Highly Coordinated Gene Regulation in Mouse Skeletal Muscle Regeneration
2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês
10.1074/jbc.m209879200
ISSN1083-351X
AutoresZhen Yan, Sangdun Choi, Xuebin Liu, Mei Zhang, Jeoffrey Schageman, Sun Young Lee, Rebecca Hart, Ling Lin, Frederick A. Thurmond, R. Sanders Williams,
Tópico(s)GDF15 and Related Biomarkers
ResumoMammalian 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. Biol. 1997; 17: 5227-5235Google Scholar) and up-regulation of a variety of E2f-responsive genes encoding proteins directly involved in DNA replication and cell cycle progression (13Galaktionov K. Chen X. Beach D. Nature. 1996; 382: 511-517Google Scholar, 14Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Google Scholar). On the other hand, myogenic differentiation is controlled by interactions of a network of myogenic transcription factors (15Olson E.N. Brennan T.J. Chakraborty T. Cheng T.C. Cserjesi P. Edmondson D. James G. Li L. Mol. Cell. Biochem. 1991; 104: 7-13Google Scholar). Studies of myogenesis during embryonic development and in cultured myogenic cell lines have provided much insight into the functional role of these transcription factors (16Braun T. Rudnicki M.A. Arnold H.H. Jaenisch R. Cell. 1992; 71: 369-382Google Scholar, 17Rudnicki M.A. Braun T. Hinuma S. Jaenisch R. Cell. 1992; 71: 383-390Google Scholar, 18Hasty P. 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Chem. 2001; 276: 19033-19039Google Scholar), whereas primary basic helix-loop-helix myogenic regulatory factors (MRFs), MyoD and Myf5 (25Davis 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), and secondary MRFs, myogenin and MRF4 (27Edmondson D.G. Olson E.N. Genes Dev. 1990; 4: 1450Google Scholar, 28Rhodes S.J. Konieczny S.F. Genes Dev. 1989; 3: 2050-2061Google Scholar), function downstream in terminal differentiation. MADS box transcription factors, such as myocyte enhancer factor 2, cooperate with MRFs in muscle-specific gene expression (29Cserjesi P. Olson E.N. Mol. Cell. Biol. 1991; 11: 4854-4862Google Scholar, 30Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Cell. 1995; 83: 1125-1136Google Scholar). However, the functional roles of these regulatory proteins in adult skeletal muscle have not been well defined. Several animal models of muscle regeneration have been described, but there has not been a comprehensive analysis of gene regulation in any model. In this study, we have taken advantage of high density cDNA microarray to assess global gene expression followed by detailed semiquantitative reverse transcription (RT)-PCR analysis in a mouse skeletal muscle regeneration model. Expression of some genes directly related to cell cycle control and myogenic differentiation was compared in the presence and absence of satellite cell activities. We have identified genes previously unknown to be regulated during skeletal muscle regeneration. We have also uncovered differential functional roles of E2f1 and E2f2in vivo using mice with targeted mutations. We modified a previously described muscle injury model (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) by injecting cardiotoxin into the tibialis anterior (TA) muscles of 6-week-old male C57BL/6 mice (Harlen). The muscles were harvested at various times (1, 2, 3, 5, 10, or 14 days) after injection. Uninjected TA muscles were used as control. 6 h before muscle harvesting, 500 mg/kg bromodeoxyuridine (BrdUrd) was injected intraperitoneally to label DNA-replicating nuclei. The left TA muscle was harvested, fixed in 4% paraformaldehyde, frozen or embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) or antibodies against various antigens. Total RNA was isolated from the right TA muscle using TriPure® kit (Roche Molecular Biochemicals). To assess the contribution of satellite cell activities in global gene expression, we injected cardiotoxin into TA muscles that had been subjected to 2,200 rads of gamma radiation (31Phelan J.N. Gonyea W.J. Anat. Rec. 1997; 247: 179-188Google Scholar) 24 h earlier. To determine whether induced expression of E2f1 or E2f2 is essential for injury-induced muscle regeneration, cardiotoxin was injected in TA muscles in mice with targeted mutation of E2f1 (Jackson Laboratory) or E2f2 allele (kind gifts from J. R. Nevins). 8-μm frozen or paraffin-embedded muscle sections were permeabilized in 0.3% Triton X-100 and PBS, blocked with normal goat serum, and incubated overnight at 4 °C with rabbit anti-MyoD antibody (1:50, Santa Cruz), rat anti-Mac-1 (1:400, Serotec), or rat anti-ag 7/4 (1:400, Serotec) in 5% normal goat serum and PBS. The sections were incubated with fluorescein isothiocyanate-conjugated secondary antibody (1:50, Jackson Laboratory) in 5% normal goat serum and PBS for 30 min at room temperature. To detect BrdUrd incorporation, the sections were then fixed for 10 min in 2% formaldehyde on ice and treated with 2 n HCl for 60 min at 37 °C to denature the DNA followed by neutralization in 0.1m borate buffer (pH 8.5). The sections were then permeabilized in 0.3% Triton X-100 and PBS and blocked with 1.5% normal horse serum and PBS and incubated overnight at 4 °C with mouse monoclonal anti-BrdUrd antibodies (1:25, Roche) in 0.1% bovine serum albumin and PBS followed by an incubation with biotinylated horse anti-mouse IgG (1:200, Dako) in 1% normal horse serum and PBS. The biotinylated IgG was detected by application of fluorescein isothiocyanate and streptavidin (1:50, Vector) and examined under epifluorescent or confocal microscope. TA muscles were fixed in a solution containing 2.5% glutaldehyde, 137 mm NaCl, 2 mm CaCl2, 4 mm KCl, 100 mm MOPS (pH 7.4) for 24 h, rinsed in the fixation solution lacking glutaldehyde for 24 h, treated with 1% osmium tetroxide in 100 mmC2H6AsO2Na (cacodylate) for 2 h, stained with 0.5% uranyl acetate for 2 h, dehydrated in ethanol, and embedded in Spurr's resin. Thin sections were cut, collected on 400-mesh copper grids, and stained with uranyl acetate and lead citrate. Microscopy was carried out using a JEOL 1200 EX electron microscope at 80 kV. A National Institute on Aging mouse cDNA chip (16,000 bytes) was used for the microarray analysis. PCR products from cDNA clones prepared by the Caltech Genome Research Laboratory (date.tree.caltech.edu/local_clones.html) were spotted onto CMT-GAPS-coated slides (Corning). Probes for microarray hybridization were generated using 5 μg of pooled muscle total RNA from five mice of the same time point. The RNA was first annealed with 100 pmol of T7-(dT)24 primer (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3′) in 12 μl at 70 °C for 10 min. The first strand cDNA was synthesized at 42 °C for 1 h in the first strand cDNA buffer containing 50 mm Tris (pH 8.3), 75 mmKCl, 3 mm MgCl2, 10 mmdithiothreitol, 0.5 mm dNTP, and 10 units/μl Superscript II reverse transcriptase (Invitrogen). The second strand cDNA was synthesized at 16 °C for 2 h in the second strand cDNA buffer containing 20 mm Tris (pH 6.9), 90 mmKCl, 4.6 mm MgCl2, 0.15 mm NAD, 10 mm (NH4)2SO4, 0.2 mm dNTP, 0.07 unit/μl Escherichia coli DNA ligase (New England Biolabs), 0.27 unit/μl E. coli DNA polymerase I (New England Biolabs), and 0.013 unit/μl RNase H (Invitrogen). T4 DNA Polymerase (20 units, Invitrogen) was added and incubated at 16 °C for 5 min. To stop the reaction, 7.5 μl of 1m NaOH and 2 mm EDTA (pH 8.0) was added, and the sample was heated at 65 °C for 10 min. The cDNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ammonium acetate and ethanol. In vitrotranscription was carried out using a T7 Megascript kit (Ambion). To generate a Cy3- or Cy5-labeled probe, 10 μg of amplified antisense RNA and 6 μg of random hexamer primers were annealed in 14 μl at 70 °C for 10 min followed by incubation at 42 °C for 2 h in 50 mm Tris (pH 8.3), 75 mm KCl, 3 mm MgCl2, 10 mm dithiothreitol, 10 μm dATP/dCTP/dGTP, 4 μm dTTP, 13.3 units/μl Superscript II reverse transcriptase (Invitrogen), and 3 μl Cy5 or Cy3 dUTP. The probes were purified by filtering through Microcon-30 filters (Millipore) before hybridization at 42 °C in a water bath overnight according to the instructions for CMT-GAPS-coated slides. The slides were scanned with a GenePix 4000A scanner (Axon Instruments) and analyzed using the GENEPIX PRO 3.0 (Axon Instruments). The raw data were normalized using a total intensity normalization method under the assumption that the total quantities of messages from both channels should be the same. Briefly, the average fold difference of all elements of the array was calculated and used as a normalization factor. This normalization factor was then used to adjust the fold for each gene in the array. We then eliminated spots that had median intensities less than the mean plus three times S.D. of the background Cy3 or Cy5 intensity. Once the normalized data were obtained and lower than background data points were removed, we processed the data further by removing any gene element that had not shown a change >2.5-fold at any time point. This cutoff level was set after we repeatedly tested the reproducibility of the microarray hybridization and found an average of only 10 gene elements with changes greater than 2.5-fold (maximal change of 2.8-fold) among 16,267 elements assayed (0.061%) when unstimulated control samples were compared (not shown). For clustering analysis, we converted the Cy5:Cy3 ratio to a log ratio (base 2), analyzed with GeneCluster 2.0 available at www-genome.wi.mit.edu, and generated a 4 × 4 self-organization map (32Tamayo P. Slonim D. Mesirov J. Zhu Q. Kitareewan S. Dmitrovsky E. Lander E.S. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2907-2912Google Scholar). To confirm the microarray findings and to survey additional genes pertinent to satellite cell proliferation and differentiation, semiquantitative RT-PCR analysis was performed as described (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). Each data point was normalized by the abundance of glyceraldhyde-3-phosphate dehydrogenase mRNA and expressed as a log2 ratio to the uninjected control in the randomly preassigned time course group. PCR primer pairs were designed using a Primer3 search engine at www-genome.wi.mit.edu. The screened genes and the oligonucleotide primer pairs used for each of the genes in this study corresponded to the following nucleotides: glyceraldhyde-3-phosphate dehydrogenase, 114–136 and 403–383 (NM_008084); cell division cycle 6 homolog (Saccharomyces cerevisiae) (Cdc6), 203–224 and 927–907 (NM_011799); origin recognition complex, subunit 1 homolog (S. cerevisiae) (Orc1), 866–885 and 1238–1219 (NM_011015); Orc2, 2381–2400 and 2780–2761 (NM_008765); minichromosome maintenance-deficient 2 homolog (S. cerevisiae) (Mcmd2), 2580–2599 and 2903–2884 (NM_008564); Mcmd3, 1445–1464 and 1866–1847 (X62154);E2f1, 2030–2050 and 2147–2128 (L21973); E2f2, 8–27 and 140–121 (AA791874); E2f3, 403–422 and 753–733 (AF015948); E2f4, 471–490 and 705–686 (AA050824);E2f5, 441–459 and 744–725 (X86925); E2f6, 42–61 and 239–220 (AW211063); DRTF-polypeptide-1 (DP1), 422–441 and 868–849 (X72310); protein-regulating cell cycle transcription factor DRTF1/E2f (DP3), 867–887 and 1204–1185 (S79780); Rb, 1608–1627 and 2089–2068 (M26391);p130, 1882–2003 and 2272–2251 (U50850); p107, 1801–1820 and 2255–2236 (U27177); cyclin D1, 183–203 and 511–481 (NM_007631); cyclin D2, 732–751 and 1065–1045 (NM_009829); cyclin D3, 981–200 and 1326–1307 (NM_007632); cyclin E, 858–879 and 1164–1142 (NM_007633); cyclin E2, 52–71 and 423–403 (NM_009830); cyclin A2, 1002–1022 and 1325–1305 (NM_009828); cyclin B, 368–387 and 844–825 (X58708); Pax3, 244–263 and 629–610 (NM_008781); Pax7, 126–145 and 450–431 (U20792); MyoD, 671–690 and 1161–1139 (M84918); myogenin (Myog), 470–492 and 850–830 (D90156); Myf5, 504–528 and 761–738 (NM_008656); myogenic factor 6 (Myf6/MRF4), 432–455 and 677–657 (NM_008657); Cdk inhibitor 2B (p15Ink4b/Cdkn2b), 49–66 and 373–354 (NM_007670); Cdk4 and Cdk6 inhibitor protein (p16Ink4a), 245–264 and 653–634 (L76150); Cdk4 and Cdk6 inhibitor p18 protein (p18Ink4c), 50–70 and 462–442 (U19596); Cdk4 and Cdk6 inhibitor p19 protein (p19Ink4d), 340–360 and 732–713 (U19597); Cdk inhibitor 1A (p21Cip1/Cdkn1a), 369–391 and 691–668 (NM_007669); Cdk inhibitor 1B (p27Kip1/Cdkn1b), 87–106 and 430–410 (NM_009875); tumor suppressor p53 (p53), 930–949 and 1233–1204 (AF161020); Cdk inhibitor 1C (p57Kip2/Cdkn1c), 903–923 and 1242–1223 (NM_009876);H19 and muscle-specific Nctc 1 (H19), 980–999 and 1344–1325 (NM_023123); insulin-like growth factor 2 (Igf2), 473–492 and 636–617 (M14951); reduced expression 3 (Rex3/Bex1), 326–345 and 631–612 (NM_009052); colony-stimulating factor 1 (Csf1), 557–576 and 961–942 (NM_007778); 18 S ribosomal RNA, 448–467 and 926–907 (X00686); sequence information for primers for mesoderm-specific transcript (Peg1/Mest), paternally expressed gene 1 (Peg3/Pw1); and zinc finger protein Zac1 (Zac1) is from a previous study (33El Kharroubi A. Piras G. Stewart C.L. J. Biol. Chem. 2001; 276: 8674-8680Google Scholar). We injected cardiotoxin into the anatomically more confined TA muscles and showed regeneration in more than 90% of the myofibers (Fig.1 A). In three independent experiments, similar morphological changes were observed repeatedly. Histological analysis demonstrated global myofiber fragmentation and edema at days 1 and 2 after injury. The number of mononucleated cells/cross-sectional area increased significantly after cardiotoxin injection with a peak around day 3. This increase in cell number is attributable to both inflammatory cell infiltration and proliferation of satellite cells. Myotubes started to appear at day 3 and became more evident at days 5 and 10 postinjection. Morphology at day 14 postinjection was not significantly different from that of the uninjected control muscles except for the presence of central nuclei, a known hallmark of recent muscle regeneration, in nearly all myofibers. The percent BrdUrd-positive nuclei increased significantly at days 2 and 3 after injury (Fig. 1, A and B), indicating active cell proliferation. These results suggest that cardiotoxin injection in TA muscle induces extensive and complete regeneration, and the regeneration process shifts morphologically from a phase of proliferation to differentiation at around day 3 after injury. To assess the functional role of satellite cell activities in skeletal muscle regeneration in vivo, transmission electron microscopy was performed. As shown in Fig. 2 A, satellite cell activation was apparent as early as 6 h after the cardiotoxin injection. The activated satellite cells were often separated from the adjacent myofiber, leaving electron-lucent gaps and exhibit more abundant cytoplasm and less condensed heterochromatin compared with quiescent satellite cells. At days 2 and 3 after injury, as many as seven or eight pairs of postmitotic satellite cells were often detected in a large cleft in degenerating myofiber beneath a single basal lamina. Myotubes with central nuclei and thick and thin filaments of nascent sarcomeres were apparent at day 5 postinjection. Adjacent to the myotubes were many small, mononucleated cells with condensed heterochromatin and little cytoplasm and organelles. These cells were universally in contact with both the basal lamina and the cell membrane of the growing myofiber and are likely to represent myogenic precursor cells that have reverted to a quiescent state. The timing of satellite cell activation, proliferation, and resumption of quiescence as evidenced by the morphological data is in agreement with the molecular events detected by cDNA microarray and RT-PCR observations in this study. Inflammatory cell infiltration, as a part of the physiological responses to muscle injury, has complicated the analysis of global gene expression. A key question is how to distinguish and ascertain the contribution of myoblast proliferation from inflammatory cell infiltration. Here we performed indirect immunofluorescence for detection of BrdUrd incorporation to mark proliferative cells on control and injured (day 3) muscle sections. We also stained the same sections for MyoD, Mac-1, or ag 7/4 as markers for proliferating myoblasts (34Koishi K. Zhang M. McLennan I.S. Harris A.J. Dev. Dyn. 1995; 202: 244-254Google Scholar, 35Cooper R.N. Tajbakhsh S. Mouly V. Cossu G. Buckingham M. Butler-Browne G.S. J. Cell Sci. 1999; 112: 2895-2901Google Scholar), peripheral macrophages (36Springer T. Galfre G. Secher D.S. Milstein C. Eur. J. Immunol. 1979; 9: 301-306Google Scholar), and infiltrating neutrophils (37Hirsch S. Gordon S. Immunogenetics. 1983; 18: 229-239Google Scholar), respectively. We predicted that a great portion of MyoD-positive cells would be positive for BrdUrd staining, and none of the Mac-1 or ag 7/4-positive cells would be positive for BrdUrd because cycling myoblasts expresses MyoD prior to terminal differentiation, whereas peripheral functional macrophages and neutrophils are terminally differentiated. Normal muscle sections showed negative results for any above mentioned staining (not shown). In any given field of a day 3 muscle section, we estimated that at least 30% of all cells were positive for MyoD, BrdUrd, or both (Fig. 2 B). Consistent with our expectation, 45% of the MyoD-positive cells (759 cells counted) were detected positive for BrdUrd, whereas none of the cells positive for Mac-1 (508 cells counted) or ag 7/4 (114 cells counted) incorporated BrdUrd. These results provided unambiguous evidence that proliferating satellite cells are the main source of cells directly involved in myogenesis in skeletal muscle regeneration, and inflammatory cells do not proliferate in injured skeletal muscle. Those cells expressing MyoD, but with no BrdUrd incorporation, may be the cycling myoblasts in phases other than S phase of DNA replication. Our BrdUrd labeling lasted 6 h, which is only a fraction of a normal cell cycle time (∼20 h) for normal mammalian cells. It is also possible that some of these MyoD-positive cells might have already exited the cell cycle and initiated the differentiation process. Consistent with this notion is the observation that many of these cells had strong staining for MyoD, which promotes myogenic differentiation (38Choi J. Costa M.L. Mermelstein C.S. Chagas C. Holtzer S. Holtzer H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7988-7992Google Scholar). To evaluate further the role of satellite cell activities in our model, gamma radiation was used to compromise the proliferative capacity of muscle stem cells, which have often been used to determine satellite cell function in skeletal muscle in vivo (31Phelan J.N. Gonyea W.J. Anat. Rec. 1997; 247: 179-188Google Scholar, 39Rosenblatt J.D. Parry D.J. J. Appl. Physiol. 1992; 73: 2538-2543Google Scholar). We subjected mouse hind limb muscles to 2,200 rads of gamma radiation 24 h before the cardiotoxin injection. Irradiated TA muscles were not morphologically different from normal TA muscles (not shown). However, myogenic cell proliferation and differentiation after cardiotoxin injection were blocked as indicated by a significantly lower increase in cell number, very few BrdUrd-positive nuclei at day 3, and the absence of newly formed myotubes at day 10 after injury (Fig. 2 C). To confirm the findings at the molecular level, we performed semiquantitative RT-PCR to quantify transcripts for Cdc6, myogenin, and p57Kip2; their expression has been shown previously to be essential for cell proliferation or myogenesis (18Hasty P. Bradley A. Morris J.H. Edmondson D.G. Venuti J.M. Olson E.N. Klein W.H. Nature. 1993; 364: 501-506Google Scholar, 40Yan Z. DeGregori J. Shohet R. Leone G. Stillman B. Nevins J.R. Williams R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3603-3608Google Scholar, 41Zhang P. Wong C. Liu D. Finegold M. Harper J.W. Elledge S.J. Genes Dev. 1999; 13: 213-224Google Scholar). Irradiated TA muscles had attenuated or delayed induction of these transcripts (Fig. 2 D), suggesting that satellite cells play a pivotal role in skeletal muscle regeneration. To investigate global gene expression during injury-induced skeletal muscle regeneration, cDNA microarray hybridizations were performed using a National Institute on Aging mouse 16,000-byte cDNA chip. Of 16,267 elements screened, 3,532 (21.7%) were altered more than 2.5-fold at one or more time points. A significant number of genes were altered during the early phase of regeneration before day 5 after cardiotoxin injection as shown by the scatter plots (Fig. 3 A). The trend became less evident as regeneration approached completion by days 10 and 14 after injury. Quantification confirmed a phasic change in the number of differentially expressed genes (>2.5-fold change) with a peak around day 2 (2,310 elements, 14.2% of total) and day 3 (2,324 elements, 14.3% of total) after injury (Fig. 3 B). By day 14 after cardiotoxin injection, only 199 gene elements (1.2% of total) showed differential expression. The self-organizing map (32Tamayo P. Slonim D. Mesirov J. Zhu Q. Kitareewan S. Dmitrovsky E. Lander E.S. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2907-2912Google Scholar) was used to assemble and analyze the data. The clustering procedure groups together cDNA elements on the basis of their common expression patterns over the time points. 16 cluster groups were used (Fig. 3 C). Fig.4 shows part of the results of a hierarchical clustering as described previously (42Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Google Scholar), and complete results are presented as supplemental data (self-organization map in supplemental data 1 and functional groups in supplemental data 2) in the Journal of Biological Chemistry on-line. In most cases, redundant cDNA probe sets were in the same cluster or in clusters with similar profiles. For example, eight redundant H2A histone family member Z gene elements were clustered in c5, and seven mouse heat shock protein 86 were clustered in c4 and c8 with a similar expression pattern. These results confirmed the fidelity of the microarray analysis used in this study. Furthermore, many functionally related genes were clustered together. For example, 48 gene elements related to energy and metabolism were clustered in c14 (26.8%, total 179 elements). There is a nearly 4-fold enrichment of these gene elements in this cluster because there are 258 gene elements related to energy and metabolism in the population (7.3%, total 3,532 elements). These findings indicate that the assay system is suitable for detecting genetic regulatory events during muscle regeneration, and we could use the hierarchical cluster analysis to identify novel genes with expression patterns similar to those well known functional genes. To identify novel genes related to myogenic differentiation, we focused our attention on genes with a peak expression pattern concurrent with muscle differentiation. For example, we noticed that three paternally imprinted genes, H19, p57Kip2, andRex3/Bex1, and four maternally imprinted genes,Igf2, Peg1/Mest,Peg3/Pw1, and Zac1, are clustered in c3 and/or c1. Because genes in these two clusters have peak induction of mRNA at day 3 or day 5, concurrent with morphological signs of myogenic differentiation, coordinated induction of these genes may play important functional roles in muscle differentiation. We also noticed similar induction of bone morphogenetic protein-6, growth arrest-specific 7 (Gas-7) and Gas
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