Cornelison, D.D.W., Wold, Barbara J.
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; c-met; Cadherins - genetics; Cells, Cultured; Gene Expression Regulation; HGF; Histocytochemistry; m-cadherin; Mice; Microscopy, Fluorescence; MRF4; Muscle Proteins - analysis; Muscle Proteins - genetics; Muscle Proteins - metabolism; Muscle, Skeletal - cytology; Muscle, Skeletal - metabolism; myf-5; MyoD; MyoD Protein - analysis; MyoD Protein - genetics; Myogenic Regulatory Factors - analysis; Myogenic Regulatory Factors - genetics; myogenin; Myogenin - analysis; Myogenin - genetics; Polymerase Chain Reaction; Proto-Oncogene Proteins c-met - genetics; Receptor Protein-Tyrosine Kinases - analysis; Receptor Protein-Tyrosine Kinases - genetics; RNA, Messenger - analysis; RNA, Messenger - genetics; RT-PCR; satellite cell; scatter factor; Transcription Factors - analysis; Transcription Factors - genetics
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1006/dbio.1997.8721

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 (MRFs) 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 (Allenet 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