Highly Coordinated Gene Regulation in Mouse Skeletal Muscle Regeneration

Mammalian skeletal muscles are capable of regeneration after injury. Quiescent satellite cells are activated to reenter the cell cycle and to differentiate for repair, recapitulating features of myogenesis during embryonic development. To understand better the molecular mechanism involved in this process in vivo, we employed high density cDNA microarrays for gene expression profiling in mouse tibialis anterior muscles after a cardiotoxin injection. Among 16,267 gene elements surveyed, 3,532 elements showed at least a 2.5-fold change at one or more time points during a 14-day time course. Hierarchical cluster analysis and semiquantitative reverse transcription-PCR showed induction of genes important for cell cycle control and DNA replication during the early phase of muscle regeneration. Subsequently, genes for myogenic regulatory factors, a group of imprinted genes and genes with functions to inhibit cell cycle progression and promote myogenic differentiation, were induced when myogenic stem cells started to differentiate. Induction of a majority of these genes, including E2f1 and E2f2, was abolished in muscles lacking satellite cell activity after gamma radiation. Regeneration was severely compromised in E2f1 null mice but not affected in E2f2 null mice. This study identifies novel genes potentially important for muscle regeneration and reveals highly coordinated myogenic cell proliferation and differentiation programs in adult skeletal muscle regeneration in vivo. Mammalian skeletal muscles are capable of regeneration after injury. Quiescent satellite cells are activated to reenter the cell cycle and to differentiate for repair, recapitulating features of myogenesis during embryonic development. To understand better the molecular mechanism involved in this process in vivo, we employed high density cDNA microarrays for gene expression profiling in mouse tibialis anterior muscles after a cardiotoxin injection. Among 16,267 gene elements surveyed, 3,532 elements showed at least a 2.5-fold change at one or more time points during a 14-day time course. Hierarchical cluster analysis and semiquantitative reverse transcription-PCR showed induction of genes important for cell cycle control and DNA replication during the early phase of muscle regeneration. Subsequently, genes for myogenic regulatory factors, a group of imprinted genes and genes with functions to inhibit cell cycle progression and promote myogenic differentiation, were induced when myogenic stem cells started to differentiate. Induction of a majority of these genes, including E2f1 and E2f2, was abolished in muscles lacking satellite cell activity after gamma radiation. Regeneration was severely compromised in E2f1 null mice but not affected in E2f2 null mice. This study identifies novel genes potentially important for muscle regeneration and reveals highly coordinated myogenic cell proliferation and differentiation programs in adult skeletal muscle regeneration in vivo. cyclin-dependent kinase(s) bromodeoxyuridine growth arrest-specific hematoxylin and eosin minichromosome maintenance deficient 4-morpholinepropanesulfonic acid myogenic regulatory factor(s) origin recognition complex paired box phosphate-buffered saline retinoblastoma reverse transcription sonic hedgehog tibialis anterior Skeletal muscles are damaged and repaired repeatedly throughout life. Muscle regeneration maintains locomotor function during aging and delays the appearance of clinical symptoms in neuromuscular diseases, such as Duchenne muscular dystrophy (1Pearce G.W. Walton J.N. J. Neurol. Bacteriol. 1962; 83: 535-550Google Scholar, 2Pearson C.M. Brain. 1962; 85: 109-118Google Scholar). This capacity for tissue repair is conferred by satellite cells located between the basal lamina and the sarcolemma of mature myofibers (3Bischoff R. Franszini-Armstrong A.G.E.a.C. Myogenesis. McGraw-Hill, Inc., New York1994: 97-118Google Scholar, 4Grounds M.D. Yablonka-Reuveni Z. Mol. Cell. Biol. Hum. Dis. Ser. 1993; 3: 210-256Google Scholar). Upon injury, satellite cells reenter the cell cycle, proliferate, and then exit the cell cycle either to renew the quiescent satellite cell pool or to differentiate into mature myofibers (5Anderson J.E. Biochem. Cell Biol. 1998; 76: 13-26Google Scholar). Understanding the molecular mechanism by which satellite cell activity is regulated could promote development of novel countermeasures to enhance muscle performance that is compromised by diseases or aging. Both the cell proliferation and differentiation programs are essential for myogenesis. Mammalian cells escape from quiescence (G0) and enter the cell cycle by activating the Cdk1/Rb/E2f signaling pathway (6Nevins J.R. Cell Growth Differ. 1998; 9: 585-593Google Scholar, 7Dyson N. Genes Dev. 1998; 12: 2245-2262Google Scholar). In general, mitogen stimulation induces expression and assembly of the G1 cyclin-dependent kinases (Cdks) (8Won K.A. Xiong Y. Beach D. Gilman M.Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9910-9914Google Scholar,9Cheng M. Sexl V. Sherr C.J. Roussel M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1091-1096Google Scholar). Activation of Cdks causes phosphorylation of the retinoblastoma protein (Rb) (10Kato J. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Google Scholar, 11Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J. Livingston D.M. Cell. 1993; 73: 487-497Google Scholar), leading to increased activities of a subset of E2f transcription factors (E2fs) (12Sears R. Ohtani K. Nevins J.R. Mol. Cell. 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R. or muscle were in and with and at with or in and were with secondary in and for at To the were then for in and with for at to the DNA by in were then in and and with and and at with mouse in and by with in and was by of and and or muscles were in a for in the lacking for with in for with for in and in were and with and was a at mouse cDNA was for the from cDNA by the were for were of muscle from mice of the time was with of in at for cDNA was at for in the cDNA and reverse cDNA was at for in the cDNA DNA E. DNA and DNA was and at for To the of and was and the was at for cDNA was with and with and In was a To a or of and of were in at for by at for in reverse and or were by at in a to the for were with a and the were a the that the of from the Briefly, the of elements of the was and as a This was then to the for gene in the then that the of the or the were and points were we the by gene that not a change at time This was after we repeatedly the of the and of gene elements with 2.5-fold change of 16,267 elements when control were we the to a with at and a P. D. J. Q. S. E. Proc. Natl. Acad. Sci. U. S. A. Scholar). To the and to genes to satellite cell proliferation and differentiation, semiquantitative analysis was as D.J. Meeson A. Elterman J. Zhao Y. Yang P. Bassel-Duby R. Williams R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5416-5421Google Scholar). was by the of and as a to the control in the time were a at genes and the for of the genes in this study to the and cell cycle and origin recognition and and minichromosome and and and E2f2, and and and and and and cell cycle transcription and and and and and and and and and and and and and and myogenin and and myogenic and and and protein and and protein and and protein and and and and and and and growth and expression and and and for for gene and protein is from a study A. G. J. Biol. Chem. 2001; 276: Scholar). cardiotoxin into the more muscles and showed regeneration in more of the myofibers In were analysis and at and after injury. of increased after cardiotoxin with a This in cell is to cell and proliferation of satellite started to at and more at and at was not from that of the control muscles for the of a of muscle in increased at and after and cell that cardiotoxin in muscle induces and and the regeneration process from a phase of proliferation to differentiation at after injury. To the functional role of satellite cell activities in skeletal muscle regeneration in vivo, was in satellite cell was as early as after the cardiotoxin injection. activated satellite cells were from the and more and with quiescent satellite and after injury, as as or of satellite cells were in a in a basal with and and of were at to the were cells with and and cells were in with the basal lamina and the cell of the and are to myogenic cells that have to a quiescent of satellite cell and of quiescence as by the is in with the molecular by cDNA and in this cell as a of the to muscle injury, the analysis of gene is to and the of proliferation from cell we for of to cells control and muscle the for or as for K. M. I.S. Dev. 1995; Scholar, S. V. G. M. J. Cell Sci. Scholar), T. G. J. 9: Scholar), and S. S. Scholar), that a of cells for and of the or cells for MyoD to terminal differentiation, whereas functional and are muscle showed for In of a muscle we that at least of cells were for or with of the cells cells were for whereas of the cells for cells or cells provided that satellite cells are the of cells directly involved in myogenesis in skeletal muscle and cells not in skeletal cells but with the in other phase of DNA which is a of a cell cycle time for is that of these cells have the cell cycle and the differentiation with this is the that of these cells for which myogenic differentiation J. S. H. Proc. Natl. Acad. Sci. U. S. A. 1990; Scholar). To the role of satellite cell activities in gamma was to the capacity of muscle stem which have to satellite cell function in skeletal muscle in J.N. 1997; Scholar, D.J. J. 1992; 73: Scholar). mouse muscles to of gamma the cardiotoxin injection. muscles were not from muscles myogenic cell proliferation and differentiation after cardiotoxin were as by a in cell at and the of at after To the at the molecular we semiquantitative to for and expression to essential for cell proliferation or myogenesis P. Bradley A. Morris J.H. Edmondson D.G. Venuti J.M. Olson E.N. Klein W.H. Nature. 1993; 364: 501-506Google Scholar, Z. DeGregori J. R. G. Nevins J.R. Williams R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: Scholar, P. D. M. S.J. Genes Dev. Scholar). muscles or induction of these that satellite cells a role in skeletal muscle regeneration. To gene expression during skeletal muscle cDNA were a mouse cDNA 16,267 elements 3,532 were more 2.5-fold at one or more time of genes were during the early phase of regeneration after cardiotoxin as by the as regeneration by and after injury. a change in the of genes with a of and of after after cardiotoxin gene elements of showed P. D. J. Q. S. E. Proc. Natl. Acad. Sci. U. S. A. Scholar) was to and the cDNA elements the of expression the time cluster were of the of a as D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: Scholar), and are as in and functional in in the of In cDNA were in the cluster or in with gene elements were in and mouse protein were in and with a expression the of the analysis in this genes were gene elements to and were in is a of these gene elements in this cluster are gene elements to and in the 3,532 that the is for regulatory during muscle and we could the cluster analysis to novel genes with expression to functional To novel genes to myogenic differentiation, we genes with a expression with muscle we that imprinted genes, and imprinted and are in genes in these have induction of at or with of myogenic differentiation, coordinated induction of these genes important functional in muscle induction of growth arrest-specific and in of which to sonic hedgehog signaling This points to the of these in myogenic of encoding of cell cycle progression and DNA replication were in and of the expression of these is early induction and expression at or and expression after injury. This is in with the that cell proliferation during the early phase of regeneration. To the and for genes with functions in the proliferation we semiquantitative for of DNA replication factors, and transcription DNA replication factors and and and early induction in at or and started to to the control after of these genes are regulated by transcription factors (6Nevins J.R. Cell Growth Differ. 1998; 9: 585-593Google Scholar, K. Dev. 1998; 8: Scholar), is to which of the transcription factors during skeletal muscle regeneration in vivo. we and E2f E2f2, and and and by semiquantitative with the we of after and in and whereas E2f1 and E2f2 were highly induced during the phase of muscle regeneration between and after injury. was and a change during regeneration. in the have expression from Induction of was with that of E2f2, and DNA replication Induction of is not after injury, in with role in induction of muscle differentiation, whereas the during the regeneration induction of genes essential for DNA replication and cell cycle control with induction of E2f2, and to that these regulatory proteins are essential for satellite cell proliferation in during muscle regeneration. in and showed induction and after injury, expression of these genes to muscle by genes or genes that are to induced during skeletal muscle differentiation were in these and skeletal muscle skeletal muscle growth growth myogenin and and skeletal muscle were in these is important to that a majority of these not a in muscles after cardiotoxin that up-regulation of these genes the of satellite cell Induction of these genes at the molecular myogenic differentiation that in regeneration To the and of myogenic transcription factors and cell cycle we semiquantitative and expression for myogenic transcription factors and in adult muscle regeneration with the control and MRF4 expression showed during muscle regeneration. was early induction of MyoD with a expression at that of Myf5 at after injury. a induction was for and myogenin at after with myogenic the analysis showed that the was induced during the phase of muscle regeneration whereas the was induced during muscle differentiation and analysis these a highly coordinated induction of for other at after injury, which and whereas and showed during the repair of the of analysis of gene expression for in study is the of the of the tissue is to which cell is for the in the expression we not induced or expression of a gene of is a of satellite cell proliferation and differentiation or of cell is not to expression for of the genes in the of the to of gamma to satellite cell activities in adult skeletal we have in this gamma of muscles at to muscle satellite cell myogenic cell proliferation and the gene expression in muscles cardiotoxin which the of gamma and in muscles at after cardiotoxin which gene expression in the of satellite cell Among 3,532 elements that were to have more a 2.5-fold change during the time and gene elements showed more 2.5-fold change in the muscles and with injury, To this is in satellite cell to in gene we analysis to gene expression between and Induction of a for was not affected by that cell was not affected by In to the gene elements that and we genes and that are to cell cycle or myogenesis and to induced during regeneration. Induction of these genes, for was abolished in the muscles at after injury. are in with the is not a study showed induction of in that role other myogenesis in vivo. analysis a subset of imprinted genes with myogenic regulatory factors, such as that coordinated expression of these imprinted genes is of to myogenic To this we the analysis for imprinted genes that to genes encoding myogenic factors, such as Induction of these imprinted genes, were abolished in muscles after injury. for not induction that we not the time when was to these that coordinated induction of these imprinted genes in muscle differentiation in vivo. we cardiotoxin in muscle for of the of satellite cell activities in gene expression in this regeneration this we to the from the muscles with the time E2f1 and E2f2 are highly induced during skeletal muscle regeneration with genes to the control of the cell cycle and DNA we that in regeneration by genes To E2f1 or E2f2 is for adult skeletal muscle we cardiotoxin in muscles in mice with of E2f1 or in mice with of E2f2 a regeneration process with whereas muscle regeneration was severely compromised in mice with of we have that but not E2f2, is essential for muscle vivo. regeneration in mouse skeletal muscle is a to study satellite cell function in (21Seale P. Sabourin L.A. Girgis-Gabardo A. Mansouri A. Gruss P. Rudnicki M.A. Cell. 2000; 102: 777-786Google Scholar, 22Garry D.J. Meeson A. Elterman J. Zhao Y. Yang P. Bassel-Duby R. Williams R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5416-5421Google Scholar, L.A. K. J.E. Rudnicki M.A. Genes Dev. 1996; Scholar). we a D.J. Meeson A. Elterman J. Zhao Y. Yang P. Bassel-Duby R. Williams R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5416-5421Google Scholar, R. A. Biol. Cell. Scholar) and showed and regeneration in as by DNA and expression and semiquantitative and between and muscles in this regeneration into molecular important for control of proliferation and differentiation in satellite cells in vivo. to and a of genes with to expression analysis to functions of and novel This to study gene in skeletal muscle C.J. J. 2001; Scholar, M. M. H. S. D. R. J. Scholar, J.A. D. J.M. J. Scholar, R. P. 51: Scholar, J. L. J. Dev. Scholar, D.G. M.A. Scholar, X. S. Scholar). In this we were to imprinted genes into with of for muscle of these have to regulated during skeletal muscle development P. D. M. S.J. Genes Dev. Scholar, X. G. Yang E. N. N. D. Dev. Biol. 1996; Scholar, M. P. P. 1995; Scholar, James M.E. P. J. Cell. 1996; Scholar). that the induction of of these imprinted genes by muscle was abolished in the the that important role in satellite cell activities during muscle regeneration gamma satellite cell proliferation and muscle regeneration. to these is these imprinted genes are regulated during the regeneration is that muscle expression of these genes as a of increased is that is to DNA or a role in gene activity M.A. Scholar). In these genes to from the imprinted in the a highly coordinated expression of imprinted genes in muscle in this analysis of expression of imprinted genes in this is to the of during muscle regeneration. induction of and with myogenic differentiation the of these to the in muscle signaling pathway is important for the of myogenesis in G. U. EMBO J. Scholar, G. S. M. 1996; 12: Scholar), and signaling is for maintenance of Myf5 and MyoD and for the of terminal differentiation in development R. Dev. Biol. 2001; Scholar). study identifies signaling in these to muscle differentiation in vivo. employed gamma to of satellite which showed of this DNA replication and and induction of and that satellite cell and proliferation are abolished by gamma radiation. cardiotoxin in muscles could for of the of satellite cell activities in gene expression during skeletal muscle regeneration. functional myogenic transcription factors in myogenic differentiation by in mouse (21Seale P. Sabourin L.A. Girgis-Gabardo A. Mansouri A. Gruss P. Rudnicki M.A. Cell. 2000; 102: 777-786Google Scholar, 23Williams B.A. Ordahl C.P. Development. 1994; 120: 785-796Google Scholar, 24Ridgeway A.G. Skerjanc I.S. J. Biol. Chem. 2001; 276: 19033-19039Google Scholar, R.L. Weintraub H. Lassar A.B. Cell. 1987; 51: 987-1000Google Scholar, 26Braun T. Buschhausen-Denker G. Bober E. Tannich E. Arnold H.H. EMBO J. 1989; 8: 701-709Google Scholar, D.G. Olson E.N. Genes Dev. 1990; 4: 1450Google Scholar, 28Rhodes S.J. Konieczny S.F. Genes Dev. 1989; 3: 2050-2061Google Scholar, P. Olson E.N. Mol. Cell. Biol. 1991; Scholar, Olson E.N. Cell. 1995; 83: Scholar). that early induction of MyoD during the phase of muscle regeneration a induction of at differentiation is with a role for a in myogenic expression of MyoD in this study is with functions in myogenic cell maintenance and in this study is the induction of expression of Myf5 was not induced after the induction of and myogenin and of differentiation, that the function of Myf5 are between embryonic development of skeletal muscle and adult muscle regeneration. this is the that of MyoD to skeletal muscle development in with expression of Myf5 M.A. Braun T. Hinuma S. Jaenisch R. Cell. 1992; 71: 383-390Google Scholar), but adult skeletal muscles lacking MyoD gene are in muscle regeneration L.A. K. J.E. Rudnicki M.A. Genes Dev. 1996; Scholar, Rudnicki M.A. Dev. Biol. 2000; Scholar). is for the satellite cell lineage specification and Myf5 is in quiescent satellite cells J.R. L. S. A. M.E. J. Cell Biol. 2000; Scholar, Dev. Biol. 1997; Scholar), we that the a role in and the satellite cell pool during skeletal muscle regeneration in vivo. from this study that the transcription factors myogenic differentiation during adult muscle regeneration from that during myogenesis in embryonic development. are for cell cycle with muscle that are capable of the cell cycle and myogenesis such a In the we expression of during muscle regeneration in vivo. highly coordinated induction of for at the time of myogenic differentiation, which and are for and and and are and are to essential for myogenesis myogenin P. D. M. S.J. Genes Dev. Scholar), and MyoD by a L.A. K. M. J. Biol. Chem. 2000; Scholar) and by of K. M. Mol. Cell. Biol. Scholar). and have in myogenic differentiation of of and M. G. M. Y. K. J. Scholar, Li Y. N. E. Xiong Y. Mol. Cell. Biol. 1998; Scholar, J. Lassar A.B. 1995; Scholar). highly coordinated induction of inhibit cell cycle progression and cell cycle whereas myogenesis regulatory between the proliferation and differentiation programs in vivo. cell into the cell cycle from quiescence of the signaling regulatory that E2f transcription factors is in this process E2f genes involved in DNA replication and cell cycle progression N. Genes Dev. 1998; 12: 2245-2262Google Scholar, 14Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Google Scholar, J. T. Nevins J.R. Mol. Cell. Biol. 1995; Scholar, E. Mol. Cell. Biol. 1994; Scholar, K. DeGregori J. G. T.J. Nevins J.R. Mol. Cell. Biol. 1996; Scholar). In this we induction of and as as of the with a to transcription factors and This induction of genes myogenic stem cells in cell cycle during regeneration in vivo. are to the functional role of of these in the control of cell proliferation during muscle regeneration. E2f1 and E2f2 are for muscle satellite proliferation and regeneration. of cardiotoxin in muscles of mice with of E2f1 in severely compromised muscle regeneration as by the of and and mature at the phase of muscle regeneration. the of induction of E2f1 to in which in to muscle to that E2f1 induces a pathway in cells DeGregori J. G. Jakoi L. Nevins J.R. Cell Growth Differ. 1998; 9: Scholar). regeneration process in E2f2 null mice is but that of E2f2 expression is not to of muscle regeneration. this study not the that the other E2f transcription factors important regulatory such as protein phosphorylation the that E2f1 and E2f2 in adult skeletal muscle regeneration. E. N. R. and for J. R. Nevins for E2f2 null and A. R. E. J. M. S. and J. M. for with