DEVELOPMENTAL DYNAMICS 19334-39 (1992)
MyoD and Myogenin Are Coexpressed in Regenerating Skeletal Muscle of the Mouse ERNST-MARTIN FUCHTBAUER AND HEINER WESTPHAL Laboratory of Mammalian Genes and Deuelopment. National Institute of Child Health and H u m a n Development, Building 6, Room 338, Bethesda, Maryland 20892
ABSTRACT The differential expression of genes triggering myogenesis might cause or reflect differences among myoblasts. Little is known about the presence of MyoDl and myogenin during the process of regeneration. We therefore examined the expression of MyoDl and myogenin in muscle regeneration after grafting. Immunostaining of regenerating skeletal muscle of the mouse revealed the presence of both MyoDl and myogenin. In mononucleated cells the proteins were not detected until shortly before fusion into myotubes. They persisted in the nuclei of regenerated muscle fibers for at least 2 weeks. MyoDl and myogenin were not detected in nonregenerating control muscle.
somites of the mouse embryo, myogenin is expressed as early as day 8.5 px. whereas MyoDl is not detected before day 10.5 p.c. In the developing limb bud, both genes are expressed simultaneously after the myogenic cells are already determined starting on day 11.5 p.c. (Sassoon et al., 1989). The myogenic cells in the limb bud originate in the somites. Therefore, it is possible that these cells had been positive for myogenin before they left the somites and migrated to the developing limb. It thus appears that during embryonic development MyoDl is not expressed earlier than myogenin. By contrast, MyoDl is expressed constitutively in many established myogenic cell lines (e.g., C2), whereas a trigger for differentiation is required for myogenin expression (for review see Olson, 1990). Even though proliferating myoblasts that ultimately Key words: MyoD1, Myogenin, Muscle regenera- differentiate and fuse into muscle fibers have many properties in common, there is growing evidence for a tion, Muscle graft, Gene expression diversity among different myogenic cells, such as fetal, embryonic, and satellite myoblasts (Stockdale et al., INTRODUCTION Development of skeletal muscle is a complex process 1989). Different types of myoblasts have been shown to requiring the positive and negative regulation of a respond differently to external signals that mediate great variety of genes. In recent years, a family of four proliferation and differentiation. They also differ in the closely related genes has been identified which have onset of muscle-specificgene expression (for review see such regulatory function (for review see Weintraub et Cossu and Mollinaro, 1987). Grafting experiments al., 1991).This gene family consists of MyoDl (Davis et have shown that, in addition to this diversification dural., 1987) myogenin (Edmondson and Olson, 1989), ing development, myoblasts may also differ in their Myfs (Braun et al., 19891, and herculinlMRF4 (Miner differentiation capacities (Hoh et al., 1989). Satellite and Wold, 1990). All four genes share sequence homol- cells are believed to be the only myogenic cells remainogy in several domains with the other members of the ing in adult skeletal muscle and t o be the sole source of gene family. This family belongs to an even larger myogenesis in regenerating adult muscle (Mauro, group of genes including the vertebrate c-myc and the 1961; Lipton and Schulz, 1979). Little is known about Drosophila achaete-scute. The homology domains in- the role of muscle inducing genes like MyoDl and myoclude an area rich in basic amino acids that is respon- genin during regeneration, and how the expression of sible for the DNA binding (Davis et al., 1990).Adjacent these genes in regenerating muscle compares to their to this basic region lies the helix-loop-helix motif, that expression in embryonic development and tissue culis essential for the heterodimenzation of these proteins. ture. This study examines the expression of MyoDl and This interaction with other helix-loop-helix proteins myogenin in regenerating skeletal muscle after auinfluences very strongly the DNA binding proteins of tografting. As muscle regeneration in whole grafts progresses from the outside toward the center, many MyoDl (Murre et al., 1989; Benezra et al., 1990). Upon transfection, MyoDl, myogenin, or one of the stages of regeneration are present simultaneously other members of the family induces the expression of muscle-specific genes in a variety of cells (Schafer et al., 1990; Choi et al., 1990). This functional similarity raises the question of why there are at least four indiReceived June 19, 1991; accepted August'l2, 1991. vidual genes. The differential expression of the myoAddress reprint requestskorrespondence to Ernst-Martin Fuchtgenic proteins MyoDl and myogenin suggests that bauer, Max-Planck-Institut fur Immunbiologie, Stubeweg 51, D-Weach has specific functions in muscle development. In 7800 Freiburg, Germany. 0 1992 WILEY-LISS,
INC.
MyoD A N D MYOGENIN I N REGENERATING MUSCLE
35
was so different, simultaneous staining was not feasible. In areas where myogenic cells began to fuse into new myotubes, only a minority of cells which appeared mononucleated stained positive for MyoDl and myogenin. In contrast, all nuclei in newly fused myotubes were positive for MyoD and myogenin (Fig. 3). This observations is consistent with the assumption that RESULTS both proteins are present simultaneously in the nuclei As the aim of this study was to localize MyoDl and of myogenic cells, but only very shortly before fusion of myogenin during the process of muscle regeneration, these cells. After longer periods of regeneration (e.g., 2 weeks) we first tested the specificity of our antibodies as follows. Omitting the primary antibodies eliminated all MyoDl and myogenin were still detected in the nuclei signal (Fig. 1C) showing that the fluorescein coupled of regenerated muscle fibers and more intensely in second antibody does not bind nonspecifically to the cells which appeared mononucleated (Fig. 4). As it is section. Anti-MyoD1 (Fig. 1A) and anti myogenin (not difficult to quantify the fluorescent signal and compare shown) sera did not stain the nuclei in normal nonre- the two antibodies used, we could not determine generated adult skeletal muscle but tend to adhere to whether the signal is diminishing a t this stage. The immunostaining results were supported by in the sarcoplasma of type IIA fibers. The absence of a MyoDl or myogenin specific signal may be explained situ hybridization with a MyoDl antisense probe (Fig. by the very low expression of these genes in adult mus- 5).In areas where myogenic cells began to fuse all myocle as seen by Northern blotting (Davis et al., 1987; tubes, but only a minority of cells which appeared Edmondson and Olson, 1989). This might either be be- mononucleated were labeled (Fig. 5A, compare to Fig. low the limit of detection or concentrated in very few 3). In areas of degeneration, where only few of these nuclei which might not be present in all sections. Fi- mononucleated cells were seen, MyoDl RNA could not nally, we used an antibody directed against proteogly- be detected (Fig. 5B). Hybridization with a MyoDl can (Fig. 1E) to prove that an immunostaining for an sense probe did not produce a signal (Fig. 5C). unrelated antigen produces a specific and distinct signal. In regenerating whole grafts many stages of regenDISCUSSION eration are present simultaneously. We will therefore The persistence of MyoDl and myogenin proteins in refer to the stage of regeneration rather than the period postoperation. For the actual regeneration time of regenerating muscle fibers was surprising considering the examples shown, see figure legends. As all studies the in situ hybridization results of Grounds et al. were done on serial sections, there was no problem in (1991). These experiments show that, in crush induced verifying the multinuclear nature of newly fused myo- regeneration, the RNAs of MyoD and myogenin are tubes. In contrast to that, we classified cells as mono- present in mononucleated cells a few hours after innucleated if they appeared only on one or two sections. jury, but are undetectable in newly formed myotubes. Furthermore, the nuclei of so called mononucleated This result could indicate that the MyoDl and myogecells were surrounded by only a small line of cytoplasm nin protein outlives its RNA by more than a week. compared to myotubes. However, it is important to More likely, the fusion of myotubes with the end of keep in mind that without electron microscopy, we can- innervated myofibers, which occur in crush injured not be certain that these cells are in fact mononucle- muscle but not in muscle grafts, might account for the ated. As many kinds of mononucleated cells are present rapid down-regulation of MyoDl and myogenin. This in a regenerating muscle graft, only a fraction of these explanation is not only supported by our in situ hybridization results, but also by the observation that MyoDl will be myogenic. In areas of early muscle fiber degeneration where and myogenin are down-regulated by electrical activity only very few cells which appeared mononucleated (Eftimie et al., 1991).The early detection of MyoDl and were seen, neither MyoDl nor myogenin could be de- myogenin RNA might be explained by the faster onset of cell proliferation in crush induced injury compared tected (not shown). In areas of predominantly degenerating muscle fi- to that of whole muscle grafts as shown by L3H1thybers with many cells which appeared mononucleated, midine uptake. In addition, it should be noted that at only a fraction (an estimated 10-20%) of the nuclei the site of the injury which best resembles the situation were positive for MyoDl or myogenin (Fig. 2). Since in a whole graft, expression of MyoDl and myogenin both antibodies were produced in rabbit, we could not was significantly less compared to more distant regions use double staining to determine conclusively whether in the muscle (Grounds et al., 1991). MyoDl and myogenin are found only in very few the same cells were positive for MyoDl and myogenin or whether these represented independent populations cells which appeared mononucleated, but in all nuclei of cells. Since the staining intensity of the two antisera of regenerated fibers. This suggests that in muscle re-
(Carlson, 1976). Biochemical studies performed with RNA or protein extracted from total tissue cannot distinguish these stages. Therefore we decided to use immunof luorescence and in situ hybridization to study the presence of MyoDl and myogenin in different stages of muscle regeneration.
Fig. 1. (A) Anti-MyoD1 staining of control muscle. The background signal is due to unspecific binding to the sarcoplasma of type IIA fibers. (C) Mononucleated cells and newly fused myotubes 6 days after grafting. The first antibody was omitted. Note the absence of specific staining of the fiber nuclei (open arrowhead) as well as the orange autofluorescence that is seen frequently in regenerating muscle. (E) Regenerated muscle
fibers 2 weeks after grafting, stained with antiproteoglycan-specific antibodies. Note the absence of signal over nuclei (open arrowhead). This is a section adjacent to those of Figure 4. (B), (D), and (F) Hematoxylin and eosin stain of the same sections as (A), (C), and (E),respectively. Bar =50 pm.
Fig. 2. Degenerating muscle fibers 6 days after grafting. Only a few nuclei are positive for MyoDl (A) and myogenin (C) (arrows), although many mononucleated cells are visible after staining with hematoxylin and eosin (6and D) that are negative in the immunostain (open arrows). Bar = 50 p.m.
Fig. 3. Newly fused myotubes 4 days after grafting. All myotube nuclei are positive for MyoDl (A) and myogenin (C) (arrowheads), but only a few mononucleated cells are stained (arrows). Open arrows indicate examples of unstained nuclei. (B) and (D) Hematoxylin and eosin stain of the same section as (A) and (C). Bar = 50 bm.
Fig. 4. Regenerated muscle fibers 2 weeks after grafting. All fiber nuclei are positive for MyoDl (A) and myogenin (C) (arrowheads). Some mononucleated cells are positive (arrows), whereas others are negative (open arrows). (B) and (D) Hematoxylin and eosin stain of the same section as (A) and (C). Bar=50 bm.
38
FUCHTBAUER AND WESTPHAL
Fig. 5. Regenerating muscle 5 days after grafting hybridized with MyoDl antisense (A and 6 ) and sense RNA (C), stained with hematoxylin and eosin. (A) Newly fused myotubes (arrowhead) and some mononucleated cells (arrow) are labeled, whereas many mononucleated cells show no signal (open arrow). (B) No label was found in areas of degen-
erating fibers with very few mononucleated cells (open arrow). (C) Hybridization with MyOD7 sense RNA probe did not produce a signal in myotubes (open arrowhead) or mononucleated cells (open arrow). Bar = 50 pm.
generation from satellite cells, MyoDl and myogenin play a n active role in the terminal differentiation of muscle fibers. It should be mentioned, however, that the presence of MyoDl and myogenin does not prove that these proteins are in their active form as heterodimers with a protein like E l 2 (Davis et al., 1990). The expression of MyoDl and myogenin in regenerating muscle resembles the expression in the developing limb bud of the mouse embryo. In both cases the genes are expressed simultaneously and relatively late in the process of myogenesis, i.e., after the cells are obviously determined to be muscle precursor cells (Sassoon et al., 1989). It is possible that one or both genes are expressed earlier in these cells, e.g., in satellite cells before they became quiescent. As nothing is known about the expression of the other two myogenic helix-loop-helix genes Myfs (Braun et al., 1989) and herculinlMRF4 (Miner and Wold, 1990; Rhodes and Konieczny, 1989) we cannot exclude the possibility that one of these is responsible for the determination of the myogenic cells in embryonic limb bud and in adult skeletal muscle. The result of Yutzey et al. (1990) that herculinlMRF4 does not transactivate muscle-specific genes even though it efficiently converts tissue culture fibroblasts into muscle cells could, in fact, be a n indication that this is a n earlier expressed gene. These considerations raise the question of whether the observed differences among the different myogenic cells (Cossu and Mollinaro, 1987) might be created by differential expression of muscle differentiation genes.
C57BL6 mice were removed. The muscles were soaked for 1-2 min in 0.5% marcain HC1 (Winthrop-Breon Lab., New York) to induce degeneration of surface f i bers which would otherwise have survived (Carlson 1976). Subsequently the muscles were orthotopically regrafted a s described (Fuchtbauer et al., 1988). After 2 , 4 , 6 , or 15 days of regeneration, mice were sacrificed by cervical dislocation. The grafted muscles were snap frozen in melting 2-methyl butane. For in situ hybridization, muscles were removed 5 days after grafting and fixed in 4% paraformaldehyde in Ca2+ and Mg2+free phosphate-buffered saline (CMF-PBS).
EXPERIMENTAL PROCEDURES Surgery In order to induce muscle regeneration, the left tibialis anterior muscles of 8- to 12-week-old female
Immunohistology Frozen sections (8 pm) were air dried for 1h r a t room temperature and fixed for 15 min in 4% formaldehyde in CMF-PBS. Sections were permeabilized by incubating for 10 min in 0.25% Triton X-100 in CMF-PBS. First antibodies were diluted in 1% normal goat serum (Sigma) 0.1% Triton X-100 in CMF-PBS. After incubating for 1h r a t room temperature the sections were washed 3 times for 10 min each in CMF-PBS and incubated for 1 h r with FITC-labeled goat anti-rabbit antibodies at room temperature. After washing 3 times for 10 min they were embedded in Elvanol and covered with a coverslip. After taking photomicrographs of the immunostained sections, the coverslips were removed in CMF-PBS a t 40°C and the Elvanol was washed off. Subsequently the sections were stained with hematoxylin and eosin. All micrographs were taken with a Zeiss Axiophot on Kodak Ektachrome 400 pushed to 800 Asa and Kodak Ektachrome 160 tungsten for fluorescence and brightfield, respectively. Primary magnification was 63 x .
MyoD AND MYOGENIN IN REGENERATING MUSCLE
Antibodies Anti-MyoD1 antibodies (kindly provided by A. Lassar and S. Tapscott) were raised against a fusion protein consisting of E . coli trp E protein and the entire MyoD sequence except for the first three amino acids (for characterization and description of the preparation see Tapscott e t al., 1988). The serum was diluted 1:200. Antimyogenin antibodies (kindly provided by E. Olson) were raised against synthetic peptides and affinity purified 1:25. Antiproteoglycan antibodies (kindly provided by J. Hassell) were raised in rabbits a s described (Hassel et al., 1980). Secondary antibody was fluorescein-coupled goat anti-rabbit IgG (Cappel #12120231).
In Situ Hybridization MyoDl antisense and sense RNA probes were transcribed from a full length cDNA (pV2CIIa kindly provided by A. Lassar) with T3 and T7 RNA polymerase, respectively, and labeled with [35S]UTP. The probes were alkali-hydrolyzed to a n approximate size of 150 nucleotides and used with a specific activity of 7 x lo4 cpmlpl. In situ hybridization was performed according to Nakamura et al. (1989) with the following modifications. After deparafinization and rehydration sections were postfixed in 3.7% formaldehyde in CMF-PBS for 5 min and proteinase K digestion was done with 20 pgl ml for 10 min at room temperature. The stringent wash was done at 65°C in 0.1 x SSC. Slides were exposed for 3 weeks. ACKNOWLEDGMENTS We thank Dr. M. Grounds for unpublished data, Dr. K. Mahon and Dr. R. Wade for discussion and suggestions, and K. L. Rubin and I. Kronbach for typing the manuscript. Ernst-Martin Fuchtbauer was supported by a Feodor-Lynen-Fellowship of the Alexander v. Humboldt Foundation, Bonn, FRG. REFERENCES Benezra R., Davis R.L., Lockshon D., Turner D.L., and Weintraub H. (1990)The Protein Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49-59. Braun T., Buschhausen-Denker G., Bober E., Tannich E., and Arnold H.H. (1989) A novel human muscle factor related to but distinct from MyoDl induces myogenic conversion in 10T1/2 fibroblasts. EMBO J . 8:701-709. Brennan T.J., and Olson E.N. (1990) Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimenzation. Genes Dev. 4:582-595. Carlson B.M. (1976)A quantitative study of muscle fiber survival and regeneration in normal, predenervated, and Marcaine-treated free muscle grafts in the rat. Exp. Neurol. 52:421-432. Choi J., Costa M.L., Mermelstein, C.S., Chagas C., Holtzer S., and Holtzer H. (1990) MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proc. Natl. Acad. Sci. U.S.A. 87:7988-7992. Cossu G., and Molinaro M. (1987) Cell heterogeneity in the myogenic lineage. Curr.Top. Dev. Biol. 23:185-208. Davis R.L., Weintraub H., and Lassar A.B. (1987) Expression of a
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single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000. Davis R.L., Cheng P.-G., Lassar A.B., and Weintraub H. (1990) The MyoD DNA binding domain contains a recognition code for musclespecific gene activation. Cell 60:733-746. Edmondson D.G., and Olson E.N. (1989) A gene with homology to the myc similarity of MyoDl is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev. 3:628-640. Eftimie R., Brenner H.R., and Buonanno A. (1991) Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity. Proc. Natl. Acad. Sci U.S.A. 88:1349-1353. Fiichtbauer E.-M., Reininghaus J., and Jockusch H. (1988) Developmental control of the excitability of muscle: Transplantation experiments on a myotonic mouse mutant. Proc. Natl. Acad. Sci U.S.A. 85:3880-3884. Grounds, M.D., Garrett K.L., Lai M.C., Wright W.E., and Beilharz M.W. (1991) Identification of skeletal muscle precursor cells in vivo using MyoDl and myogenin probes. Cell Tissue Res., in press. Hassell J.R., Gehron-Robey P., Barrach H.J., Wilczek J., Rennard S.I., and Martin G.R. (1980) Isolation of a heparin sulfate-containing proteoglycan from basement membrane. Proc. Natl. Acad. Sci U.S.A. 77:4494-4498. Hoh J.F.Y., Hughes S., Hugh G., and Pozgaj I. (1989) Three hierarchies in skeletal muscle fiber classification allotype, isotype and phenotype. In: “Cellular and Molecular Biology of Muscle Development,” Kedes LH, and Stockdale FE (eds). New York Alan R. Liss, pp 15-26. Lipton B.H., and Schultz B. (1979) Developmental fate of skeletal muscle satellite cells. Science 205:1292-1294. Mauro A. (1961) Satellite cells of skeletal muscle fibers. J . Biophys. Biochem. Cytol. 9:493-495. Miner J.H., and Wold B. (1990) Herculin, a fourth member of the MyoD family of myogenic regulatory genes. Proc. Natl. Acad. Sci U.S.A. 87:1089-1093. Murre C., McCaw P.S., Vaessin H., Caudy M., J a n L.Y., J a n Y.N., Cabrera C.V., Buskin J.N., Hauschka S.D., Lassar A.B., Weintraub H., and Baltimore D. (1989) Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58:537-544. Nakamura T., Mahon K.A., Miskin R., Dey A,, Kuwabara T., and Westphal H. (1989) Differentiation and oncogenesis: Phenotypically distinct lens tumors in transgenic mice. New Biol. 1:193-204. Olson E.N. (1990) MyoD family: A paradigm for development? Genes Dev. 4:1454-1461. Rhodes S.I., and Konieczny S.F. (1989) Identification of MRF4: A new member of the muscle regulatory factor gene family. Genes Dev. 3:2050-2061. Sassoon D., Lyons G., Wright W.E., Lin V., Lassar A,, Weintraub H., and Buckingham M. (1989) Expression of two myogenic regulatory facors myogenin and MyoDl during mouse embryogenesis. Nature (London) 341:303-307. Schafer B.W., Blakely B.T., Darlington G.J., and Blau H.M. (1990) Effect of cell history on response to helix-loop-helix family of myogenic regulators. Nature (London) 344:454-458. Stockdale F.E., Miller J.B., Feldman J.L., Lamson G., and Hager J . (1989) Myogenic cell lineages: Commitment and modulation during differentiation of avian muscle. In: “Cellular and Molecular Biology of Muscle Development,” Kedes L.H., and Stockdale F.E. (eds.) New York: Alan R. Liss, pp 3-13. Tapscott S.J., Davis R.L., Thayer M.J., Cheng P-F, Weintraub H., and Lassar A.B. (1988) MyoD1: A nuclear phosphoprotein requiring a myc homology region to convert fibroblasts to myoblasts. Science 242:405-411. Weintraub H., Davis R., Tapscott S., Thayer M., Krause M., Benezra R., Blackwell T.K., Turner D., Rupp R., Hollenberg S., Zhuang Y., and Lassar A.B. (1991) The MyoD gene family: Nodal point during specification of the muscle cell lineage. Science 251361-766. Yutzey K.E., Rhodes S.J., and Konieczny S.F. (1990) Differential trans activation associated with the muscle regulatory factors MyoD1, myogenin, and MRF4, Mol. Cell. Biol. 10:3934-3944.
MyoD and Myogenin Are Coexpressed in Regenerating Skeletal Muscle of the Mouse ERNST-MARTIN FUCHTBAUER AND HEINER WESTPHAL Laboratory of Mammalian Genes and Deuelopment. National Institute of Child Health and H u m a n Development, Building 6, Room 338, Bethesda, Maryland 20892
ABSTRACT The differential expression of genes triggering myogenesis might cause or reflect differences among myoblasts. Little is known about the presence of MyoDl and myogenin during the process of regeneration. We therefore examined the expression of MyoDl and myogenin in muscle regeneration after grafting. Immunostaining of regenerating skeletal muscle of the mouse revealed the presence of both MyoDl and myogenin. In mononucleated cells the proteins were not detected until shortly before fusion into myotubes. They persisted in the nuclei of regenerated muscle fibers for at least 2 weeks. MyoDl and myogenin were not detected in nonregenerating control muscle.
somites of the mouse embryo, myogenin is expressed as early as day 8.5 px. whereas MyoDl is not detected before day 10.5 p.c. In the developing limb bud, both genes are expressed simultaneously after the myogenic cells are already determined starting on day 11.5 p.c. (Sassoon et al., 1989). The myogenic cells in the limb bud originate in the somites. Therefore, it is possible that these cells had been positive for myogenin before they left the somites and migrated to the developing limb. It thus appears that during embryonic development MyoDl is not expressed earlier than myogenin. By contrast, MyoDl is expressed constitutively in many established myogenic cell lines (e.g., C2), whereas a trigger for differentiation is required for myogenin expression (for review see Olson, 1990). Even though proliferating myoblasts that ultimately Key words: MyoD1, Myogenin, Muscle regenera- differentiate and fuse into muscle fibers have many properties in common, there is growing evidence for a tion, Muscle graft, Gene expression diversity among different myogenic cells, such as fetal, embryonic, and satellite myoblasts (Stockdale et al., INTRODUCTION Development of skeletal muscle is a complex process 1989). Different types of myoblasts have been shown to requiring the positive and negative regulation of a respond differently to external signals that mediate great variety of genes. In recent years, a family of four proliferation and differentiation. They also differ in the closely related genes has been identified which have onset of muscle-specificgene expression (for review see such regulatory function (for review see Weintraub et Cossu and Mollinaro, 1987). Grafting experiments al., 1991).This gene family consists of MyoDl (Davis et have shown that, in addition to this diversification dural., 1987) myogenin (Edmondson and Olson, 1989), ing development, myoblasts may also differ in their Myfs (Braun et al., 19891, and herculinlMRF4 (Miner differentiation capacities (Hoh et al., 1989). Satellite and Wold, 1990). All four genes share sequence homol- cells are believed to be the only myogenic cells remainogy in several domains with the other members of the ing in adult skeletal muscle and t o be the sole source of gene family. This family belongs to an even larger myogenesis in regenerating adult muscle (Mauro, group of genes including the vertebrate c-myc and the 1961; Lipton and Schulz, 1979). Little is known about Drosophila achaete-scute. The homology domains in- the role of muscle inducing genes like MyoDl and myoclude an area rich in basic amino acids that is respon- genin during regeneration, and how the expression of sible for the DNA binding (Davis et al., 1990).Adjacent these genes in regenerating muscle compares to their to this basic region lies the helix-loop-helix motif, that expression in embryonic development and tissue culis essential for the heterodimenzation of these proteins. ture. This study examines the expression of MyoDl and This interaction with other helix-loop-helix proteins myogenin in regenerating skeletal muscle after auinfluences very strongly the DNA binding proteins of tografting. As muscle regeneration in whole grafts progresses from the outside toward the center, many MyoDl (Murre et al., 1989; Benezra et al., 1990). Upon transfection, MyoDl, myogenin, or one of the stages of regeneration are present simultaneously other members of the family induces the expression of muscle-specific genes in a variety of cells (Schafer et al., 1990; Choi et al., 1990). This functional similarity raises the question of why there are at least four indiReceived June 19, 1991; accepted August'l2, 1991. vidual genes. The differential expression of the myoAddress reprint requestskorrespondence to Ernst-Martin Fuchtgenic proteins MyoDl and myogenin suggests that bauer, Max-Planck-Institut fur Immunbiologie, Stubeweg 51, D-Weach has specific functions in muscle development. In 7800 Freiburg, Germany. 0 1992 WILEY-LISS,
INC.
MyoD A N D MYOGENIN I N REGENERATING MUSCLE
35
was so different, simultaneous staining was not feasible. In areas where myogenic cells began to fuse into new myotubes, only a minority of cells which appeared mononucleated stained positive for MyoDl and myogenin. In contrast, all nuclei in newly fused myotubes were positive for MyoD and myogenin (Fig. 3). This observations is consistent with the assumption that RESULTS both proteins are present simultaneously in the nuclei As the aim of this study was to localize MyoDl and of myogenic cells, but only very shortly before fusion of myogenin during the process of muscle regeneration, these cells. After longer periods of regeneration (e.g., 2 weeks) we first tested the specificity of our antibodies as follows. Omitting the primary antibodies eliminated all MyoDl and myogenin were still detected in the nuclei signal (Fig. 1C) showing that the fluorescein coupled of regenerated muscle fibers and more intensely in second antibody does not bind nonspecifically to the cells which appeared mononucleated (Fig. 4). As it is section. Anti-MyoD1 (Fig. 1A) and anti myogenin (not difficult to quantify the fluorescent signal and compare shown) sera did not stain the nuclei in normal nonre- the two antibodies used, we could not determine generated adult skeletal muscle but tend to adhere to whether the signal is diminishing a t this stage. The immunostaining results were supported by in the sarcoplasma of type IIA fibers. The absence of a MyoDl or myogenin specific signal may be explained situ hybridization with a MyoDl antisense probe (Fig. by the very low expression of these genes in adult mus- 5).In areas where myogenic cells began to fuse all myocle as seen by Northern blotting (Davis et al., 1987; tubes, but only a minority of cells which appeared Edmondson and Olson, 1989). This might either be be- mononucleated were labeled (Fig. 5A, compare to Fig. low the limit of detection or concentrated in very few 3). In areas of degeneration, where only few of these nuclei which might not be present in all sections. Fi- mononucleated cells were seen, MyoDl RNA could not nally, we used an antibody directed against proteogly- be detected (Fig. 5B). Hybridization with a MyoDl can (Fig. 1E) to prove that an immunostaining for an sense probe did not produce a signal (Fig. 5C). unrelated antigen produces a specific and distinct signal. In regenerating whole grafts many stages of regenDISCUSSION eration are present simultaneously. We will therefore The persistence of MyoDl and myogenin proteins in refer to the stage of regeneration rather than the period postoperation. For the actual regeneration time of regenerating muscle fibers was surprising considering the examples shown, see figure legends. As all studies the in situ hybridization results of Grounds et al. were done on serial sections, there was no problem in (1991). These experiments show that, in crush induced verifying the multinuclear nature of newly fused myo- regeneration, the RNAs of MyoD and myogenin are tubes. In contrast to that, we classified cells as mono- present in mononucleated cells a few hours after innucleated if they appeared only on one or two sections. jury, but are undetectable in newly formed myotubes. Furthermore, the nuclei of so called mononucleated This result could indicate that the MyoDl and myogecells were surrounded by only a small line of cytoplasm nin protein outlives its RNA by more than a week. compared to myotubes. However, it is important to More likely, the fusion of myotubes with the end of keep in mind that without electron microscopy, we can- innervated myofibers, which occur in crush injured not be certain that these cells are in fact mononucle- muscle but not in muscle grafts, might account for the ated. As many kinds of mononucleated cells are present rapid down-regulation of MyoDl and myogenin. This in a regenerating muscle graft, only a fraction of these explanation is not only supported by our in situ hybridization results, but also by the observation that MyoDl will be myogenic. In areas of early muscle fiber degeneration where and myogenin are down-regulated by electrical activity only very few cells which appeared mononucleated (Eftimie et al., 1991).The early detection of MyoDl and were seen, neither MyoDl nor myogenin could be de- myogenin RNA might be explained by the faster onset of cell proliferation in crush induced injury compared tected (not shown). In areas of predominantly degenerating muscle fi- to that of whole muscle grafts as shown by L3H1thybers with many cells which appeared mononucleated, midine uptake. In addition, it should be noted that at only a fraction (an estimated 10-20%) of the nuclei the site of the injury which best resembles the situation were positive for MyoDl or myogenin (Fig. 2). Since in a whole graft, expression of MyoDl and myogenin both antibodies were produced in rabbit, we could not was significantly less compared to more distant regions use double staining to determine conclusively whether in the muscle (Grounds et al., 1991). MyoDl and myogenin are found only in very few the same cells were positive for MyoDl and myogenin or whether these represented independent populations cells which appeared mononucleated, but in all nuclei of cells. Since the staining intensity of the two antisera of regenerated fibers. This suggests that in muscle re-
(Carlson, 1976). Biochemical studies performed with RNA or protein extracted from total tissue cannot distinguish these stages. Therefore we decided to use immunof luorescence and in situ hybridization to study the presence of MyoDl and myogenin in different stages of muscle regeneration.
Fig. 1. (A) Anti-MyoD1 staining of control muscle. The background signal is due to unspecific binding to the sarcoplasma of type IIA fibers. (C) Mononucleated cells and newly fused myotubes 6 days after grafting. The first antibody was omitted. Note the absence of specific staining of the fiber nuclei (open arrowhead) as well as the orange autofluorescence that is seen frequently in regenerating muscle. (E) Regenerated muscle
fibers 2 weeks after grafting, stained with antiproteoglycan-specific antibodies. Note the absence of signal over nuclei (open arrowhead). This is a section adjacent to those of Figure 4. (B), (D), and (F) Hematoxylin and eosin stain of the same sections as (A), (C), and (E),respectively. Bar =50 pm.
Fig. 2. Degenerating muscle fibers 6 days after grafting. Only a few nuclei are positive for MyoDl (A) and myogenin (C) (arrows), although many mononucleated cells are visible after staining with hematoxylin and eosin (6and D) that are negative in the immunostain (open arrows). Bar = 50 p.m.
Fig. 3. Newly fused myotubes 4 days after grafting. All myotube nuclei are positive for MyoDl (A) and myogenin (C) (arrowheads), but only a few mononucleated cells are stained (arrows). Open arrows indicate examples of unstained nuclei. (B) and (D) Hematoxylin and eosin stain of the same section as (A) and (C). Bar = 50 bm.
Fig. 4. Regenerated muscle fibers 2 weeks after grafting. All fiber nuclei are positive for MyoDl (A) and myogenin (C) (arrowheads). Some mononucleated cells are positive (arrows), whereas others are negative (open arrows). (B) and (D) Hematoxylin and eosin stain of the same section as (A) and (C). Bar=50 bm.
38
FUCHTBAUER AND WESTPHAL
Fig. 5. Regenerating muscle 5 days after grafting hybridized with MyoDl antisense (A and 6 ) and sense RNA (C), stained with hematoxylin and eosin. (A) Newly fused myotubes (arrowhead) and some mononucleated cells (arrow) are labeled, whereas many mononucleated cells show no signal (open arrow). (B) No label was found in areas of degen-
erating fibers with very few mononucleated cells (open arrow). (C) Hybridization with MyOD7 sense RNA probe did not produce a signal in myotubes (open arrowhead) or mononucleated cells (open arrow). Bar = 50 pm.
generation from satellite cells, MyoDl and myogenin play a n active role in the terminal differentiation of muscle fibers. It should be mentioned, however, that the presence of MyoDl and myogenin does not prove that these proteins are in their active form as heterodimers with a protein like E l 2 (Davis et al., 1990). The expression of MyoDl and myogenin in regenerating muscle resembles the expression in the developing limb bud of the mouse embryo. In both cases the genes are expressed simultaneously and relatively late in the process of myogenesis, i.e., after the cells are obviously determined to be muscle precursor cells (Sassoon et al., 1989). It is possible that one or both genes are expressed earlier in these cells, e.g., in satellite cells before they became quiescent. As nothing is known about the expression of the other two myogenic helix-loop-helix genes Myfs (Braun et al., 1989) and herculinlMRF4 (Miner and Wold, 1990; Rhodes and Konieczny, 1989) we cannot exclude the possibility that one of these is responsible for the determination of the myogenic cells in embryonic limb bud and in adult skeletal muscle. The result of Yutzey et al. (1990) that herculinlMRF4 does not transactivate muscle-specific genes even though it efficiently converts tissue culture fibroblasts into muscle cells could, in fact, be a n indication that this is a n earlier expressed gene. These considerations raise the question of whether the observed differences among the different myogenic cells (Cossu and Mollinaro, 1987) might be created by differential expression of muscle differentiation genes.
C57BL6 mice were removed. The muscles were soaked for 1-2 min in 0.5% marcain HC1 (Winthrop-Breon Lab., New York) to induce degeneration of surface f i bers which would otherwise have survived (Carlson 1976). Subsequently the muscles were orthotopically regrafted a s described (Fuchtbauer et al., 1988). After 2 , 4 , 6 , or 15 days of regeneration, mice were sacrificed by cervical dislocation. The grafted muscles were snap frozen in melting 2-methyl butane. For in situ hybridization, muscles were removed 5 days after grafting and fixed in 4% paraformaldehyde in Ca2+ and Mg2+free phosphate-buffered saline (CMF-PBS).
EXPERIMENTAL PROCEDURES Surgery In order to induce muscle regeneration, the left tibialis anterior muscles of 8- to 12-week-old female
Immunohistology Frozen sections (8 pm) were air dried for 1h r a t room temperature and fixed for 15 min in 4% formaldehyde in CMF-PBS. Sections were permeabilized by incubating for 10 min in 0.25% Triton X-100 in CMF-PBS. First antibodies were diluted in 1% normal goat serum (Sigma) 0.1% Triton X-100 in CMF-PBS. After incubating for 1h r a t room temperature the sections were washed 3 times for 10 min each in CMF-PBS and incubated for 1 h r with FITC-labeled goat anti-rabbit antibodies at room temperature. After washing 3 times for 10 min they were embedded in Elvanol and covered with a coverslip. After taking photomicrographs of the immunostained sections, the coverslips were removed in CMF-PBS a t 40°C and the Elvanol was washed off. Subsequently the sections were stained with hematoxylin and eosin. All micrographs were taken with a Zeiss Axiophot on Kodak Ektachrome 400 pushed to 800 Asa and Kodak Ektachrome 160 tungsten for fluorescence and brightfield, respectively. Primary magnification was 63 x .
MyoD AND MYOGENIN IN REGENERATING MUSCLE
Antibodies Anti-MyoD1 antibodies (kindly provided by A. Lassar and S. Tapscott) were raised against a fusion protein consisting of E . coli trp E protein and the entire MyoD sequence except for the first three amino acids (for characterization and description of the preparation see Tapscott e t al., 1988). The serum was diluted 1:200. Antimyogenin antibodies (kindly provided by E. Olson) were raised against synthetic peptides and affinity purified 1:25. Antiproteoglycan antibodies (kindly provided by J. Hassell) were raised in rabbits a s described (Hassel et al., 1980). Secondary antibody was fluorescein-coupled goat anti-rabbit IgG (Cappel #12120231).
In Situ Hybridization MyoDl antisense and sense RNA probes were transcribed from a full length cDNA (pV2CIIa kindly provided by A. Lassar) with T3 and T7 RNA polymerase, respectively, and labeled with [35S]UTP. The probes were alkali-hydrolyzed to a n approximate size of 150 nucleotides and used with a specific activity of 7 x lo4 cpmlpl. In situ hybridization was performed according to Nakamura et al. (1989) with the following modifications. After deparafinization and rehydration sections were postfixed in 3.7% formaldehyde in CMF-PBS for 5 min and proteinase K digestion was done with 20 pgl ml for 10 min at room temperature. The stringent wash was done at 65°C in 0.1 x SSC. Slides were exposed for 3 weeks. ACKNOWLEDGMENTS We thank Dr. M. Grounds for unpublished data, Dr. K. Mahon and Dr. R. Wade for discussion and suggestions, and K. L. Rubin and I. Kronbach for typing the manuscript. Ernst-Martin Fuchtbauer was supported by a Feodor-Lynen-Fellowship of the Alexander v. Humboldt Foundation, Bonn, FRG. REFERENCES Benezra R., Davis R.L., Lockshon D., Turner D.L., and Weintraub H. (1990)The Protein Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49-59. Braun T., Buschhausen-Denker G., Bober E., Tannich E., and Arnold H.H. (1989) A novel human muscle factor related to but distinct from MyoDl induces myogenic conversion in 10T1/2 fibroblasts. EMBO J . 8:701-709. Brennan T.J., and Olson E.N. (1990) Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimenzation. Genes Dev. 4:582-595. Carlson B.M. (1976)A quantitative study of muscle fiber survival and regeneration in normal, predenervated, and Marcaine-treated free muscle grafts in the rat. Exp. Neurol. 52:421-432. Choi J., Costa M.L., Mermelstein, C.S., Chagas C., Holtzer S., and Holtzer H. (1990) MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proc. Natl. Acad. Sci. U.S.A. 87:7988-7992. Cossu G., and Molinaro M. (1987) Cell heterogeneity in the myogenic lineage. Curr.Top. Dev. Biol. 23:185-208. Davis R.L., Weintraub H., and Lassar A.B. (1987) Expression of a
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