Isolation and Characterization of Terminally ... - Science Direct

DEVELOPMENTAL

BIOLOGY

91,

11-26 (1982)

Isolation and Characterization of Terminally Differentiated Rat Skeletal Muscle Myoblasts STEPHEN Divisim

F. KONIECZNY,

of Biology

and Medicine,

JOHN

MCKAY,

Bmwn

University,

AND JOHN Prmidence,

Chicken and

R. COLEMAN Rhode Island 02912

Received August 17, 1982; accepted in revised fawn January

8, 1982

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chic’ken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48-96 hr of culture in a low-Ca” (25 pm), Me-dep leted medium, chicken myoblasts exhibited only 3-5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of M2+ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg’:+, suggesting that M$+ plays a role in sarcomere assembly. Conditions of Ca2+ and Mg2f depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [3H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both (chicken and rat contain 2cDNA levels, suggesting arrest in the Go stage of the cell cycle.

Stockdale and Holtzer, 1961) demonstrated that the first myoblasts to appear in embryonic chicken somites differentiate prior to fusion. Although this sequence is reversed in older embryonic skeletal muscle differentiation, mononucleated myoblasts which are postmitotic and express a variety of muscle products have been observed in cultures derived from embryonic leg and breast muscle (Okazaki and Holtzer, 1965; Coleman and Coleman, 1968; Linkhart and Hauschka, 1979; Kligman and Nameroff, 1980) as well as from a cell line derived from lizard tail muscle (Bayne and Simpson, 1977). Following the demonstration by Shainberg et al. (1969) that low Ca2+ levels reversibly inhibit rat myoblast fusion, several laboratories reported that avian myoblasts cultured under these conditions withdraw from the cell cycle and synthesize muscle-specific products as mononucleated cells (Fambrough and Rash, 1971; Paterson and Prives, 1973; Emerson and Beckner, 1975; Turner et al., 1976a,b; Moss and Strohman, 1976; Vertel and Fischman, 1976). Other studies have demonstrated that chicken embryo myoblasts will also proceed through terminal differentiation when spontaneous fusion is inhibited by growth in cytochalasin B (CB) (Sanger, 1974; Holtzer et ah, 1975) or phospholipase C (Trotter and Nameroff, 1976).

INTRODUCTION

Skeletal myogenesis in viva and in vitro normally involves the proliferation of individual mononucleated myoblasts, spontaneous cell fusion to form multinucleated muscle fibers, and the subsequent elaboration of a variety of products characteristic of skeletal muscle such as myosin, acetylcholine receptors, and the M subunit of creatine kinase (M-CK)I (Coleman and Coleman, 1968; Fambrough and R,ash, 1971; Turner et ak, 1976a). These processes have been studied extensively in vitro using chick, quail, and rat primary cultures, as well as established myoblast cell lines such as L6 and L8 (see reviews in Buckingham, 1977; Merlie et al., 1977). A number of investigators have examined whether fusion per se is an obligate step in this sequence. Early work by Holtzer and co-workers (Holtzer et al., 1957; 1 Abbreviations used: Ap5A, P’,p-di(adenosine-5’)pentaphosphate; CB, cytochalasin B; CD, cytochalasin D; CK, creatine kinase; DAPI, 4’,6-diamidino-2-phenylindol; IEDTA, ethylenediaminetetraacetic acid; EE, embryo extract; EGTA, et.hylene glycol bis(aminoethyl)tetraacetic acid; EU, enzyme units; HS, horse serum; IgG, immunoglobulin G; MEM, minimal essential medium; MOPS, 3-(n-morpholino)-propanesulfonic acid; PBS, phosphate-buffered saline, pH 7.2; r3H]Tdr, [3H]thymidine. 11

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0 1982 by Academic Press, Inc. of reproduction in any form reserved.

12

DEVELOPMENTAL BIOLOGY

The response of mammalian myoblasts to fusion inhibition is not as consistent as is the case with avian cells. Shainberg et al. (1969, 1971) and Shainberg and Brik (1978) reported that while growth in low Ca2+inhibited fusion in cultures of neonatal rat myoblasts and the L6 myoblast line, the accumulation of characteristic skeletal muscle proteins failed to occur until the fusion block was released by the restoration of normal Ca” levels. By contrast, Wahrmann et al. (1976) found that cultures of rat L6 myoblasts, inhibited from fusing by growth in CB, expressed muscle proteins in amounts comparable to control cultures containing multinucleated skeletal muscle fibers, and similar results were reported by Merlie and Gros (1976) with fetal calf skeletal muscle cells grown in low Ca2+ medium. No information is available on whether individual mammalian myoblasts withdraw from cell cycle activity following fusion inhibition, as do avian myoblasts, and no direct evidence for the production of muscle proteins by individual mammalian myoblasts has been reported. In the work presented here, we (a) confirm earlier findings that individual fusion-inhibited chicken embryo myoblasts undergo terminal differentiation and express a variety of muscle-specific proteins, (b) demonstrate definitively that individual rat L6 and L8 myoblasts differentiate under appropriate fusion-inhibition conditions, (c) describe procedures for the isolation of nearly homogeneous populations of terminally differentiated chicken and rat myoblasts, and (d) present direct evidence from DNA measurements on individual nuclei that these cells are arrested in the Go/G1 phase of the cell cycle. MATERIALS

AND METHODS

Materials Fertile White Leghorn chicken eggs were purchased from Spafas, Balb/c mice from Charles River Laboratories, and the G8 mouse myoblast cell line from The American Type Culture Collection. Falcon and Corning tissue culture plates were used throughout, and medium components were obtained from Gibco Laboratories and Microbiological Associates. Chelex-100 resin was purchased from BioRad, [3H]thymidine ([3H]Tdr) from New England Nuclear, rhodamine goat anti-rabbit IgG from Miles, rabbit antibodies to chicken skeletal muscle myosin from Antibodies Incorporated, and 4’,6-diamidino-2-phenylindol (DAPI) from Boehringer-Mannheim. Quantitative CK assays utilized Sigma Kit No. 45-5, and CK isoenzyme analysis was performed using cellulose acetate strips and reagents from Gelman. Nonidet P-40 was a gift from Dr. Arthur Landy. All other reagents were purchased from Sigma or Fisher. Rat myoblast line E63 and mouse myeloma line NSl

VOLUME 91, 1982

were gifts from Drs. Stephen Kaufman and Boris Rotman, respectively. Rabbit antibodies to chicken myomesin, skeletal muscle desmin, and rat skeletal muscle myosin were gifts from Drs. Hans Eppenberger, Howard Holtzer, and Vivian Nachmias, respectively. Cell Culture Primary cultures of chicken myoblasts were obtained from ll- to 12-day embryos by procedures similar to those reported earlier (Coleman and Coleman, 1968; Colbert and Coleman, 1977). Leg musculature was removed, minced with scissors, and triturated several times in 10 ml of growth medium until most of the large fragments were dispersed. The suspension was filtered initially through a double layer of lens paper and then through a nylon filter of 20-pm pore size, resulting in a single cell suspension. Cultures were inoculated with 6 X lo5 cells per 60-mm gelatinized plate in growth medium which consisted of Eagle’s minimal essential medium supplemented with nonessential amino acids and 10% heat-inactivated horse serum plus 2.5% embryo extract (MEM, 10% HS, 2.5% EE = control medium). The medium also contained 100 units/ml penicillin and 100 pg/ml streptomycin. Chicken muscle fibroblasts were isolated by incubating cultures under the conditions described above until maximum muscle fiber formation had occurred (ca. 5 days), then trypsinizing and replating to select against differentiated cells. Three repeated passages under these conditions resulted in populations of cells which are fibroblastic in morphology, do not form muscle fibers even after extended culture periods, and do not stain with antibodies against chicken M-CK or skeletal muscle myosin. Rat myoblast cell lines (Yaffe, 1968; Richler and Yaffe, 1970) were grown in MEM supplemented with nonessential amino acids, 10% HS, and antibiotics, but lacking embryo extract. L6Jl is a cloned subline of L6 (Ringertz et al., 1978) and E63 is a cloned subline of L8 (Kaufman et al., 1980). Cells were plated at an initial density of 1.5 X lo5 per loo-mm nongelatinized dish and fed fresh medium every other day. Subculture was routinely performed prior to confluency using 0.05% trypsin in Puck’s Saline A. Several plates were saved at each passage, allowed to grow to eonfluency, and monitored for their ability to produce myotubes. In most cases, cells beyond passage 15 were not used. Mouse myoblast line G8-2 was cloned in our laboratory from line G8 (Christian et al., 1977) and was maintained in the same fashion as described above for L6Jl and E63. For determination of colony forming efficiency, initial plating densities ranging from 200 to 1050 cells per loo-mm dish were used. Cultures were grown for 2

KONIECZNY, MCKAY, AND COLEMAN

weeks in MEM, 10% HS, fixed in methanol, and stained with Giemsa. Colonies were counted under a dissecting microscope. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. All cell lines were free of mycoplasma contaminants according to the DAPI fluorochromasia assay (Chen, 1977). Fusion Inhibition

Media

Medium depleted in Caz+ and Mgz+ was prepared using a modification of the Chelex method described by Paterson and Prives (1973). Chelex-100 resin was sterilized overnight in 95% ethanol, the ethanol was decanted, and the Chelex resin equilibrated with Ca2+Mg2+-free MEM. A 10% slurry of Chelex resin and horse serum-embryo extract (110:2.5ratio) was prepared and allowed to stir overnight at 4°C. The resin was removed by centrifugation, Ca’+-M$+-free MEM was added to yield a final concentration of 10% HS, 2.5% EE, and CaClz was added to a final concentration of 25 pM ( =Ca2+,Mgz+-depleted medium). Medium depleted only in Ca2+ (25 PM) was prepared either by supplementing Chelex-treated medium with 0.81 mMMgS04 or by adding EGTA to control medium to a final concentration of 1.90 mM. Cytochalasin B (CB) medium consisted of the appropriate control medium to which was added 2.5 or 5.0 pg/ml CB for fusion inhibition of rodent or chicken embryo myoblasts, respectively. In some experiments, cytochalasin D (CD) at a concentration of 0.25 yg/ml was used instead of CB. Fusion Inhibition

Procedures

Ca2+,M2+-depleted medium was added to chicken cells 24 hr after plating 90%) spontaneous fusion (Table 3). CB-treated cells fed control medium on Day 7 formed networks of multinucleated myotubes by Day 8 and few single cells remained by Day 9. L6Jl and E63 myoblasts behaved similarly under all conditions tested except that cultures of E63 always developed slightly higher CK specific activities than did the L6Jl cells (Table 3). Electrophoretic analyses demonstrated that in all cases only the MM isoenzyme of CK was present (Fig. 3). The experiments described above suggested that rat myoblasts are capable of differentiation in the absence of spontaneous cell fusion. To determine whether the entire cell population expresses this capability, or whether only a subset of cells does so, the two classes of cells which accumulate during 48 hr of CB treatment were separated and analyzed for CK activities and for the presence of skeletal muscle myosin. Vortexing the plates or vigorously rinsing with PBS detached approximately one-third of the spherical cells. The remaining two-thirds were removed by brief treatment with 0.05% trypsin at room temperature, carefully monitored by phase microscopy, until most of the spherical cells came free and the flat, firmly adherent cells remained attached to the substratum. CK assays were performed on CB-treated cultures prior to trypsinization, on the spherical cell population removed by PBS rinsing and selective trypsinization, and on the population remaining attached after selective trypsinization (Table 4). The CK specific activity of the spherical cells

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DEVELOPMENTALBIOLOGY

VOLUME 91, 1982

TABLE 5 ANTI-SKELETAL MUSCLE MYOSIN IMMUNOFLUORESCENT STAINING OF CONTROL AND CYTOCHALASIN B-TREATED E63 RAT MYOBLASTS Cell treatment

Day of fixation

Control Fusion inhibited Fusion inhibited

5 8 8

Principal

cell type

Mononucleated myoblasts Isolated spherical cells Isolated adherent cells

Total No. cells scored

Percentage positive cells

1161 1293 1729

1.1 91.9 23.7

Note. Confluent cultures were fed medium containing CB to inhibit fusion on Day 5 as indicated in Table 3. On Day 7, the spherical cells were isolated as described in Table 4 and replated in control medium. Adherent cells remaining on the CB-treated plates were also fed control medium. On Day 8, both series were fixed and stained with anti-skeletal muscle myosin as described under Materials and Methods.

was approximately 3 times that of the unseparated cell population, 8 times that of the firmly adherent cells, and 100 times that of control mononucleated myoblasts. Thus, although at least some of the cells remaining after selective trypsinization have synthesized CK, the easily detached population is substantially enriched for cells exhibiting high levels of activity of this enzyme. Differentiation at the single cell level was examined by immunofluorescence studies with a monoclonal antibody directed against skeletal muscle myosin (Table 5). When cultures of E63 myoblasts were grown to confluency in the absence of CB and stained with antimyosin antibody prior to myotube formation (Day 5), only 1% of the cells were positive. The incidence of myosin-positive cells rose dramatically during the subsequent 48 hr of CB treatment, and most positive cells exhibited the spherical morphology described above. When these cells were isolated by selective detachment, replated at low cell density and stained with antibody, more than 90% were found to contain myosin (Table 5; Figs. 4C, D), regardless of whether they were mononucleated or binucleated. By contrast, when the cells remaining on the plates after trypsinization were tested, only 24% were positive, and these cells showed considerable variation in staining intensity. Identical staining patterns were seen when conventionally prepared rabbit antibodies specific for rat skeletal muscle myosin heavy chain (Fallon and Nachmias, 1980) were used (Figs. 4E, F). When the isolated spherical cell population was resuspended in normal medium lacking CB and replated at cell densities approaching confluency,

spontaneous fusion was initiated immediately after cell reattachment and few or no single cells remained after 24-48 hr. Because CB has been demonstrated both to disrupt microfilaments and to inhibit hexose transport (Spudich, 1972; Mizel and Wilson, 1972), the experiments described above were repeated using 0.25 pg/ml cytochalasin D (CD), which affects microfilaments without inhibiting hexose transport (Miranda et al., 1974). The results were indistinguishable from those obtained when CB was used; spherical cells accumulated which showed positive immunofluorescent staining for skeletal muscle myosin, and high specific activities of MMCK developed. If the spherical cell population is enriched for unfused myoblasts in the terminal stages of cell differentiation, as the presence of MM-CK and skeletal muscle myosin suggests, they should consist primarily of postmitotic cells. To examine this, the spherical fraction of CBtreated E63 cells was isolated and replated in normal medium at low cell density to provide optimum conditions for cell proliferation and to decrease the incidence of spontaneous fusion. rH]Tdr was added for 24 hr and the cultures were analyzed by autoradiography. Silver grains were found over only 13% of the nuclei, indicating that nearly 90% of the cells failed to engage in DNA synthesis during this period. When control E63 myoblasts which had not been exposed to CB were plated in the presence of [3H]Tdr, a labeling index of 85% was achieved by 24 hr. The ability of control and CB-treated E63 myoblasts

TABLE 6 COLONY-FORMING EFFICIENCY OF CONTROL AND CB-TREATED E63 RAT MYOBLASTS Cell treatment Control Fusion inhibited

Day of plating 5 7

Principal

cell type

Mononucleated myoblasts Isolated spherical cells

Total No. cells plated

Colony forming efficiency (% )

650 1950

54 3

Note. Fusion was inhibited by feeding cultures medium containing CB on day 5 as described in Table 3. Confluent control cultures were trypsinized on Day 5 and replated in control medium at 200-1050 cells/lOO-mm dish. The spherical population of fusion inhibited cells was isolated on Day ‘7 as described in Table 4 and plated in control medium as indicated above. After 2 weeks of growth, plates were fixed and stained with 2% Giemsa and the number of colonies formed was scored as described under Materials and Methods.

KONIECZNY, MCKAY, AND COLEMAN

Myoblast

Dij~erentiatiun

FIG. 4. Indirect immunofluorescence of differentiated chicken and rat muscle cells stained with antibodies to rat skeletal muscle myosin. Left side, phase contrast; right side, rhodamine immunofluorescence. (A and B) Control 96-hr culture from chicken embryo leg muscle stained with mouse monoclonal anti-rat myosin XMlb; (C and D) CB-treated rat E63 myoblasts 1 day after isolation stained with mouse monoclonal anti-rat myosin XMlb; (E and F) CB-treated rat E63 myoblasts 1 day after isolation stained with conventionally prepared rabbit anti-rat skeletal muscle myosin. The monoclonal antibody cross-reacts with chicken skeletal muscle myosin, exhibits A-band localization in crossstriated regions of muscle fibers, does not stain undifferentiated myoblasts of chick or rat origin, and does not stain the fibroblasts present in control chicken embryo muscle cultures. Conventional anti-rat myosin showed similar cross-reactivity characteristics, Note that the CBtreated E63 myoblasts are mononucleated cells with positive immunofluorescence. X267.

to give rise to viable colonies in clonal culture was also investigated (Table 6). Whereas control E63 cells showed a colony forming efficiency of 54% after 2 weeks of growth, only 3% of the spherical fraction of CB-treated cells formed colonies during the same period. Finally, the mouse myoblast line G8-2 was grown to confluency and exposed to CB in the same fashion as described above for the rat myoblast lines. Spontaneous fusion of G8-2 cells was inhibited, as was the case with

E63 and L6J1, but no increase in CK activity served. 3. Nuclear DNA Content of Cycling Differentiated Myoblasts

was ob-

and Terminally

The results described above demonstrate that rat myoblasts inhibited from fusing by exposure to CB express muscle specific properties and cease to engage in

22

DEVELOPMENTAL BIOLOGY

A r-.

VOLUME 91, 1982

Because of their larger genome size, cycling rat E63 myoblasts had higher nuclear fluorescence values than did chicken cells, but also showed a range of approximately twofold, corresponding to the nuclear DNA contents of cells in G1, S, and G2 (Fig. 6A). The spherical, differentiated cells which accumulated during CB treatment again showed values restricted to the lower end of the range (Fig. 6B), equivalent to the G1 fraction of the cycling cell population. The results demonstrate that postmitotic myoblasts of both rat and chick origin which have differentiated in the absence of fusion have withdrawn from the cell cycle in the prereplicative Go phase.

Cycling Chick Myoblosts

32 I

DISCUSSION 0

Dissociation of Myoblast Fusion Differentiation

Cat: Md’ Depleted Chick Myoblosts

and Terminal

On the basis of investigations utilizing Ca2+ depletion, Shainberg and co-workers (1969, 1971, 19’78) concluded that myoblast fusion is a causal step in rat skeletal muscle cell differentiation. Although our experiments

50 Relative

100 Fluorescence

150

2

Units

I

FIG. 5. Quantitative DNA measurements on individual chicken myoblast nuclei stained with the DNA fluorochrome DAPI. Cultures were grown, fixed, and stained as described in the text. Fluorescence intensity of individual nuclei was measured using a Zeiss microspectrophotometer. All values are from mononucleated cells only. (A) Oneday exponentially growing culture of chicken embryo leg muscle cells in control medium; (B) 5-day culture of chicken embryo leg myoblasts inhibited from fusing by growth in Ca’+, M$‘-depleted medium.

l-l B

cell cycle activity, as do chicken embryo myoblasts which differentiate in the absence of fusion. In order to determine whether these cells have withdrawn from the cell cycle in a prereplicative phase, as is the case in normal muscle differentiation, rat and chicken myoblasts were stained with DAPI and quantitative measurements of DNA fluorescence of individual nuclei were made by microspectrophotometry. A cycling population of chicken embryo myoblasts (24-hr control culture) exhibited nuclear DNA fluorescence values ranging from 55 to 125 relative units, with the lower values representing cells in G1, the higher values cells in Gz, and intermediate values reflecting S-phase cells (Fig. 5A). Unlike the cells in control cultures, the differentiated chicken myoblasts present in cultures maintained in Ca’+,Mp-depleted medium for 96 hr exhibited fluorescence values ranging only from 60 to 75 relative units (Fig. 5B), corresponding to the 2cDNA content characteristic of cells in Go or G1.

CB-Treated

Myoblosts, Population

I

50

I

100 150 Relative Fluorescence Units

E63

Spherical

n.200

i 50

FIG. 6. Quantitative DNA measurements on individual rat E63 myoblast nuclei stained with the DNA fluorochrome DAPI. Cultures were grown, fixed, and stained as described in the text, and the fluorescence intensity of individual nuclei was measured using a Zeiss microspectrophotometer. All values are from mononucleated cells only. (A) Three-day culture of exponentially growing cells; (B) isolated spherical cell population from a CB-treated rat myoblast culture 1 day after isolation.

KONIECZNY,

MCKAY,

AND COLEMAN

with low Ca2+ medium are in agreement with their findings, the results presented here utilizing cytochalasin B lead to a different conclusion and constitute the first direct demonstration that terminal differentiation of skeletal muscle can proceed in the absence of cell fusion in rat as well as in chick myogenesis. The earlier report by Wahrmann et al. (1976) that CK, myosin, and phosphorylase levels increased in cultures of L6 myoblasts inhibited from fusing by treatment with CB was suggestive, but did not establish that the proteins measured were the forms characteristic of skeletal muscle, nor were conclusive investigations on proliferative potential air differentiation at the single cell level reported. Our studies demonstrate that (1) when confluent cultures of both L6 and L8 myoblasts are inhibited from fusing by treatment with CB, CK activities increase to levels equal to or greater than those of spontaneously fusing cultures of comparable age; (2) the increase in #CK is due to the accumulation of the MM-isoenzyme to very high specific activities in a particular subset of cells, the spherical population which appears during exposure to CB; (3) these cells also contain skeletal muscle myosin in filamentous arrays, but do not become visibly cross-striated; and (4) they have withdrawn from the cell cycle in the Go or G1 phase. In addition, selective trypsinization procedures are described whereby terminally differentiated rat myoblasts can be isolated to approximately 90% homogeneity. The remaining 10% may consist of cycling, undifferentiated cells which were rounded up in mitosis at the time of isolation and therefore were easily detached from the plates. This interpretation is consistent with the proportion of cells in this fraction which can incorporate [3H]Tdr upon subsequent subculture, can proliferate to form colonies under clonal conditions, and does not exhibit immunofluorescent staining with anti-myosin. These results are critically dependent on the timing of CB treatment in relation to the transition from a proliferative to a differentiated state. No significant differentiation was seen when CB was added to subconfluent, exponentially growing cultures of L6Jl and E63 rat myoblasts, and mouse G8-2 cells did not respond under any of the regimes tested. The G8-2 line grows more slowly than the rat lines and differentiates relatively asynchronously, thus optimal conditions for fusion inhibition may not be obtainable. Primary cultures of neonatal rat and mouse myoblasts, which exhibit growth and differentiation characteristics like those of G8-2 cells, have been similarly unresponsive to CB treatment. The differentiation of fusion inhibited L6Jl and E63 rat myoblasts is not dependent on the binucleated state which results when cytokinesis is prevented by CB. Approximately 80% of the differentiated myoblasts in

23

Mgoblast Differentiation

our preparations were mononucleated cells with diploid nuclear DNA contents, and are presumed not to have undergone mitosis during CB treatment. While our results demonstrate with a variety of markers that myoblasts from rat cell lines and from chicken embryos can differentiate when fusion is inhibited by CB, it is not clear why only chicken cells do so under conditions of Ca2+ depletion. It is unlikely that our results with rat cells reflect the specific effect of Ca2+ on CK expression reported by Morris and Cole (1979) because these cells also fail to express skeletal muscle myosin. That comparable results were obtained both with CB, which inhibits hexose transport in addition to its effects on microfilaments, and with CD, which does not inhibit hexose transport, implies that microfilaments play a role in myoblast fusion. The recent report by Swierenga et al. (1981) that low Ca”+ conditions lead to cytoskeleton disorganization in cultured human carcinoma cells suggests a common basis for fusion inhibition by these two seemingly disparate procedures, perhaps related to the role of Ca2+ influx recently demonstrated by David et al. (1981) to precede membrane union in fusion competent chick myoblasts. The failure of rat myoblasts to differentiate in low Ca2+ may reflect a more critical dependence on extracellular Ca”+ for intracellular regulatory processes in rat cells than in chicken, rather than reflecting species differences in the relationship between myoblast fusion and differentiation as suggested by Shainberg and Brik (1978). Selective Pm-i&cation Deprivation

of Avian

Myoblasts by M$

In recent years, fusion inhibition of avian myoblasts by Ca2+ depletion has become a routine method for the production of differentiated single cells in many laboratories. Until now, however, only 60-70% of the cells in such preparations from chicken embryo muscle have consisted of differentiated myoblasts, the remaining 30-40% being comprised of fibroblastic cells and perhaps some undifferentiated myoblasts. In the most systematic quantitation of fusion kinetics to date, Turner et al. (1976a) found that in cultures of EGTA-treated myoblasts restored to normal Ca2+ levels, the incidence of fusion did not exceed 65%, a value almost identical to that found in untreated control cultures in their studies and in ours. Similar results were reported by Paterson and Prives (1973) utilizing Chelex-treated medium. Such heterogeneity has severely limited the usefulness of fusion-inhibited cultures for studies on the biochemistry, molecular biology, and physiology of muscle differentiation. The fusion-inhibition procedure described here for chicken embryo cells, utilizing depletion of both Ca2’

24

DEVELOPMENTAL BIOLOGY

and M$+, results in preparations in which more than 97% of the cells stain with antibodies to skeletal muscle myosin, desmin, and M-CK, and in which 90-100% of the cells fuse following restoration of normal Ca2+ and Mg2+ levels. Myomesin staining was unambiguous only in cells containing well aligned myofibrils, as reported previously by Eppenberger et al. (1981). Fusion inhibition under these conditions was virtually complete, with the incidence of nuclei in syncytia remaining at the 3-5% level found in 24-hr cultures immediately prior to the addition of Ca2’,Mg2+-depleted medium. The key to these results is M$+ depletion, to which the fibroblastic cells are differentially sensitive. The fibroblastic cells survived when fusion was inhibited by Ca2+ depletion alone, which was accomplished either by adding M$+ back to Ca2+,Mg2+-depleted medium, or by using EGTA to lower the concentration of free Ca2+ in the medium. Ca2’,Mg2+-depleted medium had no obvious detrimental effect on chicken myoblast viability, fusion competence or the expression of characteristic skeletal muscle proteins. Relatively few cells developed visible cross-striations unless MS+ was added back to the medium, however, suggesting that M$+ may be an important factor in some aspect of contractile apparatus assembly, such as myofibril alignment. Although Mgzf is a cofactor in many enzyme reactions and may play a variety of other roles in living cells, the basis for the differential sensitivity of fibroblasts and myoblasts is not known. Nuclear

DNA Determinations

and the Cell Cycle

The measurements of individual nuclear DNA content presented here represent the only direct demonstration to date that myoblasts which differentiate as single cells in the absence of fusion are arrested in the Go or G1 phase of the cell cycle. The pioneering Feulgen microspectrophotometric studies of Lash et al. (1957) demonstrated that muscle fibers formed in vivo contained only nuclei with 2cDNA contents, characteristic of Go and G1. This has been confirmed for muscle fiber formation in vitro both by Feulgen (Strehler et al., 1963; Friedlander et al., 1978) and DAPI (Coleman et al., 1981) microspectrophotometry. Because differentiated avian myoblasts, obtained either by CB- or low-Ca2+-mediated fusion inhibition, neither synthesize DNA nor divide, they have been presumed to have withdrawn from the cell cycle at the same stage as have normal, fusing myoblasts. No conclusive evidence on this issue has been presented until now, even though such studies do not require the homogeneous differentiated cell population described in this report. Our results with chicken myoblasts are unambiguous on this point: the nuclei of all the mononucleated differentiated cells measured had

VOLUME 91, 1982

2cDNA contents. The nuclear DNA contents found in the spherical, readily detachable cells from fusion inhibited rat myoblast cultures also strongly suggest that differentiation occurs in the prereplicative phase. The relative fluorescence values were higher and the range broader with rat cells than with chick because of the larger genome size of the rat. Only 1 of the 100 CBtreated rat cells measured showed a fluorescence value outside the Go/G1 region. It should be emphasized that the results presented above do not establish the sequence of events which determines whether myoblasts differentiate or continue to cycle; they demonstrate only that those cells which differentiate do so in Go or G1 and that myoblast fusion is not required for this transition to occur. The authors are deeply grateful to Drs. Hans Eppenberger, Howard Holtzer, and Vivian Nachmias for their donations of antisera to myomesin, desmin, and myosin, respectively; to Dr. Annette Coleman for use of the microscope spectrophotometer, to Ms. Jeanne Lawrence and Dr. Heide Lee for many helpful discussions, and to Ms. Maria Shaffer and Ms. Carol King for their able technical and secretarial assistance, respectively. SK. is a Predoctoral Trainee on NIH Training Grant 5 T32 GM 07601-02. The work reported here was supported by Grant PCM 7816136 from the National Science Foundation and Grant GM 24499 from the National Institutes of Health. Note Added in ProofI While this paper was in press, M. Tanaguchi and H. Ishikawa (J. Cell Biol. 92, 324-332, 1982) reported that the addition of Mg-ATP enhances the formation of regular sarcomere patterns in reconstituted myofibrils, affirming our contention that Mgz+ may be involved in sarcomere assembly.

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mononucleated cells of chick muscle cultures. Develop. Biol. 48,43X446. WAHRMANN,J. P., DRUGEON,G., DELAIN, E., and DELAIN, D. (1976). Gene expression during the differentiation of myogenic cells of the L6 line. Biochemie 58, 554-562. WALLIMANN,T., TURNER,D. C., and EPPENBERGER,H. M. (1977). Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle. J Cell Biol. 75, 297-317. WRIGHT, W. E., and GROS,F. (1981). Coexpression of myogenic functions in L6 rat X T984 mouse myoblast hybrids. Develop. Biol. 86, 236-240. YAFFE, D. (196Sj. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Nat. Acad. Sci. USA 61, 477-483.