Prostaglandin dependence of membrane order changes during myogenesis in vitro

Myogenic differentiation in vitro involves at least three events at the cell surface: binding of prostaglandin to cells, cell-cell adhesion, and fusion of the myoblast membranes into syncytia. Previous work has suggested that binding of prostaglandin is causal to the change in cell-cell adhesion and that both are accompanied by a characteristic reorganization of the myoblast membrane detected as a transient increase in membrane order by electron paramagnetic resonance. We show here that this membrane order change, which reaches a maximum at 38 h of development in vitro, was the last membrane order change before bilayer fusion which begins several hours later. This membrane order change, which accompanies the change in cell-cell adhesion, was dependent on the availability of prostaglandin. In myoblasts maintained in indomethacin, where further differentiation is known to be blocked at the prostaglandin binding step, the membrane order change did not occur. However, if myoblasts are provided with exogenous prostaglandin, the membrane order change occurred and differentiation proceeded. The results indicate that the basis of the membrane order change was the reorganization of myoblast membranes to allow increased adhesion and prepare the membrane for bilayer fusion. They also demonstrate that, like the increase in myoblast adhesion, the membrane order change was dependent on prostaglandin being available to bind to its receptor.     

Rescue of the Li+-induced delay of embryonic myogenesis in vitro by added inositol

Signaling between embryonic myoblasts to coordinate gene expression is part of normal skeletal muscle development in the embryo. An unanswered question is the nature of the second messengers carrying the information to the nucleus. We have investigated the cell membrane events associated with the binding of prostaglandin to a transient receptor on the embryonic chick myoblast membrane in vitro. The membrane events include a transient change in membrane order seen by electron paramagnetic resonance (EPR), a change in cell-cell adhesion, a rapid decrease in membrane permeability and fusion of the membrane bilayers. The addition of 20 mM Li+, an inhibitor of inositol phosphate phosphatase, perturbed the transient change in membrane order and delayed the change in cell-cell adhesion and conductivity for 2-6 h. Other alkali metal ions had no such effects. The addition of inositol to the culture medium in the continued presence of Li+ restored the normal timing of the two latter events. We interpret this as evidence for an inositol phosphate second messenger system which might connect the activation of the prostaglandin receptor with the change in cell-cell adhesion, the changes in membrane conductivity and perhaps bilayer fusion. We suggest that Li+, by blocking the regeneration of polyphosphatidylinositol from inositol phosphate, reduced the efficiency of the second messenger system such that further differentiation of the myoblast membrane was delayed. The exogenous inositol provided an alternative source and membrane differentiation was unaffected.     

Requirement for G protein activity at a specific time during embryonic chick myogenesis

Signaling between embryonic myoblasts involves prostaglandin metabolism, the activation of a membrane receptor and changes in polyphosphatidyl inositol metabolism. Many of these membrane-localized events occur between 33 to 35 h of differentiation, concomitant with a dramatic change in membrane organization, in myoblast aggregates in culture. Since many receptors affect inositol phosphate metabolism by activating a GTP-binding protein (G protein), we asked if there was evidence for such a protein in myogenic signaling. We show that during the period of differentiation in culture when prostaglandin is needed to bind to a transient receptor, a pertussis toxin-sensitive but cholera toxin-insensitive G protein must act. If this activation is blocked, the characteristic change in myoblast cell adhesion and subsequent membrane fusion do not occur. We suggest that a G protein couples the activated prostaglandin receptor and the change in polyphosphatidyl inositol metabolism and that this membrane transduction step is necessary for subsequent membrane differentiation events during myogenesis.     

Dependence of myoblast fusion on a cortical actin wall and nonmuscle myosin IIA

Cell-cell fusion is a fundamental cellular process that is essential for development as well as fertilization. Myoblast fusion to form multinucleated skeletal muscle myotubes is a well studied, yet incompletely understood example of cell-cell fusion that is essential for formation of contractile skeletal muscle tissue. Studies in this report identify several novel cytoskeletal events essential to an early phase of myoblast fusion among cultured murine myoblasts. During myoblast pairing and alignment, cortical actin filaments organize into a dense actin wall structure that parallels and extends the length of the plasma membrane of the bipolar, aligned cells. As fusion progresses, gaps appear within the actin wall at sites of vesicle accumulation, the vesicles pair across the aligned myoblasts, cell-cell contacts and fusion pores form. Inhibition of nonmuscle myosin IIA (NM-MHC-IIA) motor activity prevents formation of this cortical actin wall, as well as the appearance of vesicles at a membrane proximal location, and myoblast fusion. These results suggest that early formation of a subplasmalemmal actin wall during myoblast alignment is a critical event for myoblast fusion that supports bipolar membrane alignment and temporally regulates trafficking of vesicles to the nascent fusion sites during skeletal muscle myoblast differentiation.     

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.     

Electron spin resonance detection of free radicals in the mercaptan-activation and UV-inactivation of neocarzinostatin

Differentiation of chick embryo myoblasts $\text{in}\text{,}\text{vitro}$ requires both cell-cell recognition and cell-cell fusion. Prostaglandin E₁ is known to play a role in controlling fusion, and a specific receptor has been postulated. We demonstrate two peaks of specific binding activity for prostaglandin E₁ during myoblast differentiation $\text{in}\text{,}\text{vitro}$: one at 36 hours and one at 44 hours of culture. The prostaglandin binding activity of both peaks is sensitive to the inhibitors of prostaglandin synthesis, indomethacin and aspirin, and to the antibiotic tunicamycin. The 36 hour peak of binding activity occurs at the same time as the process of cell-cell recognition (24–36 hours) and recognition and prostaglandin binding exhibit similar sensitivity to indomethacin, aspirin and tunicamycin.     

Changes in myoblast membrane order during differentiation as measured by EPR

The events which make possible the characteristic fusion of the cell membranes of embryonic myoblasts are known to involve modification of the cell membrane (Hausman, R.E., Dobi, E.T., Woodford, E.J., Petrides, S., Ernst, M. and Nichols E.B. (1986) Dev. Biol. 113, 40-48). Myoblasts from chick embryos were allowed to differentiate in gyrotory aggregate culture and the order of their membranes was measured by EPR. Two spin-labels which insert at different depths into the lipid bilayer were used. Measurement with the 5-nitroxystearate label showed an increase in myoblast membrane order (2T' parallel) from 0-15 h of culture and again from 26-38 h of culture. Measurement with the 12-nitroxystearate label showed the 0-15 h increase in order but the second increase was greatly reduced and shifted in time. While the specific sources of these changes in membrane order cannot yet be identified, the changes observed correlated well with known events of myogenic differentiation in vitro. The initial increase in membrane order occurred while the myoblasts were recovering from the effects of trypsin dissociation and undergoing gyrotory aggregation. The second increase in membrane order occurred during the known period of prostaglandin receptor activity and increased cell-cell adhesion.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Neural cell adhesion molecule (NCAM) and myoblast fusion

Considerable evidence points to an involvement of neural cell adhesion molecule (NCAM) in myoblast fusion. Changes in the level of NCAM expression, isoform specificity, and localization in muscle cells and tissues correspond to key morphogenetic events during muscle differentiation and repair. Furthermore, anti-NCAM antibodies have been shown by others to reduce the rate of myoblast fusion, whereas overexpression of NCAM cDNAs increases the rate of myoblast fusion compared to controls. In this study we have used a novel fusion assay based on intracistronic complementation of lacZ, in combination with fluorescent X-gal histochemistry and immunocytochemistry to assess levels of NCAM expression in individual muscle cells. Our results indicate that a substantial proportion of newly fused myoblasts have NCAM expression levels unchanged from the levels of the surrounding unfused population suggesting that increased expression of NCAM is not required for wild-type myoblasts to fuse. Moreover, pure populations of primary myoblasts isolated from mice homozygous null for NCAM and therefore lacking the molecule, when placed in differentiation medium, consistently fused to form contractile myotubes with kinetics equivalent to wild-type primary myoblasts. We conclude that the increase in expression of NCAM, although typically observed during myogenesis, is not essential to myoblast fusion to form myotubes.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Calpain and calpastatin in myoblast differentiation and fusion: Effects of inhibitors

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca²⁺-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-β, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-β and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of μ-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.     

CD44 regulates myoblast migration and differentiation

CD44 is a transmembrane protein that plays a role in cell-cell interactions and motility in a number of cell types. Cell-cell interactions are critical for myoblast differentiation and fusion but whether CD44 regulates myogenesis is unknown. Here, we show that CD44 plays a functional role in early myogenesis. Analyses of myofiber cross-sectional area, after local injury in mouse tibialis anterior (TA) muscles, revealed that growth was transiently delayed in the absence of CD44. A muscle-intrinsic role for CD44 is suggested as primary myoblasts from CD44(-/-) mice displayed attenuated differentiation and subsequent myotube formation at early times in a differentiation-inducing in vitro environment. Chemotaxis of CD44(-/-) myoblasts toward hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) was totally abrogated, although expression of their respective receptors did not appear to differ from wild-type. Furthermore, motility of CD44(-/-) myoblasts was decreased at early stages of differentiation as determined by time-lapse microscopy. Wild-type myoblasts contained two subpopulations of slow- and fast-migrating cells, whereas CD44(-/-) myoblasts were composed predominantly of the slower migrating subpopulation. Taken together, these data suggest that myoblast migration and differentiation are closely linked and CD44 is a key regulator.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

Inhibition of myoblast differentiation in vitro by a protein isolated from liver cell medium

We have recently discovered that cells of Coon's Buffalo rat liver (BRL) line secrete a protein which is a potent inhibitor of skeletal myoblast differentiation in vitro. Using ion exchange and molecular exclusion chromatography, we have prepared this protein, which we designate "differentiation inhibitor" (DI), from the materials secreted by BRL cells maintained in serum-free medium. It is a relatively heat- stable protein which is inactivated by treatment with trypsin and mercaptoethanol and has an apparent molecular weight in the range 30,000--36,000. It exhibits no detectable mitogenic or lectin activity and differs from previously reported inhibitors of myoblast differentiation in several respects. It is active in all skeletal myoblast systems tested (Yaffe's L6 line as well as primary cultures of rat, chick, and Japanese quail myoblasts), and it blocks fusion, elevation of creatine kinase, and increased binding of alpha- bungarotoxin. Parallel fractionation of fetal bovine serum (FBS) and chick embryo extract (CEE) yields a peak of activity which similarly inhibits myoblast differentiation. We suggest that the differentiation inhibitor from BRL cells may correspond to the differentiation- inhibiting component(s) of FBS and CEE, and we call attention to the possibility that such a substance could play a role in embryonic growth of myoblasts and in satellite cell formation.     

The role of hormones and prostanoids in the in vitro proliferation and differentiation of human myoblasts

Fetal human myoblasts have been employed to examine the role of hormonal factors in human myogenesis. The results show that human myoblast proliferation is stimulated by insulin, hydrocortisone, and prostaglandin F2 alpha (PGF2 alpha). Exposure of human myoblasts preparing to differentiate to either PGE2 or isoproterenol results in the precocious initiation of differentiation (i.e., cell fusion and increase in creatine kinase activity). Three antagonists of prostanoid synthesis, indomethacin, aspirin, and DL-6-chloro-alpha-methylcarbozole-2-acetic acid, inhibit cell number increase with complete inhibitions of proliferation at 5 X 10(-5) M indomethacin and 6 X 10(-4) M aspirin. Reversal of the indomethacin-imposed block is achieved by prostaglandin F2 alpha. The same antagonists of prostanoid synthesis, when added to older cultures, depress prostaglandin E (PGE) levels and inhibit human myoblast differentiation. During differentiation, PGE is present in both the intracellular compartment (0.47 to 0.66 pmol/microgram DNA) and the culture medium (1.83 to 4.53 nmol PGE). The results suggest a role for prostanoids in the regulation of both human myoblast proliferation and differentiation. They also demonstrate that the active cyclooxygenase products are produced endogenously by the in vitro myogenic population. The findings are discussed within the context of what is known of the relationship between growth factor and prostanoid actions and the roles of these two categories of hormones in the regulation of myogenesis.     

Quantal Division and a Postmitotic State in Myoblast Differentiation

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Quantal division and a postmitotic state in myoblast differentiation.

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.     

Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation

alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.