DEVELOPMENTAL
BIOLOGY
48, 438-446
(1976)
Myosin Accumulation BARBARA Departments
in Mononucleated Cultures
September
INTRODUCTION
One of the unique characteristics of the skeletal muscle cell is its multinuclearity. Evidence is now conclusive that this morphological feature arises developmentally from the cytoplasmic fusion of mononucleated cells termed myoblasts (see reviews of Konigsberg, 1963; Holtzer, 1970; Fischman, 1972). In regions of presumptive muscle within the embryo, the appearance of multinucleated cells (myotubes) has served as a useful sign of muscle differentiation, for the bulk of myofibrillar protein synthesis and assembly generally occurs after myotube formation. In recent years, improved methods have been developed for synchronizing and/or arresting myogenic cell fusion in monolayer culture (Paterson and Strohman, 1972; O’Neill and Stockdale, 1972; Doering and Fischman, 1974). With both rat and chicken material a temporal correlation has been demonstrated between the onset of cell fusion and the increased synthesis and accumulation of certain marker proteins, such as myosin, actin, creatine kinase (Coleman and Coleman, 1968; Shainberg et al., 1971; Morris and Cole, 1972; Turner et al., 1974). Furthermore, when fusion has been arrested by Ca*+-depletion, one can prevent the increased biosynthetic rates of these proteins, yet release this inhibition by readdi438 Inc. reserved.
Biology,
19,1975
The biosynthesis and accumulation of the myosin heavy examined in embryonic chick skeletal muscle cultures under cell fusion. When compared with primary chick fibroblasts, significantly more MHC, even while mononucleated. Electron cultures revealed the presence of myosinlike thick filaments that cell fusion is not a urereauisite for mvosin accumulation _ embryonic chick muscle differentiation,
0 1976 by Academic Press, of reproduction in any form
Muscle
M. VERTEL AND DONALD A. FISCHMAN
of Biology and Anatomy and The Committee on Developmental The University of Chicago, Chicago, Illinois 60637 Accepted
Copyright All rights
Cells of Chick
chain (MHC) peptide has been conditions of normal or arrested the myogenic cells accumulated microscopy of the fusion-blocked in the myoblasts. It is concluded or myofilament assembly during
tion of Ca*+ to the cultures (Shainberg et al., 1971; Paterson and Strohman, 1972; Turner et al., 1974). Based on these in uiuo and in vitro studies, the concept has arisen that cell fusion not only precedes muscle differentiation but may in fact trigger gene expression. However, the fusion event cannot be considered the differentiative trigger in all muscle systems for, as Holtzer has emphasized, chick somitic musculature assumes a cross-striated contractile state while the cells are mononucleated (Holtzer et al., 1957; Pryzbylski and Blumberg, 1966; Holtzer, 1970) and under certain conditions one can observe myofibrillogenesis in mononucleated cells of primary monolayer cultures of both chick and rat muscle (Okazaki and Holtzer, 1965; Coleman and Coleman, 1968; Fambrough and Rash, 1971; Dienstman, 1974; Moss and Strohman, 1976). Perhaps myoblast fusion is only one early and easily recognized phenotypic manifestation of muscle differentiation, rather than a triggering event for subsequent myogenesis. As part of a larger study of the relationship of mitochondrial differentiation to myofibrillogenesis (Vertel, 1974; Vertel and Fischman, in preparation) we have examined the biosynthesis and accumulation of MHC in chick muscle and fibroblast cul-
VERTEL
AND
FISCHMAN
Myosin
tures. Our findings demonstrate that, on a per cell or per unit protein basis, there is a differential accumulation of MHC in control and fusion-blocked myogenic cultures, but not in fibroblast cultures. These results indicate that fusion is not an obligatory prerequisite for the initiation of synthesis or accumulation of myosin during muscle differentiation. MATERIALS
AND METHODS
Tissue culture. Cell cultures were prepared from the thigh muscle of 12-day-old White Leghorn chicken embryos by methods standard in this laboratory (Shimada et al ., 19671,at a density of 1 x lo6 tells/60mm Falcon tissue culture dish coated with 1% gelatin. The culture medium consisted of 83% Eagle’s Basal Medium, 10% horse serum, 5Yc embryo extract, 1% L-glutamine, and 1% penicillin-streptomycin. All medium components, with the exception of embryo extract, were purchased from Grand Island Biological Company. Solutions were equilibrated in 95% air, 5% CO,. Fibroblast cultures were prepared from cells isolated from the hindlimb perimuscular connective tissue of 1Zday chicken embryos. Trypsin dissociation and cell culture conditions were identical to those employed for muscle. Fusion of myogenic cells was blocked reversibly by varying the levels of extracellular calcium using modifications of the Chelex method described by Ozawa (1972) and by Paterson and Prives (1973). Identical results have been obtained using ethylene glycol-bis(amino ethyl ether)-l\r,N-tetraacetic acid (EGTA) to lower the levels of extracellular Ca2+ (Paterson and Strohman, 1972). During the course of these experiments, culture medium was changed at 24 and 72 hr postplating (p.p.). Protein determination. Protein concentration of whole cell homogenates were determined by the Lowry method (Lowry et
Accumulation
in
Muscle
Cells
439
al., 1951) using bovine serum albumin as a standard. Electron microscopy. Cultures were fixed in situ in one half strength Karnovsky’s fixative (Karnovsky, 19651,postfixed in 1% osmium tetroxide, stained in block with 0.5% uranyl acetate, and embedded by the method of Brinkley et al. (1967). Cell counts and fusion index. The scoring of myogenic cell fusion was performed on glutaraldehyde-fixed cultures stained with Giemsa-Toluidine Blue, by standard procedures (O’Neill and Stockdale, 1972; Doering and Fischman, 1974). A minimum of 1000 nuclei from randomly selected fields were counted for each determination. Myosin synthesis and accumulation. Myosin heavy chain (MHC) synthesis was measured by a modification of the method of Paterson and Strohman (1972) (see legend to Table 1). Coomassie Blue stained SDS-polyacrylamide gels, prepared from the myosin-containing high salt extract, were assayed for myosin content by quantitative scanning densitometry at 550 nm. The amount of protein in the 200,000 dband was established by a method similar to the one described by Bullard et al. (1973) and by Morimoto and Harrington (1974). Total protein synthesis. Protein synthesis was measured by the method described by Skosey et al. (1972). RESULTS
As noted by Coleman and Coleman (19681, the rise in myosin content per culture follows soon after, and correlates with, the formation of myotubes in vitro (Figs. 1A and lC, Tables 1 and 2). Under our usual conditions (Fig. lA, Table 2, Experiment l), 75% of the myogenic nuclei are incorporated into syncytia by 48 hr p.p., and a twentyfold increase over the 24hr value of MHC per culture can be detected by 72 hr p.p. In one experiment (Fig. lC, Table 2, Experiment 2) in which fusion occurred at an unusually slow rate,
440
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE PROTEIN
Time (hr)
AND MYOSIN
SYNTHESIS
Sample
48,
1976
1
AND ACCUMULATION DURING BLOCKED CULTURES
MYOGENESIS
A B C Total pro- Acid ppt cpm Acid ppt tein per per culture cpm per culture total pro(cpm)” tein b-4
IN CONTROL
D WIyupr (Pkd
AND FUSION-
E WIIu;~ (cpml”
(cpm/&
Experiment 24 48 72
24 38 48 68 89
F MHC per total protein X 10-z
(PdPIcLg)
1
Control Fusion-blocked Control Fusion-blocked Control Fusion-blocked Reversal*
145 145 335 299 520 426 510
8,400 7,300 18,500 16,500 33,000 18,500 28,000 Experiment 2
58
0.41
51
0.41
55 55 63 43 55
2.45 1.35 7.6 3.1 4.3
Control Fusion-blocked Control Control Fusion-blocked Control Fusion-blocked Reversal Control Fusion-blocked Reversal
112 98 202 271 213 367 300 338 655 353 480
3,400 3,400 10,800 15,400 15,600 32,000 18,600 33,000 56,000 28,000 47,000
30 34 53 57 73 87 62 97 89 86 98
72 60 330 205 609 185 280
0.28 0.28 0.73 0.45 1.5 0.73 0.83
44 0.26 0.36 0.31 2.7 1.4 2.1 9.6 3.6 5.7
120 128
0.13 0.13
101
0.15
234 121 137 673 106 315
0.74 0.47 0.58 1.5 1.0
1.2
’ Cultures were pulse labeled for 2 hr with 12.5 &i/plate of [4,5-H311eucine (Schwarz-Mann; 30-50 Ci/mmole) in 3 ml complete medium to determine acid precipitable cpm, and MHC cpm. Incorporation into MHC was determined as described by Paterson and Strohman (1972) without the addition of carrier MHC. * Reversal of the fusion block was accomplished by a switch to control complete medium at 55 hr p.p.
there was a parallel lag in the accumulation of MHC, although a thirtyfold increase did occur by 90 hr p.p. The increase in MHC per unit total protein in these two sets of experiments (Fig. 2) appeared to correlate with the kinetics of cell fusion. The incorporation of i3H11eu into the MHC rose markedly after cell fusion (Table l), and in Experiment 2, in which myotube formation occurred more slowly, there was a parallel lag in apparent MHC synthesis. These experiments confirmed the observations of Paterson and Strohman (1972) and again suggested a potential coupling of cell fusion to myosin synthesis. To examine this relationship further, myosin synthesis and accumulation were
examined in fusion-blocked cultures under conditions in which greater than 90% of the cells were mononucleated for at least 72 hr p.p. Within 7 hr after restoring normal extracellular Caz+ concentrations, 7075% of the myoblast nuclei had become incorporated into myotubes. Total cell protein, nuclear number, and MHC increased in the fusion-blocked cultures, but to a lesser extent than in control cultures (Fig. 1). Both in the low-calcium and control proportionally more muscle cultures, myosin accumulated than total cell protein; hence, the increase in fractional percent of total protein present as MHC, even under conditions of fusion arrest (Fig. 2, Table 1). The growth curves (expressed as num-
VERTEL
AND
Myosr in Accumulation
FISCHMAN
ber of nuclei per plate) for Experiments 1 and 2 are presented in graphic (Fig. 1) and tabular (Table 2) form. Up to 48 hr p-p., the cell doubling was similar in control and low Ca2+ cultures. Later in culture, however, there appeared to be a slower rate of increase in cell numbers in the low Ca2+ cultures (see also Shainberg, 1969; Paterson and Strohman, 1972). In fact,
in
Muscle
Cells
441
some myoblasts may have been lost (Table 2). This apparent cessation of myoblast replication in low Ca2+ medium suggests a correlation between mitotic inhibition and gene expression in the myogenic cell population. Fibroblasts continued to divide in low Ca’+ medium, albeit at a slower rate than in control medium (Table 2, Experiment 1; see also Balk, 1971). The atypi-
BOO
soot I 400I c zoo-
100
E
F
600
500
20
40
60
60 HOURS
20 OF
40
60
60
CULTURE
FIG. 1. The increase in number of nuclei, total protein, and myosin content in control and fusion-blocked myogenic cultures and in tibroblast cultures. (A) Typical control myogenic cultures, Expt 1. At 48 hr, 73% of the myogenic nuclei were contained in myotubes. (B) Companion fusion-blocked myogenic cultures, Expt 1. (C) Control myogenic cultures with slow rate of myotube formation, Expt 2. At 48 hr, 27% of the myogenic nuclei were contained in myotubes. (D) Companion fusion-blocked myogenic cultures, Expt 2. (E) Fibroblast cultures. (F) Fibroblast cultures grown in low calcium medium.
442
DEVELOPMENTAL
BIOLOGY
TABLE CELL
Time (hours)
TYPE
PROFILE
Sample
FOR
Total 9% mb
CONTROL
AND
% fib
Experiment 24 48 72
Control Control Fusion-blocked Control Fusion-blocked
77 22 77 4 52
4 60 4 54 5
21 18 19 42 43 Experiment
24 38 48 68 89
Control Control Control Fusion-blocked Control Fusion-blocked Control Fusion-blocked
” mb, myoblast;
mt,
66 57 53 67 14 64 10 49 myotube;
7 12 21 7 47 9 60 18
27 31 26 27 39 27 30 33
1976
2 FUSION-BLOCKED
cell population B mt
48,
VOLUME
MYOCENIC
MY\%g”npoP-
g-l;;;
%mb
per plate x 10-G
%mt
CULTURES”
I-$!;p ogenic nuclei per plate X 10-G
pln& in syncytia per P1$=TdX
1 95 27 94 6 90
5 73 6 94 10
1.2 2.35 2.35 3.8 2.5
0.95 1.9 1.95 2.15 1.4
0.05 1.4 0.08 1.95 0.135
10 18 28 9 77 12 86 27
0.89 1.0 1.8 1.75 2.5 2.1 2.8 1.6
0.67 0.69 1.3 1.3 1.5 1.5 1.9 1.1
0.064 0.12 0.38 0.11 1.2 0.18 1.7 0.28
2 90 82 72 91 23 88 14 73
fib, fibroblast.
tally slow rate of cell replication observed in Experiment 2 under both low Ca2+ and control conditions (Figs. 1C and lD, Table 2) is unexplained. It might be suggested that all of the MHC accumulation in the low Ca2+ cultures occurred within the relatively few myotubes of such cultures. If all of the myosin is ascribed to such cells and expressed as picograms of MHC per syncytial nucleus (Table 31, then there would have to be substantially more MHC per myotube of low Caz+ cultures than in controls. Morphological analysis of such cultures does not support such a contention; rather, the myotubes in the low Ca2+ cultures contain fewer myofibrils and appear more attenuated than myotubes of comparable age in control cultures. These observations strongly suggest that cells in addition to the myotubes are accumulating myosin in the fusion-blocked cultures. Since myosin has been isolated and puritied from fibroblast cell cultures (Adelstein et al., 19721, and since fibroblasts represent a significant fraction of the total cell
population in muscle cultures (Table 21, MHC accumulation was measured in primary fibroblast cultures derived from the chick perimuscular connective tissue (Figs. 1E and 1F). Although MHC content per culture did increase with time, there was no preferential accumulation of this protein, for the amount of MHC per unit total cell protein remained constant for 96 hr of culture (Fig. 2). Finally, mononucleated cells of fusionarrested cultures contained 150 A diameter thick filaments in association with 50 A thin filaments, and many large polysomes were observed (Fig. 3). Rather diffuse electron-dense material could be seen in association with the myofilament bundles, but myofibril formation was much less extensive than in corresponding myotubes of control cultures. DISCUSSION
In confirmation of prior work (Coleman and Coleman, 1968; Paterson and Strohman, 19721, this study has demonstrated that in primary cultures of chick limb mus-
VERTEL
OL
AND
I 40
i 20
Myosin
FISCHMAN
HOURS
OF
1 60 CULTURE
HOURS
OF
CULTURE
I 60
FIG. 2. Myosin accumulation in control, fusionblocked, and Ca*+-reversed myogenic cultures and fibroblast cultures. (A) Percent myosin per unit total protein. (B) Myosin content per culture. Reversal of fusion-blocked cultures was accomplished by the addition of control complete medium at 55 hr p.p. Typical control myogenic cultures;Expt 1(0-O); companion fusion-blocked myogenic cultures, Expt 1 (0-O); slowly fused control myogenic cultures, Expt 2 (0-O); companion fusion-blocked myogenic cultures, Expt 2 (W-W; Ca*+-reversed myogenic cultures, Expt 2 (A----A); fibroblast cultures (A----A).
cle there is an increased incorporation of 13Hlleu into the MHC, a rise in the relative concentration of MHC and an absolute accumulation of this protein, all of which normally accompany myotube formation
Accumulation
in Muscle
443
Cells
and subsequent myofibrillogenesis. Thus, under normal culture conditions there occurs a temporal sequence in which DNA synthesis and mitosis decline, cell fusion becomes prominent and then MHC synthesis and accumulation increase dramatically. Of importance in this manuscript, and in the accompanying report by Moss and Strohman (19761, is the observation that the rise in MHC accumulation can occur under conditions in which the myogenic cells are fusion-blocked by depletion of extracellular Caz+. Evidence has been presented that this increase in MHC content cannot be attributed to the fibroblast cell population or the relatively few multinucleated cells that are usually seen in fusion-arrested cultures. By thin-section electron microscopy, thick (150 A diameter) filaments, and nascent myofibrils have been demonstrated in the long, spindle-shaped myoblasts of Ca*+-depleted cultures. We have the impression that increasing numbers of such thick filaments appear upon accumulation of MHC, suggesting that the form of MHC that is appearing in vitro is composed primarily of the muscle specific protein. As seen in Table 1, and in confirmation of Paterson and Strohman (19721, i3Hlleu incorporation into MHC during a 2-hr pulse, is inhibited by approximately 40% in Ca2+-depleted cultures by 68 hr p.p. The accumulation of MHC in these same culTABLE MYOSIN
PER SYNCYTIAL FUSION-BLOCKED
Hours
3
NUCLEUS MUSCLE
48 68 89
1
1.75 3.9 Experiment 0.95 2.25 5.6 ~~-.-.-...-~_
AND
Fusion-blocked myosinlsyncytial nucleus (pg)
Control myosin/syncytial nucleus (pg) Experiment
48 72
IN CONTROL CULTURES
16.9 23.0 2 2.8 7.8 12.9
~~~~-
444
DEVELOPMENTAL
BIOLOGY
VOLUME
48,
1976
FIG. 3. (A) Electron micrograph of a fusion-blocked mononucleated myoblast, 90 hr p.p. Note the numerous large polysomes (p), thin-filament skeins (fs), and thick filaments (t). x 20,000. (B) Higher magnification micrograph of selected area, demonstrating thick, 150 A filaments (t) in parallel array. x 40,000. Bar, 1 pm.
tures is depressed by 50-60%, suggesting that the labeling experiments provide a reasonably good index of MHC biosynthetic rates and that increasing levels of MHC content during these culture periods probably reflect variations in synthetic rate rather than alterations in protein degradation. However, firm conclusions will have to await a more complete analysis of amino acid uptake, precursor-product pool relationships, and a more precise estimate of MHC turnover in these cultures. The data presented in this report are compatible with the observations of Emerson and Beckner (19751, who have shown that myosin can be detected within mononucleated quail muscle cells, provided such cells are inactive in DNA synthesis. Their conclusion that gene expression may
be triggered by a repression in DNA synthesis rather than the initiation of cell fusion is consistent with our observation that myoblasts in Ca*+-depleted cultures are mitotically inhibited. The presence of myosin and thick filaments in mononucleated muscle cells in vitro has been noted by other investigators (Coleman and Coleman, 1968; Fambrough and Bash, 1971), and Dienstman (1974) has recently observed that the number of such myosin-containing, mononucleated cells increases with prolonged culture under fusion-blocked conditions. The precise number of myoblasts that accumulate MHC during the short-term culture periods of our experiment cannot be stated at this time; additional histochemical studies will be necessary to settle that question. How-
VERTEL
AND FISCHMAN
hfyosi in Accumulation
ever, the observations of Moss and Stroh(1976) in the accompanying report suggest that most, if not all, mononucleated muscle cells are in fact capable of accumulating sufficient myosin to be detected by immunofluorescence. Finally, there is increasing evidence to suggest that other enzymatic transitions can occur in muscle cultures independently of cell fusion. The isozymic conversions of creatine phosphokinase (CPK) and aldolase are not tightly coupled to cell fusion (Turner et al., 19741, and increased levels of CPK have been reported in a nonfusing muscle cell line (Tarakis et al., 1974). In addition, the appearance of the acetylcholine receptor sites and increased levels of acetylcholinesterase occur in vitro with or without myogenic cell fusion (Paterson and Prives, 1973). In light of the preceding evidence, we would suggest that muscle cell fusion be considered one of the many morphological manifestations of muscle differentiation, rather than a regulatory event which triggers that process.
man
During the course of these investigations we became aware of complementary studies (Moss and Strohman, 1976) on secondary cultures of fusionblocked chick muscle in which MHC accumulation was demonstrated in mononucleated myogenic cells. Both laboratories have exchanged data and agreed upon coordinate publication of the two manuscripts. This work was supported by an N.D.E.A. Title IV fellowship, the Public Health Service Traineeship 5TOI-HD 00174-07, USPHS (HLI-13505-051, USPHS contract 43 (NHLI-6813341, and the University of Chicago Cancer Research Grant No. 1 PO1 CA 14599-02. REFERENCES ADELSTEIN, R. S., CONTI, M. A., JOHNSON, G. S., PASTAN, I., and POLLARD, T. D. (1972). Isolation and characterization of myosin from cloned mouse fibroblasts. Proc. Nat. Acad. Sci. USA 69, 36933697. BALK, S. D. (1971). Calcium as a regulator of the proliferation of normal but not transformed, chicken fibroblasts in a plasma containing medium. Proc. Nat. Acad. Sci. USA 68, 271-275. BRINKLEY, B. R., MURPHY, P., and RICHARDSON, C. L. (1967). Procedure for embedding in situ selected cells cultured in uitro. J. Cell Biol. 35, 279-283. BULLARD, B., and REDDY, M. K. (1973). How many
in
Muscle
Cells
445
myosins per cross-bridge? II. Flight muscle myosin from the blowfly Sarcophaga bullata. Cold Spring Harbor Symp. Quant. Biol. 37,423-428. COLEMAN, J. R., and COLEMAN, A. M. (1968). Muscle differentiation and macromolecular synthesis. J. Cell Physiol. 72, 19-34. DIENSTMAN, S. R. (1974). Skeletal myogenesis without fusion in vitro. J. Cell Biol. 63, 83a. DOERING, J. L., and FISCHMAN, D. A. (1974). The in vitro cell fusion of embryonic chick muscle without DNA synthesis. Develop. Biol. 36, 225-235. EMERSON, C. P., JR., and BECKNER, S. K. 119751. Activation of myosin synthesis in fusing and mononucleated myoblasts. J. Mol. Biol. 93, 431-447. 1975. FAMBROUCH, D., and RASH, J. E. 11971). Development of acetylcholine sensitivity during myogenesis. Develop. Biol. 26, 55-68. FISCHMAN, D. D. (1972). Development of striated muscle. In “The Structure and Function of Muscle” (G. H. Bourne, ed.), 2nd ed. Vol. 1, pp. 75-148. Academic Press, New York. HOLTZER, H. (1970). Myogenesis. In “Cell Differentiation” (0. Schjeide and J. de Vellis, eds.1. Chap. 17, pp. 476-502. HOLTZER, H., MARSHALL, J., and FINK, H. I 1957 1. An analysis of myogenesis by the use of fluorescent antimyosin. J. Biophys. Biochem. Cytol. 3, 705723. KARNOVSKY, M. J. (1965). A glutaraldehyde-paraformaldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137a. LOWRY, 0. H., ROSEBROUGH, N. J. FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265275. MORIMOTO, K., and HARRINGTON, W. F. I 19741. Evidence for structural changes in vertebrate thick filaments induced by calcium. J. Mol. Biol. X8, 693-709. MORRIS, G. E., and COLE, R. J. t1972). Cell fusion and differentiation in cultured chick muscle cells. Expt. Cell Res. 75, 191-199. Moss, P., and STROHMAN, R. (1976). Myosin synthesis by fusion arrested chick embryo myoblasts in cell culture. Develop. Biol. 48, 431-437. OKAZAKI, K., and HOLTZER, H. (1965). An analysis of myogenesis in vitro using fluorescein-labeled antimyosin. J. Histochem. Cytochem. 13, 7266739. O’NEILL, M., and STOCKDALE, F. (19721. A kinetic analysis of myogenesis in vitro. J. Cell Biol. 52, 52-65. OZAWA, E. (1972). The role of calcium ion In avian myogenesis in u&o. Biol. Bull. 143, 43-439. PATERSON, B., and PRIVES, J. (19731. Appearance of acetylcholine receptor in differentiating cultures of embryonic chick breast muscle. J. Cell Biol. 59, 241-245. PATERSON, B., and STROHMAN, R. 11972). Myosin
DEVELOPMENTAL BIOLOGY
446
synthesis in cultures of differentiating chick embryo skeletal muscle. Develop. Biol. 29, 113-138. PRZYBYLSKI, R. J., and BLUMBERG, J. M. (1966). Ultrastructural aspects of myogenesis in the chick. Lab. Znuest. 15, 839-863. SHAINBERG, A., YAGIL, G., and YAFFE, D. (1969). Control of myogenesis in vitro by Ca++ concentration in nutritional medium. Exptl. Cell Res. 58, 163-167. SHAINBERG, A., YAGIL, G., and YAFFE, D. (1971). Alterations of enzyme activities during muscle differentiation in vitro. Develop. Biol. 25, l-29. SHIMADA, Y., FISCHMAN,.D. A., and MOSCONA, A. A. (1967). The tine structure ofembryonic chick skeletal muscle cells differentiated in vitro. J. Cell Biol.
35, 445-453.
SKOSEY,J. L., ZAK, R., MARTIN, A. F., ASCHENBRENNER, V., and RABINOWITZ, M. (1972). Biochemical
VOLUME 48, 1976
correlates of cardiac hypertrophy 5. Labeling of collagen, myosin, and DNA during experimental myocardiac hypertrophy in the rat. Circ. Res. 31, 145-157. TARIKAS, H., and SCHUBERT, D. (1974). Regulation of adenylate kinase and creatine kinase activities in myogenic cells. Proc. Nat. Acad. Sci. USA 71, 2377-2381.
TURNER, D. C., MAIER, V., and EPPENBERGER, H. (1974). Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells. Deuelop. Biol. 37, 63-89. VERTEL, B. M. (1974). Mitochondrial development during myogenesis. Ph.D. Thesis. University of Chicago, Chicago, Ill. VERTEL, B. M., and FISCHMAN, D. A. (1976). In preparation.
BIOLOGY
48, 438-446
(1976)
Myosin Accumulation BARBARA Departments
in Mononucleated Cultures
September
INTRODUCTION
One of the unique characteristics of the skeletal muscle cell is its multinuclearity. Evidence is now conclusive that this morphological feature arises developmentally from the cytoplasmic fusion of mononucleated cells termed myoblasts (see reviews of Konigsberg, 1963; Holtzer, 1970; Fischman, 1972). In regions of presumptive muscle within the embryo, the appearance of multinucleated cells (myotubes) has served as a useful sign of muscle differentiation, for the bulk of myofibrillar protein synthesis and assembly generally occurs after myotube formation. In recent years, improved methods have been developed for synchronizing and/or arresting myogenic cell fusion in monolayer culture (Paterson and Strohman, 1972; O’Neill and Stockdale, 1972; Doering and Fischman, 1974). With both rat and chicken material a temporal correlation has been demonstrated between the onset of cell fusion and the increased synthesis and accumulation of certain marker proteins, such as myosin, actin, creatine kinase (Coleman and Coleman, 1968; Shainberg et al., 1971; Morris and Cole, 1972; Turner et al., 1974). Furthermore, when fusion has been arrested by Ca*+-depletion, one can prevent the increased biosynthetic rates of these proteins, yet release this inhibition by readdi438 Inc. reserved.
Biology,
19,1975
The biosynthesis and accumulation of the myosin heavy examined in embryonic chick skeletal muscle cultures under cell fusion. When compared with primary chick fibroblasts, significantly more MHC, even while mononucleated. Electron cultures revealed the presence of myosinlike thick filaments that cell fusion is not a urereauisite for mvosin accumulation _ embryonic chick muscle differentiation,
0 1976 by Academic Press, of reproduction in any form
Muscle
M. VERTEL AND DONALD A. FISCHMAN
of Biology and Anatomy and The Committee on Developmental The University of Chicago, Chicago, Illinois 60637 Accepted
Copyright All rights
Cells of Chick
chain (MHC) peptide has been conditions of normal or arrested the myogenic cells accumulated microscopy of the fusion-blocked in the myoblasts. It is concluded or myofilament assembly during
tion of Ca*+ to the cultures (Shainberg et al., 1971; Paterson and Strohman, 1972; Turner et al., 1974). Based on these in uiuo and in vitro studies, the concept has arisen that cell fusion not only precedes muscle differentiation but may in fact trigger gene expression. However, the fusion event cannot be considered the differentiative trigger in all muscle systems for, as Holtzer has emphasized, chick somitic musculature assumes a cross-striated contractile state while the cells are mononucleated (Holtzer et al., 1957; Pryzbylski and Blumberg, 1966; Holtzer, 1970) and under certain conditions one can observe myofibrillogenesis in mononucleated cells of primary monolayer cultures of both chick and rat muscle (Okazaki and Holtzer, 1965; Coleman and Coleman, 1968; Fambrough and Rash, 1971; Dienstman, 1974; Moss and Strohman, 1976). Perhaps myoblast fusion is only one early and easily recognized phenotypic manifestation of muscle differentiation, rather than a triggering event for subsequent myogenesis. As part of a larger study of the relationship of mitochondrial differentiation to myofibrillogenesis (Vertel, 1974; Vertel and Fischman, in preparation) we have examined the biosynthesis and accumulation of MHC in chick muscle and fibroblast cul-
VERTEL
AND
FISCHMAN
Myosin
tures. Our findings demonstrate that, on a per cell or per unit protein basis, there is a differential accumulation of MHC in control and fusion-blocked myogenic cultures, but not in fibroblast cultures. These results indicate that fusion is not an obligatory prerequisite for the initiation of synthesis or accumulation of myosin during muscle differentiation. MATERIALS
AND METHODS
Tissue culture. Cell cultures were prepared from the thigh muscle of 12-day-old White Leghorn chicken embryos by methods standard in this laboratory (Shimada et al ., 19671,at a density of 1 x lo6 tells/60mm Falcon tissue culture dish coated with 1% gelatin. The culture medium consisted of 83% Eagle’s Basal Medium, 10% horse serum, 5Yc embryo extract, 1% L-glutamine, and 1% penicillin-streptomycin. All medium components, with the exception of embryo extract, were purchased from Grand Island Biological Company. Solutions were equilibrated in 95% air, 5% CO,. Fibroblast cultures were prepared from cells isolated from the hindlimb perimuscular connective tissue of 1Zday chicken embryos. Trypsin dissociation and cell culture conditions were identical to those employed for muscle. Fusion of myogenic cells was blocked reversibly by varying the levels of extracellular calcium using modifications of the Chelex method described by Ozawa (1972) and by Paterson and Prives (1973). Identical results have been obtained using ethylene glycol-bis(amino ethyl ether)-l\r,N-tetraacetic acid (EGTA) to lower the levels of extracellular Ca2+ (Paterson and Strohman, 1972). During the course of these experiments, culture medium was changed at 24 and 72 hr postplating (p.p.). Protein determination. Protein concentration of whole cell homogenates were determined by the Lowry method (Lowry et
Accumulation
in
Muscle
Cells
439
al., 1951) using bovine serum albumin as a standard. Electron microscopy. Cultures were fixed in situ in one half strength Karnovsky’s fixative (Karnovsky, 19651,postfixed in 1% osmium tetroxide, stained in block with 0.5% uranyl acetate, and embedded by the method of Brinkley et al. (1967). Cell counts and fusion index. The scoring of myogenic cell fusion was performed on glutaraldehyde-fixed cultures stained with Giemsa-Toluidine Blue, by standard procedures (O’Neill and Stockdale, 1972; Doering and Fischman, 1974). A minimum of 1000 nuclei from randomly selected fields were counted for each determination. Myosin synthesis and accumulation. Myosin heavy chain (MHC) synthesis was measured by a modification of the method of Paterson and Strohman (1972) (see legend to Table 1). Coomassie Blue stained SDS-polyacrylamide gels, prepared from the myosin-containing high salt extract, were assayed for myosin content by quantitative scanning densitometry at 550 nm. The amount of protein in the 200,000 dband was established by a method similar to the one described by Bullard et al. (1973) and by Morimoto and Harrington (1974). Total protein synthesis. Protein synthesis was measured by the method described by Skosey et al. (1972). RESULTS
As noted by Coleman and Coleman (19681, the rise in myosin content per culture follows soon after, and correlates with, the formation of myotubes in vitro (Figs. 1A and lC, Tables 1 and 2). Under our usual conditions (Fig. lA, Table 2, Experiment l), 75% of the myogenic nuclei are incorporated into syncytia by 48 hr p.p., and a twentyfold increase over the 24hr value of MHC per culture can be detected by 72 hr p.p. In one experiment (Fig. lC, Table 2, Experiment 2) in which fusion occurred at an unusually slow rate,
440
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE PROTEIN
Time (hr)
AND MYOSIN
SYNTHESIS
Sample
48,
1976
1
AND ACCUMULATION DURING BLOCKED CULTURES
MYOGENESIS
A B C Total pro- Acid ppt cpm Acid ppt tein per per culture cpm per culture total pro(cpm)” tein b-4
IN CONTROL
D WIyupr (Pkd
AND FUSION-
E WIIu;~ (cpml”
(cpm/&
Experiment 24 48 72
24 38 48 68 89
F MHC per total protein X 10-z
(PdPIcLg)
1
Control Fusion-blocked Control Fusion-blocked Control Fusion-blocked Reversal*
145 145 335 299 520 426 510
8,400 7,300 18,500 16,500 33,000 18,500 28,000 Experiment 2
58
0.41
51
0.41
55 55 63 43 55
2.45 1.35 7.6 3.1 4.3
Control Fusion-blocked Control Control Fusion-blocked Control Fusion-blocked Reversal Control Fusion-blocked Reversal
112 98 202 271 213 367 300 338 655 353 480
3,400 3,400 10,800 15,400 15,600 32,000 18,600 33,000 56,000 28,000 47,000
30 34 53 57 73 87 62 97 89 86 98
72 60 330 205 609 185 280
0.28 0.28 0.73 0.45 1.5 0.73 0.83
44 0.26 0.36 0.31 2.7 1.4 2.1 9.6 3.6 5.7
120 128
0.13 0.13
101
0.15
234 121 137 673 106 315
0.74 0.47 0.58 1.5 1.0
1.2
’ Cultures were pulse labeled for 2 hr with 12.5 &i/plate of [4,5-H311eucine (Schwarz-Mann; 30-50 Ci/mmole) in 3 ml complete medium to determine acid precipitable cpm, and MHC cpm. Incorporation into MHC was determined as described by Paterson and Strohman (1972) without the addition of carrier MHC. * Reversal of the fusion block was accomplished by a switch to control complete medium at 55 hr p.p.
there was a parallel lag in the accumulation of MHC, although a thirtyfold increase did occur by 90 hr p.p. The increase in MHC per unit total protein in these two sets of experiments (Fig. 2) appeared to correlate with the kinetics of cell fusion. The incorporation of i3H11eu into the MHC rose markedly after cell fusion (Table l), and in Experiment 2, in which myotube formation occurred more slowly, there was a parallel lag in apparent MHC synthesis. These experiments confirmed the observations of Paterson and Strohman (1972) and again suggested a potential coupling of cell fusion to myosin synthesis. To examine this relationship further, myosin synthesis and accumulation were
examined in fusion-blocked cultures under conditions in which greater than 90% of the cells were mononucleated for at least 72 hr p.p. Within 7 hr after restoring normal extracellular Caz+ concentrations, 7075% of the myoblast nuclei had become incorporated into myotubes. Total cell protein, nuclear number, and MHC increased in the fusion-blocked cultures, but to a lesser extent than in control cultures (Fig. 1). Both in the low-calcium and control proportionally more muscle cultures, myosin accumulated than total cell protein; hence, the increase in fractional percent of total protein present as MHC, even under conditions of fusion arrest (Fig. 2, Table 1). The growth curves (expressed as num-
VERTEL
AND
Myosr in Accumulation
FISCHMAN
ber of nuclei per plate) for Experiments 1 and 2 are presented in graphic (Fig. 1) and tabular (Table 2) form. Up to 48 hr p-p., the cell doubling was similar in control and low Ca2+ cultures. Later in culture, however, there appeared to be a slower rate of increase in cell numbers in the low Ca2+ cultures (see also Shainberg, 1969; Paterson and Strohman, 1972). In fact,
in
Muscle
Cells
441
some myoblasts may have been lost (Table 2). This apparent cessation of myoblast replication in low Ca2+ medium suggests a correlation between mitotic inhibition and gene expression in the myogenic cell population. Fibroblasts continued to divide in low Ca’+ medium, albeit at a slower rate than in control medium (Table 2, Experiment 1; see also Balk, 1971). The atypi-
BOO
soot I 400I c zoo-
100
E
F
600
500
20
40
60
60 HOURS
20 OF
40
60
60
CULTURE
FIG. 1. The increase in number of nuclei, total protein, and myosin content in control and fusion-blocked myogenic cultures and in tibroblast cultures. (A) Typical control myogenic cultures, Expt 1. At 48 hr, 73% of the myogenic nuclei were contained in myotubes. (B) Companion fusion-blocked myogenic cultures, Expt 1. (C) Control myogenic cultures with slow rate of myotube formation, Expt 2. At 48 hr, 27% of the myogenic nuclei were contained in myotubes. (D) Companion fusion-blocked myogenic cultures, Expt 2. (E) Fibroblast cultures. (F) Fibroblast cultures grown in low calcium medium.
442
DEVELOPMENTAL
BIOLOGY
TABLE CELL
Time (hours)
TYPE
PROFILE
Sample
FOR
Total 9% mb
CONTROL
AND
% fib
Experiment 24 48 72
Control Control Fusion-blocked Control Fusion-blocked
77 22 77 4 52
4 60 4 54 5
21 18 19 42 43 Experiment
24 38 48 68 89
Control Control Control Fusion-blocked Control Fusion-blocked Control Fusion-blocked
” mb, myoblast;
mt,
66 57 53 67 14 64 10 49 myotube;
7 12 21 7 47 9 60 18
27 31 26 27 39 27 30 33
1976
2 FUSION-BLOCKED
cell population B mt
48,
VOLUME
MYOCENIC
MY\%g”npoP-
g-l;;;
%mb
per plate x 10-G
%mt
CULTURES”
I-$!;p ogenic nuclei per plate X 10-G
pln& in syncytia per P1$=TdX
1 95 27 94 6 90
5 73 6 94 10
1.2 2.35 2.35 3.8 2.5
0.95 1.9 1.95 2.15 1.4
0.05 1.4 0.08 1.95 0.135
10 18 28 9 77 12 86 27
0.89 1.0 1.8 1.75 2.5 2.1 2.8 1.6
0.67 0.69 1.3 1.3 1.5 1.5 1.9 1.1
0.064 0.12 0.38 0.11 1.2 0.18 1.7 0.28
2 90 82 72 91 23 88 14 73
fib, fibroblast.
tally slow rate of cell replication observed in Experiment 2 under both low Ca2+ and control conditions (Figs. 1C and lD, Table 2) is unexplained. It might be suggested that all of the MHC accumulation in the low Ca2+ cultures occurred within the relatively few myotubes of such cultures. If all of the myosin is ascribed to such cells and expressed as picograms of MHC per syncytial nucleus (Table 31, then there would have to be substantially more MHC per myotube of low Caz+ cultures than in controls. Morphological analysis of such cultures does not support such a contention; rather, the myotubes in the low Ca2+ cultures contain fewer myofibrils and appear more attenuated than myotubes of comparable age in control cultures. These observations strongly suggest that cells in addition to the myotubes are accumulating myosin in the fusion-blocked cultures. Since myosin has been isolated and puritied from fibroblast cell cultures (Adelstein et al., 19721, and since fibroblasts represent a significant fraction of the total cell
population in muscle cultures (Table 21, MHC accumulation was measured in primary fibroblast cultures derived from the chick perimuscular connective tissue (Figs. 1E and 1F). Although MHC content per culture did increase with time, there was no preferential accumulation of this protein, for the amount of MHC per unit total cell protein remained constant for 96 hr of culture (Fig. 2). Finally, mononucleated cells of fusionarrested cultures contained 150 A diameter thick filaments in association with 50 A thin filaments, and many large polysomes were observed (Fig. 3). Rather diffuse electron-dense material could be seen in association with the myofilament bundles, but myofibril formation was much less extensive than in corresponding myotubes of control cultures. DISCUSSION
In confirmation of prior work (Coleman and Coleman, 1968; Paterson and Strohman, 19721, this study has demonstrated that in primary cultures of chick limb mus-
VERTEL
OL
AND
I 40
i 20
Myosin
FISCHMAN
HOURS
OF
1 60 CULTURE
HOURS
OF
CULTURE
I 60
FIG. 2. Myosin accumulation in control, fusionblocked, and Ca*+-reversed myogenic cultures and fibroblast cultures. (A) Percent myosin per unit total protein. (B) Myosin content per culture. Reversal of fusion-blocked cultures was accomplished by the addition of control complete medium at 55 hr p.p. Typical control myogenic cultures;Expt 1(0-O); companion fusion-blocked myogenic cultures, Expt 1 (0-O); slowly fused control myogenic cultures, Expt 2 (0-O); companion fusion-blocked myogenic cultures, Expt 2 (W-W; Ca*+-reversed myogenic cultures, Expt 2 (A----A); fibroblast cultures (A----A).
cle there is an increased incorporation of 13Hlleu into the MHC, a rise in the relative concentration of MHC and an absolute accumulation of this protein, all of which normally accompany myotube formation
Accumulation
in Muscle
443
Cells
and subsequent myofibrillogenesis. Thus, under normal culture conditions there occurs a temporal sequence in which DNA synthesis and mitosis decline, cell fusion becomes prominent and then MHC synthesis and accumulation increase dramatically. Of importance in this manuscript, and in the accompanying report by Moss and Strohman (19761, is the observation that the rise in MHC accumulation can occur under conditions in which the myogenic cells are fusion-blocked by depletion of extracellular Caz+. Evidence has been presented that this increase in MHC content cannot be attributed to the fibroblast cell population or the relatively few multinucleated cells that are usually seen in fusion-arrested cultures. By thin-section electron microscopy, thick (150 A diameter) filaments, and nascent myofibrils have been demonstrated in the long, spindle-shaped myoblasts of Ca*+-depleted cultures. We have the impression that increasing numbers of such thick filaments appear upon accumulation of MHC, suggesting that the form of MHC that is appearing in vitro is composed primarily of the muscle specific protein. As seen in Table 1, and in confirmation of Paterson and Strohman (19721, i3Hlleu incorporation into MHC during a 2-hr pulse, is inhibited by approximately 40% in Ca2+-depleted cultures by 68 hr p.p. The accumulation of MHC in these same culTABLE MYOSIN
PER SYNCYTIAL FUSION-BLOCKED
Hours
3
NUCLEUS MUSCLE
48 68 89
1
1.75 3.9 Experiment 0.95 2.25 5.6 ~~-.-.-...-~_
AND
Fusion-blocked myosinlsyncytial nucleus (pg)
Control myosin/syncytial nucleus (pg) Experiment
48 72
IN CONTROL CULTURES
16.9 23.0 2 2.8 7.8 12.9
~~~~-
444
DEVELOPMENTAL
BIOLOGY
VOLUME
48,
1976
FIG. 3. (A) Electron micrograph of a fusion-blocked mononucleated myoblast, 90 hr p.p. Note the numerous large polysomes (p), thin-filament skeins (fs), and thick filaments (t). x 20,000. (B) Higher magnification micrograph of selected area, demonstrating thick, 150 A filaments (t) in parallel array. x 40,000. Bar, 1 pm.
tures is depressed by 50-60%, suggesting that the labeling experiments provide a reasonably good index of MHC biosynthetic rates and that increasing levels of MHC content during these culture periods probably reflect variations in synthetic rate rather than alterations in protein degradation. However, firm conclusions will have to await a more complete analysis of amino acid uptake, precursor-product pool relationships, and a more precise estimate of MHC turnover in these cultures. The data presented in this report are compatible with the observations of Emerson and Beckner (19751, who have shown that myosin can be detected within mononucleated quail muscle cells, provided such cells are inactive in DNA synthesis. Their conclusion that gene expression may
be triggered by a repression in DNA synthesis rather than the initiation of cell fusion is consistent with our observation that myoblasts in Ca*+-depleted cultures are mitotically inhibited. The presence of myosin and thick filaments in mononucleated muscle cells in vitro has been noted by other investigators (Coleman and Coleman, 1968; Fambrough and Bash, 1971), and Dienstman (1974) has recently observed that the number of such myosin-containing, mononucleated cells increases with prolonged culture under fusion-blocked conditions. The precise number of myoblasts that accumulate MHC during the short-term culture periods of our experiment cannot be stated at this time; additional histochemical studies will be necessary to settle that question. How-
VERTEL
AND FISCHMAN
hfyosi in Accumulation
ever, the observations of Moss and Stroh(1976) in the accompanying report suggest that most, if not all, mononucleated muscle cells are in fact capable of accumulating sufficient myosin to be detected by immunofluorescence. Finally, there is increasing evidence to suggest that other enzymatic transitions can occur in muscle cultures independently of cell fusion. The isozymic conversions of creatine phosphokinase (CPK) and aldolase are not tightly coupled to cell fusion (Turner et al., 19741, and increased levels of CPK have been reported in a nonfusing muscle cell line (Tarakis et al., 1974). In addition, the appearance of the acetylcholine receptor sites and increased levels of acetylcholinesterase occur in vitro with or without myogenic cell fusion (Paterson and Prives, 1973). In light of the preceding evidence, we would suggest that muscle cell fusion be considered one of the many morphological manifestations of muscle differentiation, rather than a regulatory event which triggers that process.
man
During the course of these investigations we became aware of complementary studies (Moss and Strohman, 1976) on secondary cultures of fusionblocked chick muscle in which MHC accumulation was demonstrated in mononucleated myogenic cells. Both laboratories have exchanged data and agreed upon coordinate publication of the two manuscripts. This work was supported by an N.D.E.A. Title IV fellowship, the Public Health Service Traineeship 5TOI-HD 00174-07, USPHS (HLI-13505-051, USPHS contract 43 (NHLI-6813341, and the University of Chicago Cancer Research Grant No. 1 PO1 CA 14599-02. REFERENCES ADELSTEIN, R. S., CONTI, M. A., JOHNSON, G. S., PASTAN, I., and POLLARD, T. D. (1972). Isolation and characterization of myosin from cloned mouse fibroblasts. Proc. Nat. Acad. Sci. USA 69, 36933697. BALK, S. D. (1971). Calcium as a regulator of the proliferation of normal but not transformed, chicken fibroblasts in a plasma containing medium. Proc. Nat. Acad. Sci. USA 68, 271-275. BRINKLEY, B. R., MURPHY, P., and RICHARDSON, C. L. (1967). Procedure for embedding in situ selected cells cultured in uitro. J. Cell Biol. 35, 279-283. BULLARD, B., and REDDY, M. K. (1973). How many
in
Muscle
Cells
445
myosins per cross-bridge? II. Flight muscle myosin from the blowfly Sarcophaga bullata. Cold Spring Harbor Symp. Quant. Biol. 37,423-428. COLEMAN, J. R., and COLEMAN, A. M. (1968). Muscle differentiation and macromolecular synthesis. J. Cell Physiol. 72, 19-34. DIENSTMAN, S. R. (1974). Skeletal myogenesis without fusion in vitro. J. Cell Biol. 63, 83a. DOERING, J. L., and FISCHMAN, D. A. (1974). The in vitro cell fusion of embryonic chick muscle without DNA synthesis. Develop. Biol. 36, 225-235. EMERSON, C. P., JR., and BECKNER, S. K. 119751. Activation of myosin synthesis in fusing and mononucleated myoblasts. J. Mol. Biol. 93, 431-447. 1975. FAMBROUCH, D., and RASH, J. E. 11971). Development of acetylcholine sensitivity during myogenesis. Develop. Biol. 26, 55-68. FISCHMAN, D. D. (1972). Development of striated muscle. In “The Structure and Function of Muscle” (G. H. Bourne, ed.), 2nd ed. Vol. 1, pp. 75-148. Academic Press, New York. HOLTZER, H. (1970). Myogenesis. In “Cell Differentiation” (0. Schjeide and J. de Vellis, eds.1. Chap. 17, pp. 476-502. HOLTZER, H., MARSHALL, J., and FINK, H. I 1957 1. An analysis of myogenesis by the use of fluorescent antimyosin. J. Biophys. Biochem. Cytol. 3, 705723. KARNOVSKY, M. J. (1965). A glutaraldehyde-paraformaldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137a. LOWRY, 0. H., ROSEBROUGH, N. J. FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265275. MORIMOTO, K., and HARRINGTON, W. F. I 19741. Evidence for structural changes in vertebrate thick filaments induced by calcium. J. Mol. Biol. X8, 693-709. MORRIS, G. E., and COLE, R. J. t1972). Cell fusion and differentiation in cultured chick muscle cells. Expt. Cell Res. 75, 191-199. Moss, P., and STROHMAN, R. (1976). Myosin synthesis by fusion arrested chick embryo myoblasts in cell culture. Develop. Biol. 48, 431-437. OKAZAKI, K., and HOLTZER, H. (1965). An analysis of myogenesis in vitro using fluorescein-labeled antimyosin. J. Histochem. Cytochem. 13, 7266739. O’NEILL, M., and STOCKDALE, F. (19721. A kinetic analysis of myogenesis in vitro. J. Cell Biol. 52, 52-65. OZAWA, E. (1972). The role of calcium ion In avian myogenesis in u&o. Biol. Bull. 143, 43-439. PATERSON, B., and PRIVES, J. (19731. Appearance of acetylcholine receptor in differentiating cultures of embryonic chick breast muscle. J. Cell Biol. 59, 241-245. PATERSON, B., and STROHMAN, R. 11972). Myosin
DEVELOPMENTAL BIOLOGY
446
synthesis in cultures of differentiating chick embryo skeletal muscle. Develop. Biol. 29, 113-138. PRZYBYLSKI, R. J., and BLUMBERG, J. M. (1966). Ultrastructural aspects of myogenesis in the chick. Lab. Znuest. 15, 839-863. SHAINBERG, A., YAGIL, G., and YAFFE, D. (1969). Control of myogenesis in vitro by Ca++ concentration in nutritional medium. Exptl. Cell Res. 58, 163-167. SHAINBERG, A., YAGIL, G., and YAFFE, D. (1971). Alterations of enzyme activities during muscle differentiation in vitro. Develop. Biol. 25, l-29. SHIMADA, Y., FISCHMAN,.D. A., and MOSCONA, A. A. (1967). The tine structure ofembryonic chick skeletal muscle cells differentiated in vitro. J. Cell Biol.
35, 445-453.
SKOSEY,J. L., ZAK, R., MARTIN, A. F., ASCHENBRENNER, V., and RABINOWITZ, M. (1972). Biochemical
VOLUME 48, 1976
correlates of cardiac hypertrophy 5. Labeling of collagen, myosin, and DNA during experimental myocardiac hypertrophy in the rat. Circ. Res. 31, 145-157. TARIKAS, H., and SCHUBERT, D. (1974). Regulation of adenylate kinase and creatine kinase activities in myogenic cells. Proc. Nat. Acad. Sci. USA 71, 2377-2381.
TURNER, D. C., MAIER, V., and EPPENBERGER, H. (1974). Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells. Deuelop. Biol. 37, 63-89. VERTEL, B. M. (1974). Mitochondrial development during myogenesis. Ph.D. Thesis. University of Chicago, Chicago, Ill. VERTEL, B. M., and FISCHMAN, D. A. (1976). In preparation.
