DEVELOPMENTAL
BIOLOGY
Myoblast
49,
548-555 (1976)
Differentiation in Vitro: Morphological Mononucleated Myoblasts JOHN
Department
ofBiological
Structure,
A. TROTTER School
AND MARK
of Medicine,
Accepted
University
December
Differentiation
of
NAMEROFF of Washington,
Seattle,
Washington
98195
1, 1975
Phospholipase C from Clostridium perfringens has been shown previously to inhibit the fusion of cultured chick myoblasts without affecting recognition or cell cycle parameters. In this paper we report that the mononucleated myoblasts, in phospholipase C, synthesize thick and thin filaments and organize them into myofibrils, and that T-tubules and sarcoplasmic reticulum differentiate and join in morphologically typical junctions. The structurally differentiated mvoblasts can then fuse with one another to form myotubes. We conclude that cell fusion is not necessary for muscle differentiation. INTRODUCTION
When maintained in an appropriate culture medium, single cells isolated either mechanically or enzymatically from embryonic muscle of chick, as well as from other sources, attach to the substratum, migrate, divide, line up into strings of adherent cells, and fuse to form syncytia. Concomitant with fusion there is a rapid rise in the activities of several enzymes characteristic of muscle cells (2, 5, 18, 21, 32, 33) and in the rate of myosin synthesis (6, 27). Evidence has been put forward to support the notion that fusion per se is the “trigger” that turns on the synthesis of these muscle-specific proteins (2, 21, 27, 29, 30). The evidence has been that there was little or no increase in muscle-specific molecules when fusion was blocked by some alteration of the culture medium. Upon restoration of fusion-permissive conditions, both fusion and muscle-specific protein synthesis began at about the same time. Recently, however, evidence has been published to support the contrary notion that fusion and the other aspects of terminal muscle differentiation need have no causal relation to one another. Thus, it has been shown that creatine phosphokinase activity increases at the same rate as in fusing controls in cultures of myoblasts
kept from fusing by ethylene glycol (aminoethylether), tetraacetic acid (EGTA), or phospholipase C (PLC; 18), and at a lower rate in cultures treated with bromodeoxyuridine (BUdR; 14, 18). Creatine phosphokinase and phosphorylase also have been found to increase in cultures of a myoblast cell line in which fusion was inhibited by the presence of the drug cytochalasin B (3). In addition, myosin synthesis has been reported to occur at the same rate as in myotubes in mononucleated myoblasts cultured in EGTA (6). Skeletal muscle myosin also has been detected immunologically in mononucleated myoblasts in EGTA and cytochalasin B (4, 15), and thick and thin filaments have been seen with the electron microscope in mononucleated myoblasts cultured in the presence of low calcium (9), EGTA (6), and cytochalasin B (15-17). Thus, the literature is divided on the issue of the relation of cell fusion to cytoplasmic differentiation. We have explored the relation of cell fusion to other aspects of the differentiative program of myogenic cells using PLC, which reversibly blocks the fusion step without interfering with recognition (24) or cell cycle parameters (23). We now report that the mononucleated postmitotic myoblasts, which arise in medium containing PLC, synthesize myofilaments and 548
Copyright 0 1976 by Academic press, Inc. All rights of reproduction in any form reserved.
BRIEF NOTES
organize them into myofibrils and that the sarcoplasmic reticulum and T-tubules also differentiate and form typical junctions in these mononucleated cells. Such differentiated cells fuse with one another when they are cultured in fresh medium without PLC. We regard these observations as evidence that cell fusion is not causally related to the other major steps of terminal skeletal muscle differentiation. MATERIALS
AND
METHODS
Minced breast muscles from 11-day embryonic chicks were exposed to 0.25% trypsin for 30 min at 37”C, mechanically dispersed in complete culture medium, and filtered through two layers of lens tissue. Cultures were established on gelatincoated 35mm plastic dishes by plating 0.5 x 10” cells in 1.5 ml of medium (Eagle’s MEM, 82.5%; horse serum, 10.3%; embryo extract, 5.2%; penicillin-streptomycin solution, 1.0%; Fungizone, 1.0%; referred to as medium 1621). The culture environment was a water-saturated atmosphere of 5% CO, in air at 37.5”C. The medium was changed on the first day after plating and on every second day thereafter. Phospholipase C (phosphatidylcholine: choline phosphohydrolase, E.C.3.1.4.3, referred to as PLC) from Clostridium perfringens was obtained from Worthington Biochemical Corp., Freehold, N. J., and was purified by chromatography on Sephadex G-100 (Pharmacia, Piscataway, N.J.) as described previously (23). The purified enzyme was used in medium 1621 at concentrations of 0.4-1.0 pg/ml. n-arabinofuranosylcytosine (Ara C) was obtained from Calbiochem, La Jolla, Calif., and was used at a concentration of 10 pglml in medium 1621 (3.6 x lo-: M). To determine the effect, if any, of PLC on the ultrastructure of already fused myotubes, PLC was added on Day 3, when the cultures were mostly fused, at up to 4x the dose required to inhibit fusion. Such cultures were maintained in PLC-medium on a normal feeding schedule up to Day 7,
549
when they and controls without PLC were fixed for electron microscopy. Morphological differentiation of unfused cells was determined by fixing 7-day cultures in which fusion was prevented by the presence of PLC on Days l-7. PLC does not appear to affect the doubling time of cycling cells in these cultures (231, while the myoblasts withdraw from the cell cycle at the time at which fusion would normally occur (24). The 7-day cultures in PLC from Days l-7 are, therefore, overgrown with cycling, presumably largely nonmyogenic, cells which are seen, in the electron microscope, to resemble authentic fibroblasts and to be surrounded by typical collagen fibers (unpublished observations). To examine mononucleated myoblasts on Day 7, Ara C, which kills cycling cells (lo), was added to PLC-treated cultures on Day 3, when controls were well fused, and was either removed on Day 5 or continued to Day 7. The cultures were identical whether Ara C was present for 2 or 4 days. For determining whether unfused cells in PLC develop a muscle-specific morphology, cells grown in PLC from Day 1 and Ara C from Day 3 were trypsinized from their dishes on Day 5 and were subcultured at low (5000 tells/35-mm dish) or at high (1.5 x 10” tells/35-mm dish) density. The low-density cultures were set up in both the presence and absence of PLC. These subcultures were fixed for electron microscopy 2, 3, or 5 days after inoculation. For electron microscopy, cultures were rinsed once in a buffered salt solution and were then fixed for 1 hr in 1.2% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.3. This and all subsequent steps were carried out at room temperature. After a brief rinse in cacodylate buffer, cultures were postfixed in 1.33% osmium tetroxide in the same buffer, stained for 1 hr in 0.5% uranyl acetate in water, dehydrated through graded alcohols, and embedded in Epon. Thin sections, either stained with uranyl acetate and lead citrate or with lead cit-
550
DEVELOPMENTAL
rate alone, were examined electron microscope.
BIOLOGY
in a JEOL 1OOB
RESULTS
Three days after plating in medium 1621, the control cultures were fused extensively and some loosely organized myofilament bundles were discernible in the electron microscope. Seven days after plating, the myotubes showed extensive crossstriations in the phase microscope and contracted spontaneously. When Ara C was added to cultures on Day 3 and either continued for the duration or removed on Day 5, myotubes in 7-day cultures appeared identical to those in medium 1621 alone, but the population of single cells was dramatically reduced and the cultures were littered with the debris of the killed cycling cells (10). Addition of PLC (at up to 4 x the dose required to inhibit fusion, i.e., at up to 4.0 pg/ml) to the fused myotubes
VOLUME
49, 1976
on Day 3 had no discernible effect on their developing structures (Fig. 1). Thus, myotubes in 7-day cultures, which were grown in the presence or absence of PLC or Ara C from Day 3, were all ultrastructurally identical. When PLC was added to the cultures on Day 1 and continued through Day 7, the myoblasts withdrew from the cell cycle between Days 2 and 3 (23) while most of the nonmyogenic cells continued to divide. Addition of Ara C at 10 pg/ml on Day 3 resulted in 7-day cultures containing largely lined-up, needle-like, mononucleated cells and a few large, flattened, fibroblasts which had apparently withdrawn from the cell cycle. In the electron microscope, the elongate, postmitotic, mononucleated myoblasts were seen to contain thick and thin filaments in varying degrees of myofibrillar organization, but always less well organized than in fused con-
FIG. 1. Myotube in a 7-day culture maintained in the presence of PLC at a concentration of 0.8 pg/ml the dose required to stop fusion) from Day 3 to Day 7. Well-organized sarcomeres are apparent. Note triadic junction (arrow) and M-bands (open arrow). x 17,000.
(2 x the
551
BRIEF NOTES
trol cultures of the same age (Fig. 2). A count of the number of cell profiles containing thick filaments in sections parallel to the dish surface and either toward the bottom, middle, or top of the cell layer indicated that more than 75% of the cells in each case contained thick filaments. Because the absence of thick filaments in a single section does not imply their absence in the cell, this figure must be considered a minimum estimate. In cross sections of single cells cultured in PLC from Day 1 to 7, the thick filaments were in typical hexagonal array (not shown). In parallel sections through many different mononucleated cells, the myofilaments appeared to be at different formative stages, all of which could also be seen in myotubes of different ages. Figure 2, for example, apparently represents a stage of early sarcomere formation: Z-material is present and the sarcoplasmic reticulum is
FIG. myofibril nascent
2. Mononucleated is apparently I-band (arrow).
preferentially located in the region of the I-band, but M-bands are absent and the overall organization appears loose. Mbands were very rarely seen in mononucleated cells in 7-day cultures though they were quite common in myotubes in cultures of the same age. In many sections, the T-tubules were observed apparently invaginating from the cell surface and branching, as described by Ezerman and Ishikawa (81, and on several occasions typical triadic junctions were observed (Fig. 3) as well as sarcoplasmic reticulum-T-tubule like junctions between the sarcoplasmic reticulum and the cell surface (not shown). Although it seemed unlikely, there remained the possibility that the apparently mononucleated cells in 7-day PLC-treated cultures were covertly fused at hidden and infrequent sites. To further substantiate that the differentiated cells in PLC were
myoblast maintained in the presence of PLC at the stage of forming sarcomeres. SR tubules Z-bands are indicated by open arrows. x 19,000.
from Day 1 to Day 7. The are present in the region
single of the
552
DEVELOPMENTAL
BIOLOGY
VOLUME
49, 1976
FIG. 3. Mononucleated myoblast in a T-day culture as in Fig, 2. The myofibrils formation. Note the well-formed triadic junction (arrow). x 21,500.
mononucleated, cells from 5-day cultures grown in PLC from Day 1 and in Ara C from Day 3 were removed from their dishes with 0.05% trypsin and were replated at either 5000 or 1.5 x lo6 tells/35mm dish. We assumed that sites of true cell fusion would be resistant to the action of trypsin and that we were not artifactually creating mononucleated cells from syncytia. Half of the low-density cultures were replated into medium 1621, the other half into the same medium containing PLC. All the cultures on Days l-2 after replating consisted totally of mononucleated cells. On Day 3 after subculturing, the low-density cultures still consisted of highly elongate mononucleated cells and some very large flattened cells. The high density subcultures were beginning to fuse on Day 3 and on Day 5 were extensively fused. When mononuclear cells, selected under the phase contrast microscope to be free of contacts with any other cells, were
are in an early
stage
of
examined in the electron microscope, they were seen to be similar to cells in the primary 7-day PLC-treated cultures described above. The cells which were subcultured into medium containing PLC were identical to those without PLC (Fig. 4). When the cells grown from Day 1 to 5 in PLC-medium containing Ara C from Day 3 were subcultured at high density in medium 1621, they fused to form branching myotubes similar to those formed in control cultures grown without drugs. Thus, it is clear that the postmitotic myoblasts containing myofibrils and other morphological entities characteristic of muscle cells still retained the capacity to fuse with one another. When examined in the electron microscope 2-3 days after fusing, these cells appeared to be at the stage of early myofibril formation (Fig. 51, with few wellformed sarcomeres but with many oriented thick and thin filaments and abun-
BRIEF NOTES
myoblast from a low-density subculture made on Day 5 from FIG. 4. Mononucleated PLC-1621. The myotilaments have reached a stage of organization equivalent to that in the in PLC on Day 7. x 27,000. FIG. 5. Myotube which formed in a high density subculture from a 5-day PLC+Ara C ture was for 5 days in medium 1621. Fusion occurred at Days 2-3. Myofibrils are in formation. x 20,000.
dant dense (presumptive Z) material. Thus, those cells which had been in culture a total of 10 days and had been fused for about 2 days, morphologically resembled control myotubes 1 or 2 days after fusion. We do not know whether any of the myofibrils in these fused cells were carried over intact from the mononucleated cells or whether the process of fusion required a breakdown of preexisting myofibrils. DISCUSSION
From the time it became apparent that skeletal muscle syncytia arose by the fusion of mononucleated cells in uiuo (20, 22) and in vitro (13, 19, 31), the question of what relation cell fusion might have to the rest of the myogenic differentiation program has vexed investigators. Although it
PLC+Ara primary
C into cultures
primary. an early
Subculstage of
has been clear that at least some mononucleated cells in culture elongate and synthesize myofibrils (1, 7, 9, 25), twitch (11, and develop acetylcholine (ACh) sensitivity (91, and that many mononucleated cells in somites also develop myofibrils (12), it has been uncertain how to interpret the presence of these differentiated mononuclear cells. Fambrough and Rash (9) considered them to be “pioneer myoblasts” and speculated, on the basis of combined morphological and electrophysiological data, that “every cell fusion during myogenesis involves at least one cell which is already synthesizing actin, myosin, and ACh receptors” (9, p. 66). Having shown that ACh receptors, cholinesterase activity, and adenylate cyclase activity all increased at the same rate in fusion-arrested
554
DEVELOPMENTAL
BIOLOGY
as in fusing cells, Prives and Paterson (28) contrasted these results with those obtained for myosin (27) and creatine phosphokinase (5, 301, both of which were reported to increase only in fused cultures. They concluded that differentiation of the cell surface precedes cytoplasmic differentiation and is independent of cell fusion, whereas cytoplasmic differentiation depends on prior fusion. This conclusion is in contrast to that reached by Fambrough and Rash (9) who found that every AChsensitive mononucleated cell they tested had myofibrillar material in its cytoplasm. At this time, then, there appears to be general agreement that the cell surface differentiates independently of fusion, but disagreement concerning cytoplasmic differentiation. Recent data indicating that muscle-specific macromolecules are synthesized at the same rate in unfused myoblasts as in fused controls (3, 6, 18) are in clear conflict with other reports (2, 5, 27, 30, 33). The results described in this paper support the view that differentiation proceeds in postmitotic myoblasts independently of cell fusion. We have shown that fusion is not required for myofilament synthesis, myofibril assembly, T-tubule invagination, sarcoplasmic reticulum differentiation, or the formation of SR-T-tubule junctions. The morphologically differentiated mononuclear cells, which represent the overwhelming majority of the postmitotic population in our myogenic cultures, can fuse subsequently to form syncytia. A similar sequence of events has also been described by Holtzer and his colleagues for cells cultured in EGTA (15, 17) and in cytochalasin B (15-17). Why the single cells appear to develop the muscle-specific morphology at a slower rate than fused cells is an open question,’ 1 The slower rate of morphological differentiation in mononucleated cells explains the discrepancy between this paper and an earlier one (Nameroff et al., 1973) in which we stated that no 150 A filaments were seen in PLC-treated cells. In the earlier experiments without Ara C, the cultures old enough to
VOLUME
49,
1976
which we are presently approaching using the PLC-Ara C system described in this paper. This work was supported by Grants HD-06392 and GM-00136 from the National Institutes of Health and by a grant from the Muscular Dystrophy Associations of America, Inc. We thank Dr. R. D. Lund for the use of the electron microscope and Drs. J. Luft, S. D. Hauschka, and J. M. Keller for criticisms of the manuscript. REFERENCES 1. CAPERS, C. R. (1960). J. Biophys. Biochem. Cytol. 7, 559-579. 2. COLEMAN, J. R., and COLEMAN, A. W. (19681. J. Cell. Physiol. 72, Suppl. 1, 19-34. DELAIN, D., and WAHRMANN, J. P. (1975). Exp. Cell Res. 93, 495-498. DIENSTMAN, S. R. (1974). J. Cell Biol. 63, 83a. EASTON, T., G., and REICH, E. (1972). J. Biol. Chem. 247, 6420-6431. EMERSON, C. P., and BECKNER, S. K. (19751. J. Mol. Biol. 93, 431-447. 7. ENGEL, W. K., and HORVATH, B. (19601. J. Exp. Zool. 144, 209-223. 8. EZERMAN, E. B., and ISHIKAWA, H. (1967). J. Cell Biol. 35, 405-420. 9. FAMBROUGH, D., and RASH, J. E. (1971). Deuelop. Biol. 26, 55-68. G. D. (1972). Develop. Biol. 28, 40710. FISCHBACH, 429. 11. HOLTZER, H. (1970). In “Cell Differentiation” (0. A. Schjeide and J. de Vellis, eds.), pp. 476503. Van Nostrand Reinhold, New York. H., MARSHALL, J. M., and FINCK, H. 12. HOLTZER, (1957). J. Biophys. Biochem. Cytol. 3, 705-723. H., ABBOTT, J., and LASH, J. (1958). 13. HOLTZER, Anat. Rec. 131, 567. H., WEINTRAUB, H., MAYNE, R., and 14. HOLTZER, MOCHAN, B. (1972). Curr. Topics Dev. Biol. 7, 229-256. H., RUBINSTEIN, N., DIENSTMAN, S., 15. HOLTZER, CHI, J., BIEHL, J., and SOMLYO, A. P. (1974). Biochemie 56, 1575-1580. 16. HOLTZER, H., CROOP, J., DIENSTMAN, S., ISHIKAWA, H., and SOMLYO, A. P. (1975). Proc. Nat. Acad. Sci. USA 72, 513-517. 17. HOLTZER, H., STRAHS, K., BIEHL, J., SOMLYO, A. P., and ISHIKAWA, H. (1975). Science 188, 943945. J. M., and NAMEROFF, M. (1974). Differ18. KELLER, entiation 2, 19-23. have numerous thick filaments were overgrown with nonmyogenic cells, making their examination in the EM very difficult. In the earlier paper, thick filaments were scanty enough to be missed.
BRIEF 19. KONIGSBERG, I. R., MCELVAIN, N., TOOTLE, M., and HERRMANN, H. (1960). J. Biophys. Biothem. Cytol. 8, 333-343. 20. LASH, J., Swxa, H., and HOLTZER, H. (1956). Anat. Rec. 124, 324. 21. LOOMIS, W. F., WAHRMANN, J. P., and LUZZATI, D. (1973). Proc. Nat. Acad. Sci. USA 70, 425429. 22. MINTZ, B., and BAKER, W. W. (1967). Proc. Nat. Acad. Sci. USA 58, 592-598. 23. NAMEROFF, M., TROTTER, J. A., KELLER, J. M., and MUNAR, E. (1973). J. Cell Biol. 58, 107118. 24. NAMEROFF, M., and MUNAR, E. (1976). Develop. Biol., in press. 25. OKAZAKI, K., and HOLTZER, H. (1965). J. Histothem. Cytochem. 13, 726-738.
NOTES
555
26. PATERSON, B., and PRIVES, J. (1973). J. Cell Biol. 59, 241-245. 27. PATERSON, B., and STROHMAN, R. C. (1972). Develop. Biol. 29, 113-138. 26. PRIVES, J., and PATERSON, B. (1974). Proc. Nat. Acad. Sci. USA 71, 3208-3211. 29. PRZYBYLA, A., and STROHMAN, R. C. (1974). Proc. Nat. Acad. Sci. USA 71, 662-666. 30. SHAINBERG, A., YAGIL, G., and YAFFE, D. (1971). Develop. Biol. 25, l-29. 31. STOCKDALE, F. E., and HOLTZER, H. (1961). Exp. Cell Res. 24, 508-520. 32. TURNER, D. C., MAIER, J., and EPPENBERGER, H. M. (1974). Deuelop. Biol. 37, 63-89. 33. YAFFE, D., and DYM, H. (1972). Cold Spring Harbor Symp. Quant. Biol. 37, 543-547.
BIOLOGY
Myoblast
49,
548-555 (1976)
Differentiation in Vitro: Morphological Mononucleated Myoblasts JOHN
Department
ofBiological
Structure,
A. TROTTER School
AND MARK
of Medicine,
Accepted
University
December
Differentiation
of
NAMEROFF of Washington,
Seattle,
Washington
98195
1, 1975
Phospholipase C from Clostridium perfringens has been shown previously to inhibit the fusion of cultured chick myoblasts without affecting recognition or cell cycle parameters. In this paper we report that the mononucleated myoblasts, in phospholipase C, synthesize thick and thin filaments and organize them into myofibrils, and that T-tubules and sarcoplasmic reticulum differentiate and join in morphologically typical junctions. The structurally differentiated mvoblasts can then fuse with one another to form myotubes. We conclude that cell fusion is not necessary for muscle differentiation. INTRODUCTION
When maintained in an appropriate culture medium, single cells isolated either mechanically or enzymatically from embryonic muscle of chick, as well as from other sources, attach to the substratum, migrate, divide, line up into strings of adherent cells, and fuse to form syncytia. Concomitant with fusion there is a rapid rise in the activities of several enzymes characteristic of muscle cells (2, 5, 18, 21, 32, 33) and in the rate of myosin synthesis (6, 27). Evidence has been put forward to support the notion that fusion per se is the “trigger” that turns on the synthesis of these muscle-specific proteins (2, 21, 27, 29, 30). The evidence has been that there was little or no increase in muscle-specific molecules when fusion was blocked by some alteration of the culture medium. Upon restoration of fusion-permissive conditions, both fusion and muscle-specific protein synthesis began at about the same time. Recently, however, evidence has been published to support the contrary notion that fusion and the other aspects of terminal muscle differentiation need have no causal relation to one another. Thus, it has been shown that creatine phosphokinase activity increases at the same rate as in fusing controls in cultures of myoblasts
kept from fusing by ethylene glycol (aminoethylether), tetraacetic acid (EGTA), or phospholipase C (PLC; 18), and at a lower rate in cultures treated with bromodeoxyuridine (BUdR; 14, 18). Creatine phosphokinase and phosphorylase also have been found to increase in cultures of a myoblast cell line in which fusion was inhibited by the presence of the drug cytochalasin B (3). In addition, myosin synthesis has been reported to occur at the same rate as in myotubes in mononucleated myoblasts cultured in EGTA (6). Skeletal muscle myosin also has been detected immunologically in mononucleated myoblasts in EGTA and cytochalasin B (4, 15), and thick and thin filaments have been seen with the electron microscope in mononucleated myoblasts cultured in the presence of low calcium (9), EGTA (6), and cytochalasin B (15-17). Thus, the literature is divided on the issue of the relation of cell fusion to cytoplasmic differentiation. We have explored the relation of cell fusion to other aspects of the differentiative program of myogenic cells using PLC, which reversibly blocks the fusion step without interfering with recognition (24) or cell cycle parameters (23). We now report that the mononucleated postmitotic myoblasts, which arise in medium containing PLC, synthesize myofilaments and 548
Copyright 0 1976 by Academic press, Inc. All rights of reproduction in any form reserved.
BRIEF NOTES
organize them into myofibrils and that the sarcoplasmic reticulum and T-tubules also differentiate and form typical junctions in these mononucleated cells. Such differentiated cells fuse with one another when they are cultured in fresh medium without PLC. We regard these observations as evidence that cell fusion is not causally related to the other major steps of terminal skeletal muscle differentiation. MATERIALS
AND
METHODS
Minced breast muscles from 11-day embryonic chicks were exposed to 0.25% trypsin for 30 min at 37”C, mechanically dispersed in complete culture medium, and filtered through two layers of lens tissue. Cultures were established on gelatincoated 35mm plastic dishes by plating 0.5 x 10” cells in 1.5 ml of medium (Eagle’s MEM, 82.5%; horse serum, 10.3%; embryo extract, 5.2%; penicillin-streptomycin solution, 1.0%; Fungizone, 1.0%; referred to as medium 1621). The culture environment was a water-saturated atmosphere of 5% CO, in air at 37.5”C. The medium was changed on the first day after plating and on every second day thereafter. Phospholipase C (phosphatidylcholine: choline phosphohydrolase, E.C.3.1.4.3, referred to as PLC) from Clostridium perfringens was obtained from Worthington Biochemical Corp., Freehold, N. J., and was purified by chromatography on Sephadex G-100 (Pharmacia, Piscataway, N.J.) as described previously (23). The purified enzyme was used in medium 1621 at concentrations of 0.4-1.0 pg/ml. n-arabinofuranosylcytosine (Ara C) was obtained from Calbiochem, La Jolla, Calif., and was used at a concentration of 10 pglml in medium 1621 (3.6 x lo-: M). To determine the effect, if any, of PLC on the ultrastructure of already fused myotubes, PLC was added on Day 3, when the cultures were mostly fused, at up to 4x the dose required to inhibit fusion. Such cultures were maintained in PLC-medium on a normal feeding schedule up to Day 7,
549
when they and controls without PLC were fixed for electron microscopy. Morphological differentiation of unfused cells was determined by fixing 7-day cultures in which fusion was prevented by the presence of PLC on Days l-7. PLC does not appear to affect the doubling time of cycling cells in these cultures (231, while the myoblasts withdraw from the cell cycle at the time at which fusion would normally occur (24). The 7-day cultures in PLC from Days l-7 are, therefore, overgrown with cycling, presumably largely nonmyogenic, cells which are seen, in the electron microscope, to resemble authentic fibroblasts and to be surrounded by typical collagen fibers (unpublished observations). To examine mononucleated myoblasts on Day 7, Ara C, which kills cycling cells (lo), was added to PLC-treated cultures on Day 3, when controls were well fused, and was either removed on Day 5 or continued to Day 7. The cultures were identical whether Ara C was present for 2 or 4 days. For determining whether unfused cells in PLC develop a muscle-specific morphology, cells grown in PLC from Day 1 and Ara C from Day 3 were trypsinized from their dishes on Day 5 and were subcultured at low (5000 tells/35-mm dish) or at high (1.5 x 10” tells/35-mm dish) density. The low-density cultures were set up in both the presence and absence of PLC. These subcultures were fixed for electron microscopy 2, 3, or 5 days after inoculation. For electron microscopy, cultures were rinsed once in a buffered salt solution and were then fixed for 1 hr in 1.2% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.3. This and all subsequent steps were carried out at room temperature. After a brief rinse in cacodylate buffer, cultures were postfixed in 1.33% osmium tetroxide in the same buffer, stained for 1 hr in 0.5% uranyl acetate in water, dehydrated through graded alcohols, and embedded in Epon. Thin sections, either stained with uranyl acetate and lead citrate or with lead cit-
550
DEVELOPMENTAL
rate alone, were examined electron microscope.
BIOLOGY
in a JEOL 1OOB
RESULTS
Three days after plating in medium 1621, the control cultures were fused extensively and some loosely organized myofilament bundles were discernible in the electron microscope. Seven days after plating, the myotubes showed extensive crossstriations in the phase microscope and contracted spontaneously. When Ara C was added to cultures on Day 3 and either continued for the duration or removed on Day 5, myotubes in 7-day cultures appeared identical to those in medium 1621 alone, but the population of single cells was dramatically reduced and the cultures were littered with the debris of the killed cycling cells (10). Addition of PLC (at up to 4 x the dose required to inhibit fusion, i.e., at up to 4.0 pg/ml) to the fused myotubes
VOLUME
49, 1976
on Day 3 had no discernible effect on their developing structures (Fig. 1). Thus, myotubes in 7-day cultures, which were grown in the presence or absence of PLC or Ara C from Day 3, were all ultrastructurally identical. When PLC was added to the cultures on Day 1 and continued through Day 7, the myoblasts withdrew from the cell cycle between Days 2 and 3 (23) while most of the nonmyogenic cells continued to divide. Addition of Ara C at 10 pg/ml on Day 3 resulted in 7-day cultures containing largely lined-up, needle-like, mononucleated cells and a few large, flattened, fibroblasts which had apparently withdrawn from the cell cycle. In the electron microscope, the elongate, postmitotic, mononucleated myoblasts were seen to contain thick and thin filaments in varying degrees of myofibrillar organization, but always less well organized than in fused con-
FIG. 1. Myotube in a 7-day culture maintained in the presence of PLC at a concentration of 0.8 pg/ml the dose required to stop fusion) from Day 3 to Day 7. Well-organized sarcomeres are apparent. Note triadic junction (arrow) and M-bands (open arrow). x 17,000.
(2 x the
551
BRIEF NOTES
trol cultures of the same age (Fig. 2). A count of the number of cell profiles containing thick filaments in sections parallel to the dish surface and either toward the bottom, middle, or top of the cell layer indicated that more than 75% of the cells in each case contained thick filaments. Because the absence of thick filaments in a single section does not imply their absence in the cell, this figure must be considered a minimum estimate. In cross sections of single cells cultured in PLC from Day 1 to 7, the thick filaments were in typical hexagonal array (not shown). In parallel sections through many different mononucleated cells, the myofilaments appeared to be at different formative stages, all of which could also be seen in myotubes of different ages. Figure 2, for example, apparently represents a stage of early sarcomere formation: Z-material is present and the sarcoplasmic reticulum is
FIG. myofibril nascent
2. Mononucleated is apparently I-band (arrow).
preferentially located in the region of the I-band, but M-bands are absent and the overall organization appears loose. Mbands were very rarely seen in mononucleated cells in 7-day cultures though they were quite common in myotubes in cultures of the same age. In many sections, the T-tubules were observed apparently invaginating from the cell surface and branching, as described by Ezerman and Ishikawa (81, and on several occasions typical triadic junctions were observed (Fig. 3) as well as sarcoplasmic reticulum-T-tubule like junctions between the sarcoplasmic reticulum and the cell surface (not shown). Although it seemed unlikely, there remained the possibility that the apparently mononucleated cells in 7-day PLC-treated cultures were covertly fused at hidden and infrequent sites. To further substantiate that the differentiated cells in PLC were
myoblast maintained in the presence of PLC at the stage of forming sarcomeres. SR tubules Z-bands are indicated by open arrows. x 19,000.
from Day 1 to Day 7. The are present in the region
single of the
552
DEVELOPMENTAL
BIOLOGY
VOLUME
49, 1976
FIG. 3. Mononucleated myoblast in a T-day culture as in Fig, 2. The myofibrils formation. Note the well-formed triadic junction (arrow). x 21,500.
mononucleated, cells from 5-day cultures grown in PLC from Day 1 and in Ara C from Day 3 were removed from their dishes with 0.05% trypsin and were replated at either 5000 or 1.5 x lo6 tells/35mm dish. We assumed that sites of true cell fusion would be resistant to the action of trypsin and that we were not artifactually creating mononucleated cells from syncytia. Half of the low-density cultures were replated into medium 1621, the other half into the same medium containing PLC. All the cultures on Days l-2 after replating consisted totally of mononucleated cells. On Day 3 after subculturing, the low-density cultures still consisted of highly elongate mononucleated cells and some very large flattened cells. The high density subcultures were beginning to fuse on Day 3 and on Day 5 were extensively fused. When mononuclear cells, selected under the phase contrast microscope to be free of contacts with any other cells, were
are in an early
stage
of
examined in the electron microscope, they were seen to be similar to cells in the primary 7-day PLC-treated cultures described above. The cells which were subcultured into medium containing PLC were identical to those without PLC (Fig. 4). When the cells grown from Day 1 to 5 in PLC-medium containing Ara C from Day 3 were subcultured at high density in medium 1621, they fused to form branching myotubes similar to those formed in control cultures grown without drugs. Thus, it is clear that the postmitotic myoblasts containing myofibrils and other morphological entities characteristic of muscle cells still retained the capacity to fuse with one another. When examined in the electron microscope 2-3 days after fusing, these cells appeared to be at the stage of early myofibril formation (Fig. 51, with few wellformed sarcomeres but with many oriented thick and thin filaments and abun-
BRIEF NOTES
myoblast from a low-density subculture made on Day 5 from FIG. 4. Mononucleated PLC-1621. The myotilaments have reached a stage of organization equivalent to that in the in PLC on Day 7. x 27,000. FIG. 5. Myotube which formed in a high density subculture from a 5-day PLC+Ara C ture was for 5 days in medium 1621. Fusion occurred at Days 2-3. Myofibrils are in formation. x 20,000.
dant dense (presumptive Z) material. Thus, those cells which had been in culture a total of 10 days and had been fused for about 2 days, morphologically resembled control myotubes 1 or 2 days after fusion. We do not know whether any of the myofibrils in these fused cells were carried over intact from the mononucleated cells or whether the process of fusion required a breakdown of preexisting myofibrils. DISCUSSION
From the time it became apparent that skeletal muscle syncytia arose by the fusion of mononucleated cells in uiuo (20, 22) and in vitro (13, 19, 31), the question of what relation cell fusion might have to the rest of the myogenic differentiation program has vexed investigators. Although it
PLC+Ara primary
C into cultures
primary. an early
Subculstage of
has been clear that at least some mononucleated cells in culture elongate and synthesize myofibrils (1, 7, 9, 25), twitch (11, and develop acetylcholine (ACh) sensitivity (91, and that many mononucleated cells in somites also develop myofibrils (12), it has been uncertain how to interpret the presence of these differentiated mononuclear cells. Fambrough and Rash (9) considered them to be “pioneer myoblasts” and speculated, on the basis of combined morphological and electrophysiological data, that “every cell fusion during myogenesis involves at least one cell which is already synthesizing actin, myosin, and ACh receptors” (9, p. 66). Having shown that ACh receptors, cholinesterase activity, and adenylate cyclase activity all increased at the same rate in fusion-arrested
554
DEVELOPMENTAL
BIOLOGY
as in fusing cells, Prives and Paterson (28) contrasted these results with those obtained for myosin (27) and creatine phosphokinase (5, 301, both of which were reported to increase only in fused cultures. They concluded that differentiation of the cell surface precedes cytoplasmic differentiation and is independent of cell fusion, whereas cytoplasmic differentiation depends on prior fusion. This conclusion is in contrast to that reached by Fambrough and Rash (9) who found that every AChsensitive mononucleated cell they tested had myofibrillar material in its cytoplasm. At this time, then, there appears to be general agreement that the cell surface differentiates independently of fusion, but disagreement concerning cytoplasmic differentiation. Recent data indicating that muscle-specific macromolecules are synthesized at the same rate in unfused myoblasts as in fused controls (3, 6, 18) are in clear conflict with other reports (2, 5, 27, 30, 33). The results described in this paper support the view that differentiation proceeds in postmitotic myoblasts independently of cell fusion. We have shown that fusion is not required for myofilament synthesis, myofibril assembly, T-tubule invagination, sarcoplasmic reticulum differentiation, or the formation of SR-T-tubule junctions. The morphologically differentiated mononuclear cells, which represent the overwhelming majority of the postmitotic population in our myogenic cultures, can fuse subsequently to form syncytia. A similar sequence of events has also been described by Holtzer and his colleagues for cells cultured in EGTA (15, 17) and in cytochalasin B (15-17). Why the single cells appear to develop the muscle-specific morphology at a slower rate than fused cells is an open question,’ 1 The slower rate of morphological differentiation in mononucleated cells explains the discrepancy between this paper and an earlier one (Nameroff et al., 1973) in which we stated that no 150 A filaments were seen in PLC-treated cells. In the earlier experiments without Ara C, the cultures old enough to
VOLUME
49,
1976
which we are presently approaching using the PLC-Ara C system described in this paper. This work was supported by Grants HD-06392 and GM-00136 from the National Institutes of Health and by a grant from the Muscular Dystrophy Associations of America, Inc. We thank Dr. R. D. Lund for the use of the electron microscope and Drs. J. Luft, S. D. Hauschka, and J. M. Keller for criticisms of the manuscript. REFERENCES 1. CAPERS, C. R. (1960). J. Biophys. Biochem. Cytol. 7, 559-579. 2. COLEMAN, J. R., and COLEMAN, A. W. (19681. J. Cell. Physiol. 72, Suppl. 1, 19-34. DELAIN, D., and WAHRMANN, J. P. (1975). Exp. Cell Res. 93, 495-498. DIENSTMAN, S. R. (1974). J. Cell Biol. 63, 83a. EASTON, T., G., and REICH, E. (1972). J. Biol. Chem. 247, 6420-6431. EMERSON, C. P., and BECKNER, S. K. (19751. J. Mol. Biol. 93, 431-447. 7. ENGEL, W. K., and HORVATH, B. (19601. J. Exp. Zool. 144, 209-223. 8. EZERMAN, E. B., and ISHIKAWA, H. (1967). J. Cell Biol. 35, 405-420. 9. FAMBROUGH, D., and RASH, J. E. (1971). Deuelop. Biol. 26, 55-68. G. D. (1972). Develop. Biol. 28, 40710. FISCHBACH, 429. 11. HOLTZER, H. (1970). In “Cell Differentiation” (0. A. Schjeide and J. de Vellis, eds.), pp. 476503. Van Nostrand Reinhold, New York. H., MARSHALL, J. M., and FINCK, H. 12. HOLTZER, (1957). J. Biophys. Biochem. Cytol. 3, 705-723. H., ABBOTT, J., and LASH, J. (1958). 13. HOLTZER, Anat. Rec. 131, 567. H., WEINTRAUB, H., MAYNE, R., and 14. HOLTZER, MOCHAN, B. (1972). Curr. Topics Dev. Biol. 7, 229-256. H., RUBINSTEIN, N., DIENSTMAN, S., 15. HOLTZER, CHI, J., BIEHL, J., and SOMLYO, A. P. (1974). Biochemie 56, 1575-1580. 16. HOLTZER, H., CROOP, J., DIENSTMAN, S., ISHIKAWA, H., and SOMLYO, A. P. (1975). Proc. Nat. Acad. Sci. USA 72, 513-517. 17. HOLTZER, H., STRAHS, K., BIEHL, J., SOMLYO, A. P., and ISHIKAWA, H. (1975). Science 188, 943945. J. M., and NAMEROFF, M. (1974). Differ18. KELLER, entiation 2, 19-23. have numerous thick filaments were overgrown with nonmyogenic cells, making their examination in the EM very difficult. In the earlier paper, thick filaments were scanty enough to be missed.
BRIEF 19. KONIGSBERG, I. R., MCELVAIN, N., TOOTLE, M., and HERRMANN, H. (1960). J. Biophys. Biothem. Cytol. 8, 333-343. 20. LASH, J., Swxa, H., and HOLTZER, H. (1956). Anat. Rec. 124, 324. 21. LOOMIS, W. F., WAHRMANN, J. P., and LUZZATI, D. (1973). Proc. Nat. Acad. Sci. USA 70, 425429. 22. MINTZ, B., and BAKER, W. W. (1967). Proc. Nat. Acad. Sci. USA 58, 592-598. 23. NAMEROFF, M., TROTTER, J. A., KELLER, J. M., and MUNAR, E. (1973). J. Cell Biol. 58, 107118. 24. NAMEROFF, M., and MUNAR, E. (1976). Develop. Biol., in press. 25. OKAZAKI, K., and HOLTZER, H. (1965). J. Histothem. Cytochem. 13, 726-738.
NOTES
555
26. PATERSON, B., and PRIVES, J. (1973). J. Cell Biol. 59, 241-245. 27. PATERSON, B., and STROHMAN, R. C. (1972). Develop. Biol. 29, 113-138. 26. PRIVES, J., and PATERSON, B. (1974). Proc. Nat. Acad. Sci. USA 71, 3208-3211. 29. PRZYBYLA, A., and STROHMAN, R. C. (1974). Proc. Nat. Acad. Sci. USA 71, 662-666. 30. SHAINBERG, A., YAGIL, G., and YAFFE, D. (1971). Develop. Biol. 25, l-29. 31. STOCKDALE, F. E., and HOLTZER, H. (1961). Exp. Cell Res. 24, 508-520. 32. TURNER, D. C., MAIER, J., and EPPENBERGER, H. M. (1974). Deuelop. Biol. 37, 63-89. 33. YAFFE, D., and DYM, H. (1972). Cold Spring Harbor Symp. Quant. Biol. 37, 543-547.
