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

Cornelison, D. D. W. and Wold, Barbara J. (1997) Single-Cell Analysis of Regulatory Gene Expression in Quiescent and Activated Mouse Skeletal Muscle Satellite Cells. Developmental Biology, 191 (2). pp. 270-283. ISSN 0012-1606. https://resolver.caltech.edu/CaltechAUTHORS:20190506-133355251

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Abstract

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 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1006/dbio.1997.8721DOIArticle
ORCID:
AuthorORCID
Wold, Barbara J.0000-0003-3235-8130
Additional Information:© 1997 Academic Press. Received 25 June 1997, Accepted 19 August 1997, Available online 25 May 2002. We are very grateful to Drs. Keiko Kato and Scott Fraser for the cells multiplex single-cell RT-PCR protocol and Dr. Yinong Zhang for instruction in the patch-clamp technique. This work was supported NIH Grants AR40708 and AR42671 and an MDA grant to B.J.W. D.D.W.C. was supported by a NIH Predoctoral Training Grant and by a fellowship from the ARCS Foundation.
Funders:
Funding AgencyGrant Number
NIHAR40708
NIHAR42671
Muscular Dystrophy AssociationUNSPECIFIED
NIH Predoctoral FellowshipUNSPECIFIED
ARCS FoundationUNSPECIFIED
Subject Keywords:satellite cell; c-met; m-cadherin; MyoD; myogenin; MRF4; myf-5; HGF; RT-PCR; scatter factor
Issue or Number:2
Record Number:CaltechAUTHORS:20190506-133355251
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190506-133355251
Official Citation:D.D.W. Cornelison, Barbara J. Wold, Single-Cell Analysis of Regulatory Gene Expression in Quiescent and Activated Mouse Skeletal Muscle Satellite Cells, Developmental Biology, Volume 191, Issue 2, 1997, Pages 270-283, ISSN 0012-1606, https://doi.org/10.1006/dbio.1997.8721. (http://www.sciencedirect.com/science/article/pii/S0012160697987212)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:95252
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 May 2019 20:49
Last Modified:03 Oct 2019 21:11

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