DEVELOPMENTAL

BIOLOGY

148,249-260

(1991)

Myoblasts from Fetal and Adult Skeletal Muscle Regulate Myosin Expression Differently REBECCAS.HARTLEY,*

EVERETTBANDMAN,~ANDZIPORAYABLONKA-REUVENI*

*Department of Biological Structure, School of Medicine, and TDepartment of Food Science and Technology, Accepted

University University July

of Washington, Seattle, Washington 981.9.5; of California, Davis, CalifomLia 95616

31, 1991

We compared the expression of myosin heavy chains in myogenic cultures prepared from fetal (embryonic Day 10) and adult (12-16 week) chicken pectoralis muscle using immunofluorescence with isoform-specific monoclonal antibodies. We found that the majority of fetal myocytes (differentiated myoblasts) and myotubes coexpressed ventricular and embryonic myosin heavy chains in culture. Also, when fetal cells were plated at a clonal density most clones coexpressed both ventricular and embryonic isoforms. In contrast, all adult myocytes and newly formed adult myotubes expressed just ventricular myosin, whether plated at mass or clonal densities. Within 12-24 hr of the onset of fusion, adult myotubes began to express embryonic myosin as well. Eventually, the majority of adult myotubes coexpressed both ventricular and embryonic myosin. The delay of embryonic myosin expression until after fusion was also seen in passaged adult myoblasts and in myoblasts isolated from regenerating adult muscle. The expression of embryonic myosin can be abolished by inhibiting fusion with EGTA in adult but not in fetal cultures. We conclude that both fetal and adult myotubes express ventricular and embryonic myosins but only fetal myocytes express the embryonic isoform prior to fusion. This difference in the regulation of embryonic myosin expression between fetal and adult myoblasts supports the hypothesis that these cells may represent two distinct populations of myogenic precursors. (6 1991 Academic Press, Inc.

INTRODUCTION

The myogenic precursor cells of adult skeletal muscle (usually referred to as satellite cells) are mitotically quiescent but can reinitiate proliferative activity following injury (Mauro, 1979; Campion, 1984). More subtle stresses to the muscle such as stretch, some types of exercise, denervation, or mild compression also stimulate satellite cell division (see White and Esser, 1989, for a review). Progeny of activated satellite cells (adult myoblasts) form new myofibers or fuse into preexisting muscle fibers (Snow, 1977a,b; Mauro, 1979). Newly formed adult myofibers in regenerating muscle express developmental isoforms of muscle-specific proteins such as myosin and tropomyosin in a sequential manner which recapitulates transitions during development (Matsuda et ah, 1983; Cerny and Bandman, 1987; Saad et al, 1987). In chicken pectoralis muscle, embryonic, neonatal, and adult isoforms of fast myosin heavy chain (MHC)l are sequentially expressed during both muscle development and adult muscle regeneration (Bader et al., 1982; Bandman et ah, 1982; Matsuda et ab, 1983; Cerny and Bandman, 1987). Additionally, venr Abbreviations used: EGTA, ethylene ether) N,N’-tetraacetic acid; ElO, embryonic heavy chain; DAPI, 4,6-diamidino-2-phenylindole.

glycol Day

tricular and embryonic MHCs are coexpressed in early muscle development as well as in regenerating muscle and during nascent fiber development in overloaded chicken muscles (Kennedy et ah, 1989; Sweeney et al., 1989; Bandman et ah, 1990). Other studies document the expression of many fast MHC isoforms in myogenic cultures from chicken embryos (Cerny and Bandman, 1987; Strohman et al., 1990; Hartley and Yablonka-Reuveni, 1990). These studies suggest that the potential of myotubes derived from fetal2 myoblasts to express neonatal and adult fast myosins is similar to that of myotubes derived from adult myoblasts, even though these isoforms are not expressed in the embryo at the time of cell isolation. However, other cell culture studies have found differences between the precursor cells in developing and adult muscle, raising the possibility that the myogenic precursor cells found at these developmental stages may be distinct populations. Compared to chicken fetal myoblasts, cultured satellite cells from chicken fuse into myotubes later (Yablonka-Reuveni et al., 1987), express desmin as cycling cells more frequently (Yablonka-Reuveni and Nameroff, 1990), express more platelet-derived growth factor binding sites (Yablonka-Reuveni et al.,

bis(@aminoethyl 10; MHC, myosin ’ Fetal

249

myoblasts

refer

to cells from

Day

0012-1606/91 Copyright All rights

7 and on chicken

embryos.

$3.00

Q 1991 by Academic Press, Inc. of reproduction in any form reserved.

250

DEVELOPMENTALBIOLOGY V0~~~~148,1991

1990; in preparation); and have a different response to merocyanine 540 (Nameroff and Rhodes, 1989). Studies on mammalian myoblasts (reviewed by Cossu and Molinaro, 1987) have shown differences between fetal and adult myoblasts in their sensitivity to a tumor promoter (Cossu et ah, 1983), in expression of acetylcholine receptors (Cossu et al., 198’7), and in the form of acetylcholinesterase expressed (Senni et al., 1987). In this study we report differences in the regulation of embryonic myosin expression in fetal myoblasts from Day 10 chicken embryos (El0 myoblasts) and in adult chicken myoblasts. Immunolabeling with specific monoclonal antibodies revealed that ventricular and embryonic myosins are expressed upon differentiation in El0 cultures in both mono- and multinucleated cells. Adult cultures at first express only ventricular myosin in differentiated mononucleated cells (myocytes) and newly formed myotubes. Blocking fusion prevents embryonic myosin expression in adult but not in El0 cultures. These results identify a distinct difference in fast myosin expression in fetal and adult myoblasts, supporting the hypothesis that these cells may comprise distinct precursor populations. METHODS Animals

Embryonated chicken eggs (White Leghorn) and adult chickens (White Leghorn) were purchased from local growers. Eggs were maintained in a forced air incubator at 37.5”C. In preliminary experiments we used Hubbard chickens kindly provided by Acme Poultry (Seattle, WA). No differences were observed in the results obtained with different strains. Isolation

and Culture

of Adult Myoblasts

Satellite cells from 12- to 16-week chickens were isolated from the pectoralis muscle as previously described (Yablonka-Reuveni et ah, 1987; Yablonka-Reuveni, 1989) with some modifications. Five grams of breast muscle was finely minced into l- to 2-mm2 fragments. The fragments were treated with 0.04% collagenase (Worthington, Freehold, NJ) in a final volume of 30 ml of Eagle’s minimum essential medium (MEM) for 45 min at 37°C with gentle stirring. The treated tissue was then collected by centrifugation at approximately 300~ for 1 min. The tissue was redigested in 30 ml trypsin solution (final concentration 0.1% in MEM) for 45 min at 37°C with gentle stirring. Following trypsinization the digest was brought up to 50 ml with MEM containing 10% horse serum (Sigma, St. Louis, MO) and collected by centrifugation (300g for 10 min). The pellet was resus-

pended in 40 ml MEM and aliquots of 5 ml were triturated 30 times each with a Pasteur pipet. By maximizing the volume of the cell suspension per original amount of tissue (8 ml for every 1 g of tissue) the viscosity of the suspension was reduced, which resulted in higher cell yields (up to lo6 cells/g tissue). The cell suspension was filtered through three double nitex filters, pore size 50 pm (Tekco, Elmsford, NY) and loaded onto twelve 20/ 60% Percoll gradients (8 ml 20% layered on a 2-ml cushion of 60%) in 15 ml Sorvall polycarbonate tubes (DuPont, Wilmington, PA). The gradients were prepared and spun as previously described (Yablonka-Reuveni et aZ., 1987; Yablonka-Reuveni, 1989). The cells at the 20/ 60% interface from each gradient were pooled, and the resulting cell suspension was diluted 1:5 with MEM and centrifuged to pellet the cells (300~ for 10 min). The cell pellet was resuspended in complete medium (85% MEM, 10% horse serum, 5% chicken embryo extract, and penicillin and streptomycin at lo5 units per liter each). Cells were plated at l-2 X lo5 per 35-mm dish for mass cultures and 100-200 cells per 60-mm dish for clonal cultures using complete medium as above. Dishes (Corning, Corning, NY) were precoated with 2% gelatin (Sigma). Medium was replaced 24-30 hr after plating and every other day thereafter for mass cultures and every third day for clonal cultures. Isolation

and Culture

of El0 Myoblasts

Primary cultures were prepared from the pectoralis muscles of lo-day embryonic chicks essentially as previously described (Yablonka-Reuveni et al, 1988). The muscle was excised, finely minced, and dissociated into single cells by trypsin digestion. The crude cell suspension was enriched for myogenic cells by Percoll density centrifugation using polycarbonate tubes as for adult myoblasts (one gradient per three embryos). Cells were plated as mass and clonal cultures as described above for adult myoblasts. Collagenase was not included in routine preparations of El0 myoblasts but was included in some experiments. No significant differences were observed in results obtained with El0 myoblasts isolated with or without collagenase digestion. Subcultures

of El0 and Adult

Myoblasts

For experiments with subcultured cells, primary cultures were trypsinized for 5 min at 37°C with 0.05% trypsin in MEM, passed through a double lens paper filter to eliminate myotubes, and replated (YablonkaReuveni et al., 1987). El0 cultures were subcultured on Day 2 of culture and the secondary cultures obtained were again subcultured 24 hr later. Adult myoblasts were subcultured on Day 3 and secondary cultures were

MHC Regulation by Satellite Cells

HARTLEY,BANDMAN,ANDYABLONKA-REUVENI

subcultured again at 24 hr. All cells were plated x lo5 per 35-mm dish. Cold Injury

at 1

of Adult Muscle

Cold injury of adult pectoral muscle was performed to induce proliferation of satellite cells in the animal (Cerny and Bandman, 1987). The birds were anesthesized either by an intravenous injection of sodium pentabarbitol (about 30 mg/kg chicken) or by an intramuscular injection of first Ketalar (80 mg/kg chicken) followed by Equisthesin (15 mg/kg chicken) into the thigh. A 5-cm incision was made in the skin of the right breast and a cylindrical stainless steel rod cooled in liquid nitrogen was pressed on the muscle for 10 sec. Ten to twelve spots of approximately 1 cm in diameter were made per exposed muscle and the incision sutured. Activated adult myoblasts were isolated from the injured region 3-5 days followed injury and cultured as described above for adult myoblasts. Cells isolated from the same region of the contralateral uninjured muscle served as a control. EGTA Treatment of Cultures

When indicated, EGTA was added to El0 and adult cultures 24 hr postplating at a final concentration of 1.8-2.2 mM to block fusion of differentiated myoblasts (Paterson and Strohman, 1972). Since the calcium concentration varies depending on the lot of horse serum and embryo extract, the EGTA concentration needed to block fusion, but not interfere with attachment and spreading, was determined for each preparation. Myosin Heavy Chain Isofwm Detection Single immunojluorescent labeling. Expression of myosin in mass cultures was assayed using isoform-specific monoclonal antibodies (mAbs) in an indirect immunofluorescence assay as previously described (Hartley and Yablonka-Reuveni, 1990). Embryonic fast myosin heavy chain was detected using mAb EB165. EB165 reacts with all known fast embryonic isoforms of MHC in the chicken and also with the adult fast isoform (Cerny and Bandman, 198’7; Bandman et al., 1990). As determined with a mAb against adult MHC, the adult isoform is not expressed in l- to 7-day-old myogenic cultures from El0 (Yablonka-Reuveni et al., 1990; Hartley and Yablonka-Reuveni, 1990) and adult pectoral muscle (data not shown). Therefore, reactivity with EB165 is due to the presence of the embryonic isoform. Ventricular myosin was detected with mAb HVII, which is specific for chicken ventricular myosin (Bandman et al., 1990; Bourke et al., 1991). In some experiments mAb

251

MF20 was used as a general marker for all sarcomeric myosins (Bader et ah, 1982; Zadeh et al., 1986). MF20 was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, John Hopkins University School of Medicine (Baltimore, MD) and the Department of Biology, University of Iowa (Iowa City, IA) under contract NOl-HD-6-2915 from the NICHD. Cultures were fixed for 30-60 set in a cold solution of 70% ethanol:formalin:acetic acid (AFA, 20:2:1) and incubated for at least 1 hr in 0.05 MTris, 0.15 MNaCl, and 1% normal goat serum to block nonspecific binding. Cultures were then incubated for 1 hr at room temperature with antibodies diluted in blocking solution (EB165 and HVll in ascites fluid form diluted l:lOOO, MF20 as a hybridoma supernatent diluted 1:5). This was followed by incubation with fluorescein-conjugated goat antimouse IgG (Organon-Technika Cappel, Downington, PA) diluted 1:lOOO. Nuclei were counterstained with ethidium bromide at a concentration of 2 mg/ml in 0.05 M Tris, 0.15 M NaCl for 5 min at room temperature, followed by three rinses with the same buffer (Hartley and Yablonka-Reuveni, 1990). Cultures were viewed with a Zeiss photomicroscope equipped with phase and epifluorescence optics. Ethidium bromide-stained nuclei were visualized with rhodamine optics. Photomicrographs were taken using fluorescein optics, under which nuclear staining is still detectable but is dimmer than with rhodamine optics. Double immunojluorescent labeling. Embryonic and ventricular myosin were colocalized in El0 and adult myogenic cultures using double-immunolabeling. For double-labeling of primary mass and clonal cultures, cultures were incubated first with EB165, followed by rhodamine-conjugated goat anti-mouse IgG (OrganonTechnika Cappel) diluted 1:200 in blocking solution as described for single-labeling. Cultures were then treated with 1% normal mouse serum for 1 hr to block nonspecific binding and finally reacted with fluorescein-conjugated HVll (1:5000). Fluoresceinated HVll was prepared as described (Goding, 1976) with the following modifications: Fluorescein isothiocyanate was dissolved in acetone at -20°C (1 mg/ml) and unbound fluorochrome was removed with extensive dialysis in PBS for at least 4 days. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 1 pg/ml) for 5 min at room temperature and visualized using Hoescht filters. To ensure that HVll reacts only with its antigen and not nonspecifically with the goat anti-mouse IgG we double-labeled (as above) smooth muscle cultures prepared from El0 aortic arches with mAb 2HlO indirectly labeled with rhodamine-conjugated goat anti-mouse

DEVELOPMENTALBIOLOGY V0~~~~148,1991

252

micrograph showing reactivity of myogenic FIG. 1. A representative cultures from adult chicken with an anti-ventricular myosin mAb. Reactivity was visualized using fluorescein-conjugated goat antimouse IgG (cytoplasmic fluorescence). Nuclei were counterstained with ethidium bromide (nuclear fluorescence). Micrograph was taken using Auorescein optics. Arrows indicate myosin-negative cells. Bar, 30 fim.

IgG and with directly labeled HVll. mAb 2HlO was originally prepared by immunizing mice with chicken satellite cells (Yablonka-Reuveni, 1988) and also reacts with vascular smooth muscle cells in viva and in culture (Z. Yablonka-Reuveni, unpublished results). There was no staining of smooth muscle cultures for ventricular myosin while 2HlO specifically stained the cytoskeleton of smooth muscle cells. Quantitation of myosin-positive cells. The number of myosin-positive cells was determined by quantitating the nuclei within mononucleated and multinucleated cells reacting with the myosin mAbs. Nuclei were stained with ethidium bromide or DAPI. At least 500 nuclei in 10 or more fields were counted using duplicate plates. In some experiments the number of mononucleated myosin-positive cells was determined as well as the total myosin-positive cells. All experiments were repeated at least twice with similar results.

most differentiated myocytes and myotubes in 3- to 5day-old adult cultures were not reactive with monoclonal antibodies specific for embryonic, neonatal, or adult fast MHC. Recent studies in the chicken reported the expression of both ventricular and embryonic MHC in early muscle development of both fast and slow muscles, in nascent fiber formation, in overloaded adult muscle, and in regenerating adult muscle (Kennedy et ah, 1989; Sweeney et al., 1989; Stewart et al, 1989). In light of this we asked whether the initial myosin expressed in adult myocytes and myotubes was the ventricular isoform. Using a mAb against ventricular myosin we found that both myocytes and myotubes were reactive with this antibody (Fig. 1). The frequency of adult cells reacting with the anti-ventricular and anti-embryonic myosin mAb at different time points in culture is shown in Fig. 2. Cells reacting with the anti-ventricular mAb are first detected on Day 3 in adult cultures (Fig. Z), at the same time that the first MFZO-positive cells are present (data not shown). The frequency of these cells increases with time in culture as myoblasts differentiate and form myotubes. Myocytes reactive with the mAb against embryonic myosin are rarely seen in adult cultures (see Table 1 for double label results) but after fusion, which has begun by Day 4, myotubes begin to react with this antibody (Fig. 2). Faint staining is initially present in only a few myotubes, and myotubes may differ in stain-

RESULTS

Myosin Isoform Expression Bfers Myogenic Cultures

in El0 and Adult

Employing a monoclonal antibody against all isoforms of sarcomeric myosin (MFZO) we observed that cultured adult chicken myoblasts began expressing myosin on Day 3 of culture, 2 days later than El0 myoblasts. These results are consistent with the delayed fusion in adult compared to embryonic cultures reported et aZ., 1987). Surprisingly, previously (Yablonka-Reuveni

FIG.2. Frequency of myosin-positive cells in El0 and adult primary myogenic cultures. Duplicate cultures were labeled at each time point by indirect immunofluorescence with either anti-embryonic or antiventricular myosin mAbs. Nuclei were counterstained with ethidium bromide and the number of nuclei within myosin-positive ceils versus total nuclei was determined. At least 500 nuclei in 10 or more fields were counted per plate. The graph represents data from an average of four experiments. Error bars represent standard error of the mean (SEM); where there are no error bars, the symbols are bigger than the SEM. El0 values decrease after 3 days due to continued proliferation of nonmyogenic cells. emb, embryonic MHC; vent, ventricular MHC.

HARTLEY, BANDMAN, AND YABLONKA-REUVENI

MHC

Regulaticm

b2~ Satellite

253

Cells

TABLE 1 DISTRIBUTION OF El0 AND ADULT CELLS REACTING WITH ANTI-VENTRICULAR AND/OR ANTI-EMBRYONIC MYOSIN mAbs Nuclei in E+V+ cells Source of cells El0

Culture age (dam)

Total nuclei

(Number of fields)

1

500 501 540 514 604 755 1173 1314

(41) (45)

2 3 4 Adult

1 2 3 4 5 6

504 501 506 506 531 508 1409 1066 2802 2013 4251 3791

(16) (16) (10) (10) (10) (10) (58) (42) (40) (35) (13)

06) (10) (10) (10) (10) (10) (10)

All

Nuclei in E’ cells

Total nuclei in myosin+ cells

Nuclei in V+ cells All

Mono

All

Mono

Mono

All

Mono

20 13 94 109 225 294 359 333

18 13 65 88 60 33 15 12

5 5 3 7 0 0 0 1

5 5 3 7 0 0 0 1

2 3 18 10 27 40 47 20

2 3 14 10 15 32 20 20

27 21 115 126 252 334 506 353

25 21 x2 105 75 65 35 33

0 0 0 0 0 0 0 0 37 20 1029 958

0 0 0 0 0 0 0 0 2 0 1 1

0 0 0 0 0 0 0 3 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 1 1 4 2 33 13 1035 649 302 142

0 0 1 1 4 0 23 9 381 246 31 31

0 0 1 1 4 2 33 16 107“ 669 1474 1100

0 0 1 1 4 0 25 9 383 246 32 32

Note. Duplicate cultures were double-labeled with mAbs against ventricular and embryonic myosin as for Fig. 6. Nuclei were counterstained with DAPI and the nuclei within myosin-positive cells (total and mononucleated) were counted. Data are from one experiment. ET+, nuclei within cells positive for both ventricular and embryonic myosin; E+, nuclei within embryonic-only myosin-positive cells; V’, nuclei within ventricular-only myosin-positive cells; mono, mononucleated cells; all, mononucleated cells and myotubes.

ing intensity along their length. By the sixth culture day the majority of the myotubes are stained brightly with both embryonic and ventricular MHC mAbs. In comparison, as demonstrated in Fig. 2, 24-hr-old El0 myogenic cultures already contain differentiated cells that stain with mAbs against both embryonic and ventricular myosins (see Fig. 6 for micrographs of double label experiments). The percentage of nuclei in cells reactive with both antibodies increases until the third day of culture as myoblasts differentiate and fuse into myotubes (Fig. 2). El0 myocytes and myotubes stain with both the ventricular and the embryonic antibodies at all timepoints examined. In these experiments the maximum percentage of differentiated cells in El0 cultures is consistently higher than that of adult cultures. This is probably not due to a higher proportion of nonmyogenic cells in adult cultures but rather to the nature of adult myoblasts, since clonal studies show that adult cultures have a greater proportion of myogenic to nonmyogenic clones as well as fewer differentiated cells per myogenic clone as compared to El0 cultures (data not shown, see also Yablonka-Reuveni et al., 1987).

A Staggered Myosin Expressim Pattern Is an Inherent Property of Adult Myoblasts

To determine if the adult pattern of myosin expression is an inherent property of adult myoblasts or is due to their quiescence at the time of isolation, myosin isoforms were examined in subcultured cells and in cells isolated from cold-injured adult muscle, where satellite cell proliferation is induced in vivo prior to isolation. Figure 3 demonstrates that subcultured adult myoblasts react with the anti-ventricular myosin mAb beginning on Day 1 of the tertiary culture, and the antiembryonic myosin mAb on Day 3, after fusion has occurred, similar to adult myoblasts in primary culture. In this experiment embryonic MHC staining did not reach levels comparable to those of ventricular myosin, such as that seen in primary cultures. This may be due to either detachment of the myotubes or proliferation of nonmyogenic cells. Figure 3 also shows that subcultured El0 myoblasts react with both mAbs concomitantly as do El0 myoblasts in primary cultures. As shown in Fig. 4, adult myoblasts isolated from regenerating muscle also maintain a staggered myosin expression pattern similar to primary and subcultured

DEVELOPMENTAL Adult

El 0 cultures A-A rmb 0-n vent

70

A---A m-m

BIOLOGY

VOLUME

148. 1991

cultures emb vent

60

A

A 1

2

3 Days

4

I” Culture

FIG. 3. Frequency of myosin-positive cells in El0 and adult tertiary cultures. El0 cells were subcultured on Days 1 and 2, and adult cells were subcultured on Days 3 and 4 of culture. Duplicate cultures were labeled and data were obtained as for Fig. 2. Experiment was repeated twice with similar results. Graph shows data from one experiment using duplicate plates. Standard deviation is less than 5%. emb, embryonic MHC; vent, ventricular MHC.

adult myoblasts. In this experiment cells were isolated 5 days following injury. Similar results were obtained with cells isolated 3 days postinjury. EGTA

Treatment Prevents Expression of Embryonic Myosin in Adult but Not El0 Cultures

Embryonic myosin is expressed after fusion in adult cultures but prior to fusion in El0 cultures. Thus, we

T. 50

I

c Ll :.

Adult A-A C-G

SO

Control A-A u-0

30 fi1,

cultures

Treated

cultures

emb vent

A--A

emb vent

m-m

1

00 s Days

I” Culture

FIG. 5. Frequency of myosin-positive cells in El0 and adult myogenie cultures treated with EGTA. (A) El0 and (B) adult cultures. EGTA was added 1 day postplating (Day 1 on graph). Myosin labeling and quantitation were as for Fig. 1. Experiment was repeated twice with similar results. (A) Graph represents data from one experiment using duplicate plates. Error bars represent standard deviation; where there are no error bars, the symbols are larger than the standard deviation. (B) Graph represents one experiment using single plates. The experiment was repeated once with similar results. emb, embryonic MHC, vent, ventricular MHC.

cultures emb vent

1

20

c ~~ 0

R

IO

: 0 1

2

3

4 Days

5

6

7

8” Culture

FIG. 4. Frequency of myosin-positive cells in adult myogenic cultures prepared from regenerating muscle. Adult myoblasts were isolated 5 days postinjury from cold-injured pectoral muscle. Myosin labeling and quantitation were as for Fig. 1. Experiment was repeated twice, with cell isolations at 3 and 5 days postinjury for each experiment, with similar results. Graph represents data from one experiment using duplicate plates. Error bars represent standard deviation; where there are no error bars, the symbols are bigger than the standard deviation. emb, embryonic MHC; vent, ventricular MHC.

tested whether blocking fusion with EGTA would prevent expression of embryonic MHC in adult but not El0 cultures. In fusion-blocked El0 cultures the percentage of cells staining with anti-ventricular and anti-embryonic myosin mAbs is similar to that of untreated cultures (Fig. 5A), although fewer cells are reactive with the embryonic myosin mAb. In adult cultures, the percentage of cells positive with the anti-ventricular myosin mAb is similar to untreated cultures while reactivity with the anti-embryonic myosin mAb is essentially abolished (Fig. 5B). The highest frequency of mononucleated cells reacting with the anti-embryonic myosin mAb in adult cultures is 0.4%, even up until 10 days of culture

HARTLEY,BANDMAN,ANDYABLONKA-REUVENI

(data not shown). A rescue experiment was done in which fresh media without EGTA was added to EGTAblocked adult cultures on Day 6. Large myotubes formed within 12 hr and 6 and 62% of nuclei within myotubes were in embryonic MHC-positive myotubes by 2 and 4 days after the rescue, respectively. These results suggest that fusion may be a prerequisite for embryonic myosin expression in adult but not in El0 myogenic cultures. Double-Immunolabeling Shows That El0 Myoblasts Coexpress Embryonic and Ventricular Myosin in Mass Cultures

MHC Regulatim

by Satellite Cells

255

percentage of mononucleated cells labeling just with the mAb against ventricular myosin is low, while even fewer cells label with the anti-embryonic myosin mAb alone (Fig. 7A). The latter are seen only on the first 2 days in culture. Adult myoblasts are uniform in their myosin staining patterns (Table 2 and Fig. 7B). Until the fifth day in adult cultures all myosin-positive mononucleated cells and myotubes label with just the anti-ventricular myosin mAb. By the fifth day a small percentage of the nuclei are in myotubes colabeling with both myosin mAbs (Fig. 7B). In a 5-day adult culture 36% of the nuclei are in myosin-positive cells; of these 3% are in myotubes which colabel with both mAbs compared with 97% in cells labeling with the ventricular mAb only. Mononucleated cells account for 36% of these ventricular reactive cells whereas there are no adult myocytes labeling with just the anti-embryonic myosin mAb. On the sixth day, about 30% of total nuclei are in myosin-positive cells; of these 82% are in myotubes that colabel with both mAbs compared to 18% in cells labeling with the ventricular mAb only (the majority of which are in myotubes).

Since El0 myoblasts label concomitantly with either anti-ventricular or anti-embryonic MHC mAbs, single labeling of these cultures cannot determine if a myoblast can express both or only one isoform. We thus conducted double-immunolabeling experiments to resolve whether El0 and adult myoblasts and myotubes consist of subpopulations based on the myosin isoform(s) expressed upon differentiation. Figure 6 illustrates double labeling of 5-day (a and b) and 6-day (c and d) adult cultures and 2-day (e and f) El0 cultures with anti-ventricular and anti-embryonic Ckmal Analysis of Double-Labeled El0 and Adult MHC mAbs. These micrographs show that El0 myoCultures cytes react with both anti-ventricular and anti-embryonic myosin mAbs (Figs. 6e and 6f) as do adult myotubes Clonal studies of El0 and adult cultures were per(Figs. 6c and 6d), but not adult myocytes and newly formed to further analyze the nature of the myoblast formed adult myotubes (Figs. 6a and 6b). Cells reacting populations with respect to myosin expression. Of 51 just with the anti-ventricular mAb can be seen in El0 El0 myogenic clones examined after 5 days in culture, and adult cultures (Fig. 6a; arrows in 6c and se), while 48 contained mono- and multinucleated cells reactive cells reacting with just the anti-embryonic myosin mAb with both embryonic and ventricular myosin mAbs and are only present in El0 cultures (Fig. 6f, arrowhead). 3 were reactive with the ventricular mAb only (Table 1). Table 1 and Fig. ‘7 summarize the double labeling results After 10 and 15 days in culture all myogenic clones were for El0 and adult mass cultures. Table 1 shows the numpositive with both antibodies. In addition to double-laber of nuclei in myocytes and myotubes reacting with beled myotubes, some of the clones assayed at 15 days anti-ventricular and/or anti-embryonic myosin mAbs contained myotubes that labeled with the anti-embryfor El0 and adult duplicate cultures. Data from this ta- onic mAb but not with the anti-ventricular mAb (7 of ble were used to calculate the percentage of cells posi- 42). This suggests a possible progression from expresstive for each myosin isoform and the percentages from ing both isoforms to expressing only embryonic fast duplicate cultures were averaged and plotted in Fig. ‘7. myosins. Myotubes in individual clones varied in stainAs seen in Table 1 and Fig. 7A on the first day in El0 ing intensity, as did individual clones. cultures 4.8% of the total nuclei are in myosin-positive In 7-day clonal cultures from adult muscle, myotubes cells. Of these, 69% are in myocytes which are positive had not yet formed and myosin-positive cells were not for both antibodies; 21% in myocytes positive for the found (Table 2). Myotubes and myocytes in 11-day adult embryonic mAb only and 10% are positive with the ven- clonal cultures reacted only with the anti-ventricular tricular mAb only. By the third day 43% of the total myosin mAb (43 of 43). In 15-day clones 16 of 44 clones nuclei are in myosin-positive cells; 89% of these nuclei contained myotubes reactive with both ventricular and label with both antibodies, 0% with the embryonic mAb embryonic myosin mAbs, while 28 clones stained with only, and 11% with the ventricular mAb only. Thus, in the anti-ventricular myosin mAb only, suggesting a proEl0 cultures the majority of differentiated cells colabel gression from expressing just ventricular myosin to exwith both mAbs, whether mono- or multinucleated. The pressing both embryonic and ventricular myosins.

256

DEVELOPMENTAL

BIOLOGY

VOLUME

148.1991

FIG. 6. Representative micrographs of El0 and adult myogenic cultures double-labeled with anti-ventricular and anti-embryonic myosin mAbs. Cultures were reacted with anti-embryonic myosin mAb indirectly labeled with rhodamine-conjugated goat anti-mouse (b, d, f) followed by directly fluoresceinated anti-ventricular myosin mAb (a, c, e). (a and b) Five-day adult cultures, (c and d) 6-day adult cultures, and (e and f) 2-day El0 cultures. Arrows indicate ventricular-positive, embryonic-negative cells; arrowhead indicates embryonic-positive, ventricular-negative cell. Cells which did not stain with either myosin antibody are also present in the cultures. Bar, 30 pm.

DISCUSSION

In this study we examined the expression of ventricular and embryonic MHC in fetal (ElO) and adult chicken myogenic cultures using isoform-specific monoclonal antibodies. El0 and adult chicken myocytes differ in their reactivity with these antibodies when cultured under identical conditions. El0 myocytes that contain both ventricular and embryonic myosins are present 24 hr after culturing. Following fusion, myotubes in El0 cul-

tures also contain both ventricular and embryonic myosins. In adult cultures myosin-positive cells are not seen until the third day, and these mononucleated cells contain only ventricular myosin. Embryonic myosin is first detected in ventricular-expressing adult myotubes on the fifth culture day, subsequent to fusion. Consistent with these results, El0 clonal cultures are composed primarily of clones coexpressing both myosins while all adult clones initially express just ventricular myosin. The staggered expression of ventricular and embryonic

HARTLEY,

BANDMAN,

AND YABLONKA-REUYENI

MHC Regulation by Satellite Cells

257

FIG. 7. Frequency of myosin-positive cells in double-labeled El0 and adult mass cultures. Duplicate cultures were double-labeled at each time point by indirect immunofluorescence with the anti-embryonic myosin mAb and directly fluoresceinated with anti-ventricular antibody. Nuclei were counterstained with DAPI and the percentage of nuclei (both mononucleated and total) within myosin-positive cells was determined. The graphs are based on data from Table 1. Error bars depict standard deviation; where there are none, the symbols are bigger than the standard deviation. (A) El0 cultures. (B) Adult cultures. Upper panels of both A and B show myosin-positive cells both as mononucleated cells and in myotubes; lower panels show mononucleated myosin-positive cells only. emb/vent, cells reactive with embryonic and ventricular MHC mAbs; emb, cells reactive with embryonic MHC mAb; vent, cells reactive with ventricular MHC mAb; total, all myosin-positive cells.

myosin is also present in subcultured adult myoblasts and cultures of activated satellite cells from regenerating muscle. Thus, the lag in embryonic myosin expression is an inherent property of adult myoblasts and not due to their quiescence upon isolation. Our results are based on reactivity with mAbs EB165 and HVll, which have been shown to distinguish between embryonic fast MHC isoforms and ventricular MHC, respectively, via isoform-specific epitopes (Cerny and Bandman, 1987; Bandman et al., 1990; Bourke et ah, 1991). As a result of the mAb specificity we were able to analyze the expression of these isoforms by El0 and adult myogenic cultures and to resolve a progression of first ventricular myosin expression and then, after fusion, embryonic myosin expression in adult myogenic cultures. The difference was first apparent because

adult myotubes did not initially express embryonic MHC. Moreover, labor intensive single cell counts in combination with specific immunolabeling enabled us to detect and quantitate MHC-positive mononucleated cells. The presence of these myocytes is not surprising, as myoblasts first withdraw from the cell cycle and then terminally differentiate and become fusion capable. Since fusion and differentiation are temporally separated, it is reasonable to assume that at each time point examined myocytes will be present. The ability to detect these cells led to the identification of subpopulations of myoblasts, differing in their myosin expression pattern, e.g., those expressing ventricular MHC only (El0 and adult cultures) and those expressing both ventricular and embryonic MHC (El0 cultures). Since EB165 reacts with all known isoforms of embryonic myosin (of which

258

DEVELOPMENTAL

TABLE 2 REACWITY OF El0 AND ADULT MYOGENIC VENTRICULAR AND/OR ANTI-EMBRYONIC

CLONES WITH ANTMYOSIN mAbs

Number Source of cells

BIOLOGY

of clones

Culture age (days)

E+V+

E+

V+

El0

5 10 15

48 38 42

0 0 0

3 0 0

51 38 42

Adult

7 11 15

0 0 16

0 0 0

0 43 28

0 43 44

Total

myosin+

Note. Cells were plated at a clonal density and myogenic clones were marked prior to fixation and staining. Five cultures were fixed for each time point and double-labeled as for Table 1, except nuclei were not counterstained. E+V+, clones with cells (mononucleated cells and myotubes) positive for both ventricular and embryonic myosin; E+, clones with cells positive for embryonic myosin only; V+, clones with cells positive for ventricular myosin only.

there may be at least three, Umeda et ah, 1981; Van Horn and Crow, 1989; Lagrutta et al., 1989; Hofmann et al., 1988), it is possible that adult and fetal cultures express different isoforms of embryonic myosin. Development of probes specific for the different embryonic isoforms is needed to resolve this issue. Other myosin isoforms not analyzed by us, such as chicken slow myosin, SMl (Feldman and Stockdale, 1991), may also be present or differentially expressed in fetal and adult chicken muscle cultures. Our results suggest a correlation between fusion and embryonic myosin expression in adult myogenic cultures. To test whether expression of embryonic fast myosin is dependent on cell fusion, we cultured El0 and adult cells in the presence of EGTA. EGTA prevents fusion but not myosin expression in chicken myogenic cultures (Paterson and Strohman, 1972). In the present study EGTA-treated El0 cultures express both ventricular and embryonic myosin but EGTA-treated adult cultures express only ventricular myosin. If the expression of embryonic myosin in adult cultures is maturation-dependent, such as was shown for neonatal myosin expression (Cerny and Bandman, 1986), blocking fusion may interfere with maturation thus preventing its expression. The defect in recently characterized quail myogenie cell lines which express only the ventricular myosin isoform and do not express actin nor assemble myofibrils may also be explained by a failure to mature (Antin and Ordahl, 1991). The possibility that EGTA interferes with embryonic myosin expression directly, independent of its effects on fusion, is not likely, since embryonic myosin was expressed in EGTA-treated El0 cultures.

VOLUME

148,199l

Previous studies have shown that the embryonic myosin isoform is expressed by newly differentiated adult and fetal myoblasts from fast and slow muscle (Bandman et al., 1982; Matsuda et al., 1983). Recent findings, however, reported that in addition to the embryonic isoforms a ventricular-like isoform is also present in both developing and regenerating muscle (Sweeney et al., 1989; Kennedy et al, 1989; Bandman et al, 1990; Bourke et al., 1991). Our results document that ventricular and embryonic myosin are coexpressed by both myocytes and myotubes from chicken fetal muscle and further show that ventricular myosin is expressed prior to embryonic myosin in adult myogenic cultures. In El0 mass cultures, a small number of cells expressing ventricular myosin and not embryonic myosin were detected as were a small number of El0 clones expressing only ventricular myosin (3 of 48 clones). These cells may be the first “adult-like” myoblasts in El0 embryos. Our preliminary results indicate that El8 myogenic cultures contain even more of these adult-like myoblasts, which suggests that myoblasts with the satellite cell phenotype are present in increasing numbers during development. El0 cultures also contained a few cells which expressed embryonic but not ventricular myosin and eventually disappeared. These cells may have already undergone a progression, as mononucleated cells, from expressing both ventricular and embryonic myosins to expressing embryonic myosin alone. The presence of myotubes expressing just embryonic myosin along with myotubes coexpressing both isoforms in 15-day clones from El0 embryos is evidence for this type of myosin progression in myotubes as well. Satellite cells and fetal myoblasts originate in the somites and presumably belong to the same mesodermal lineage (Armand et al., 1983), but it is not known whether they are the same cells. Previous studies in mammals and in avia (summarized in the introduction) demonstrate differences between fetal and adult myoblasts. These differences may indicate that fetal and adult myoblasts are distinct cells. The current study supports this notion and is also consistent with the hypothesis that there are different lineages of myoblasts. This hypothesis is founded on data showing heterogeneity between myoblasts from the embryonic and fetal developmental periods based on the type of MHC expressed by their myotubes in culture (for a review see Stockdale and Miller, 1987; Miller and Stockdale, 1989; Vivarelli et ah, 1988), the response to growth factors (Seed and Hauschka, 1988), the morphology and frequency of myotubes and the media required in culture (Hauschka, 1974; White et ak, 1975; Seed and Hauschka, 1984), and their response to a tumor promoting agent (Cossu et aZ., 1988).

HARTLEY, BANDMAN, AND YABLONKA-REUVENI

Although our results support the hypothesis that El0 and adult myoblasts represent different myogenic lineages, we cannot rule out an alternate hypothesis. Namely, the hypothesis that a single cell type alters its phenotype as its in vivo environment changes during development, growth, and regeneration. The latter hypothesis could be tested by modifying culture conditions in an attempt to change the myosin expression pattern of adult myoblasts to that of El0 myoblasts or vice versa. We are grateful to Maricela V. Pier for excellent technical assistance and to Acme Poultry (Seattle, WA) for their generous donations of adult chickens. This work was supported by grants from the NIH (AR39677 to Z.Y.-R. and AGO8573 to E.B.), American Heart Association, Washington Affiliate (Z.Y.-R.), and Muscular Dystrophy Association (E.B.). R.S.H. was supported by a Predoctoral Developmental Biology Training Grant from the NIH (HD07183). REFERENCES ANTIN, P. B., and ORDAHL, C. P. (1991). Isolation and characterization of an avian myogenic cell line. Dev. Biol. 143,111-121. ARMAND, O., BOUTINEAU, A.-M., MAUGER, A., PAUTOU, M.-P., and KIENY, M. (1983). Origin of satellite cells in avian skeletal muscle. Arch.

Anaf.

72,163-181.

BADER, D., MASAKI, T., and FISCHMAN, D. A. (1982). Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J. CeZl BioL 95, 763-770. BANDMAN, E., BOURKE, D. L., and WICK, M. (1990). Regulation of myosin heavy chain expression during development, maturation, and regeneration in avian muscle: The role of myogenic and non-myogenie factors. In “The Dynamic State of Muscle Fibers” (D. Pette, Ed.), pp. 127-138. Walter de Gruyter, Berlin. BANDMAN, E., MATSUDA, R., and STROHMAN, R. C. (1982). Developmental appearance of myosin heavy and light chain isoforms in viva and in vitro in chicken skeletal muscle. Dev. BioL 3, 508-518. BOURKE, D. L., WILIE, S., WICK, R. M., and BANDMAN, E. (1991). Differentiating skeletal muscles initially express a ventricular myosin heavy chain. Basic Appl. Myol. 1, 13-21. CAMPION, D. R. (1984). The muscle satellite cell: A review. 1&. Rev. Cyfol.

87, 225-247.

CERNY,L. C., and BANDMAN, E. (1986). Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures. J. Cell Biol. 103, 2153-2161. CERNY, L. C., and BANDMAN, E. (1987). Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle. Dev. Biol. 119,350-362. Cossu, G., EUSEBI, F., GRASSI, F., and WANKE, E. (1987). Acetylcholine receptors are present in undifferentiated satellite cells but not in embryonic myoblasts in culture. Dev. Biol. 123, 43-50. COSSU, G., and MOLINARO, M. (1987). Cell heterogeneity in the myogenie lineage. Cum Top. Dev. Biol. 23,185-208. COSSU, G., MOLINARO, M., and PACIFICI, M. (1983). Differential response of satellite cells and embryonic myoblasts to a tumor promoter. De?: BioL 98, 520-524. Cossu, G., RANALDI, G. M., SENNI, I., MOLINARO, M., and VIVARELLI, E. (1988). ‘Early’ mammalian myoblasts are resistant to phorbol esterinduced block of differentiation. Development 102,65-69. FELDMAN, J. L., and STOCKDALE, F. E. (1991). Skeletal muscle satellite

MHC

Regulation

by Satellite

Cells

259

cell diversity: Satellite cells form fibers of different types m cell culture. Dev. Biol. 143,320-334. HARTLEY, R. S., and YABLONKA-REUVENI, Z. (1990). Long-term maintenance of primary myogenic cultures on a reconstituted basement membrane. In Vitro Cell. Dev. Biol. 26, 955-961. HAUSCHKA, S. D. (1974). Clonal analysis of vertebrate myogenesis III. Developmental changes in the muscle-colony-forming cells of the human fetal limb. Dev. BioL 37,345-368. HOFMANN, S., D~~STERH~FT, S., and PE?TE, D. (1988). Six myosin isoforms are expressed during chick breast muscle development. FEBS

Lett.

238,245-248.

KENNEDY, J. M., SWEENEY, L. J., and GAO, L. (1989). Ventricular myosin expression in developing and regenerating muscle, cultured myotubes, and nascent myofibers of overloaded muscle in the chicken. Med. Sci. Sports Exercise 21, S187-S197. LAGRUT~A, A. A., MCCARTHY, J. G., SCHERCZINGER,C. A., and HEYWOOD,S. M. (1989). Identification and developmental expression of a novel embryonic myosin heavy chain gene in chicken. DNA 8, 39-50.

MATSUDA, R., SPECTOR,D. H., and STROHMAN, R. C. (1983). Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms. Dev. Biol. 100, 78-88.

MAURO, A. (1979). “Muscle Regeneration.” Raven Press, New York. MILLER, J. B., and STOCKDALE, F. E. (1989). Multiple cellular processes regulate expression of slow myosin heavy chain isoforms during avian myogenesis in vitro. Dev. BioL 136, 393-404. NAMEROFF, M., and RHODES,L. D. (1989). Differential response among cells in the chick embryo myogenic lineage to photosensitization by Merocyanine 540. J. Cell. Physiol. 141,475-482. PATERSON, B., and STROHMAN, R. C. (1972). Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle. Dev. Biol. 29,113-128. SAAD, A. D., OBINATA, T., and FISCHMAN, D. A. (1987). Immunochemical analysis of protein isoforms in thick myofilaments of regenerating skeletal muscle. Dev. Biol. 119, 336-349. SEED, J., and HAUSCHKA, S. D. (1984). Temporal separation of the migration of distinct myogenic precursor populations into the developing chick wing bud. Deu Biol. 106, 389-393. SEED, J., and HAUSCHKA, S. D. (1988). Clonal analysis of vertebrate myogenesis VIII. Fibroblast growth factor (FGF)-dependent and FGF-independent muscle colony types during chick wing development. Dev. BioL 128,40-49. SENNI, M. I., CASTRIGNANO, F., POIANA, G., Cossu, G., SCARCELI,A, G., and BIAGIONI, S. (1987). Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in cuiture. Dgerentiation 36, 1944198. SNOW, M. H. (1977a). Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. I. Fine structural analysis. Anat. Rec. 188, 182-200.

SNOW, M. H. (197713).Myogenic cell formation in regeneratingrat skeletal muscle injured by mincing. II. An autoradiographic study. Anat.

Rec. 188, 201-218.

STEWART, A. F. R., KENNEDY, J. M., BANDMAN, E., and ZAK, R. (1989). A myosin isoform repressed in hypertrophied ALD muscle of the chicken reappears during regeneration following cold injury. Deu. Biol.

135, 367-375.

STOCKDALE, F. E., and MILLER, J. B. (1987). The cellular basis of myosin heavy chain isoform expression during development of avian skeletal muscles, Dev. Biol. 123, 1-9. STROHMAN, R. C., BAYNE, E., SPECTOR, D., OBINATA, T., MICOU-EASTWOOD, J., and MANIOTIS, A. (1990). Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro. In Vitro Cell. Dev. Biol.

26, 201-208.

260

DEVELOPMENTAL BIOLOGY

SWEENEY, L. J., KENNEDY, J. M., ZAK, R., KOKJOHN, K., and KELLEY, S. W. (1989). Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian myogenesis. Dev. Biol. 133,361-374. UMEDA, P. K., SINHA, A. M., JAKOVCIC, S., MERTEN, S., Hsu, H.-J., SUBRAMANIAN, K. N., ZAK, R., and RABINOWITZ, M. (1981). Molecular cloning of two fast myosin heavy chain cDNAs from chicken embryo skeletal muscle. Proc. NatL Acad. Sci. USA 78,2843-2847. VAN HORN, R., and CROW,M. T. (1989). Fast myosin heavy chain expression during the early and late embryonic stages of chicken skeletal muscle development. Den BioL 134,279-288. VIVARELLI, E., BROWN, W. E., WHALEN, R. G., and Cossu, G. (1988). The expression of slow myosin during mammalian somitogenesis and limb bud differentiation. J. Cell Biol. 107,2191-2197. WHITE, T. P., and ESSER, K. A. (1989). Satellite cell growth and growth factor involvement in skeletal muscle growth. Med Sci. Sports Exercise 21, S158-S163. WHITE, N. K., BONNER, P. H., NELSON, D. R., and HAUSCHKA, S. D. (1975). Clonal analysis of vertebrate myogenesis. IV. Medium-dependent classification of colony-forming cells. Dev. BioL 44, 346361. YABLONKA-REUVENI, Z. (1988). Identification of satellite cell-specific antigens. J. Cell Biochem. lZC(Suppl.), 334.

VOLUME 148, 1991

YABLONKA-REUVENI, Z. (1989). Application of density centrifugation and flow cytometry for the isolation and characterization of myogenie and fibroblast-like cells from skeletal muscle. In “Cellular and Molecular Biology of Muscle Development” (L. H. Kedes and F. E. Stockdale, Eds.), pp. 869-879. A. R. Liss, New York. YABLONKA-REUVENI, Z., ANDERSON, S. K., BOWEN-POPE, D. F., and NAMEROFF, M. (1988). Biochemical and morphological differences between fibroblasts and myoblasts from embryonic chicken skeletal muscle. Cell Tissue Res. 252,339-348. YABLONKA-REWENI, Z., BOWEN-POPE, D. F., and HARTLEY, R. (1990). Proliferation and differentiation of myoblasts: The role of plateletdrived growth factor and the basement membrane. In “The Dynamic State of Muscle Fibers” (D. Pette, Ed.), pp. 693-707. de Gruyter, Berlin. YABLONKA-REUVENI, Z., and NAMEROFF, M. (1990). Temporal differences in desmin expression between myoblasts from embryonic and adult chicken skeletal muscle. &$erentiatim 45,21-28. YABLONKA-REUVENI, Z., QUINN, L. S., and NAMEROFF, M. (1987). Isolation and clonal analysis of satellite cells from chicken pectoral muscle. Dev. BioL 119,252-259. ZADEH, B. J., GONZALEZ-SANCHEZ, A., FISCHMAN, D. A., and BADER, D. (1986). Myosin heavy chain expression in embryonic cardiac cell cultures. Dev. Biol. 115,204-214.