Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.     

Embryonic and fetal rat myoblasts express different phenotypes following differentiation in vitro

Myosin heavy chain (MHC) is encoded by a multigene family containing members which are expressed in developmental and fiber type-specific patterns. In developing rats, primary (1°) and secondary (2°) myotjbes can be disfinguished by differences in MHC expression: 1° myotubes coexpress embryonic and slow MHC, while 2° myotubes initially express only embryonic MHC. We have used monoclonal antibodies which recognize the embryonic, slow, neonatal, and adult fast IIB/IIX MHCs to examine MHC accumulation in myoblasts obtained from hindlimbs of embryonic day (ED) 14 and ED 20 Sprague-Dawley rats during differentiation in vitro. Embryonic myoblasts (ED 14), which develop into 1° myotubes in vivo, differentiate as myocytes or small myotubes (i.e., 1–4 nuclei) which express both embryonic and slow MHC. They do not accumulate detectable levels of neonatal or adult fast IIB/IIX MHC. Fetal myoblasts, which develop into secondary myotubes in vivo, fuse to form large myotubes (i.e., 10–50 nuclei) and express predominantly embryonic MHC at 3 days in culture. These myotubes accumulate neonatal and adult fast IIB/IIX isoforms of MHC and eventually contract spontaneously. In contrast to embryonic myotubes, they do not accumulate slow MHC. Our results demonstrate that embryonic and fetal rat myoblasts express different phenotypes in vitro and suggest that they represent distinct myoblast lineages similar to those previously described in chickens and mice. These two lineages may be responsible for the generation of distinct populations of 1° and 2° myotubes in vivo. © 1993Wiley-Liss, Inc.     

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.     

Comparison of contractile characteristics of muscle from Holstein and double-muscled Belgian Blue foetuses.

The aim of the present study was to precise the origin of the particular muscle characteristics of double-muscled cattle by comparing muscle properties of Holstein and double-muscled Belgian Blue (BB) foetuses. Ten 100-day-old foetuses of each genotype were studied. The weight and length of foetuses and the length, weight and area of the Semitendinosus (ST) muscle were analysed. Contractile differentiation of the different fibre types was studied by immunohistochemistry using several monoclonal antibodies raised against different myosin heavy chain isoforms (MHC slow, fast, foetal) and by electrophoresis. Proliferation phase of myoblasts from each genotype was analysed in primary culture. On 100 days of foetal life, the foetuses of both genotypes did not show any significant differences in their weight and length. However, BB cattle already present muscle hypertrophy, which seems to originate from a higher myoblast proliferation observed in primary culture. The use of anti-MHC antibodies shows that ST muscle of BB contained a smaller proportion of primary fibres and a higher proportion of secondary fibres which will give principally fast fibres in adult muscle. Electrophoresis analysis confirms a lower proportion of slow MHC in ST of BB.     

MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts.

Isolated chicken myoblasts had previously been utilized in many studies aiming at understanding the emergence and regulation of the adult myogenic precursors (satellite cells). However, in recent years only a small number of chicken satellite cell studies have been published compared to the increasing number of studies with rodent satellite cells. In large part this is due to the lack of markers for tracing avian myogenic cells before they become terminally differentiated and express muscle-specific structural proteins. We previously demonstrated that myoblasts isolated from fetal and adult chicken muscle display distinct schedules of myosin heavy-chain isoform expression in culture. We further showed that myoblasts isolated from newly hatched and young chickens already possess the adult myoblast phenotype. In this article, we report on the use of polyclonal antibodies against the chicken myogenic regulatory factor proteins MyoD and myogenin for monitoring fetal and adult chicken myoblasts as they progress from proliferation to differentiation in culture. Fetal-type myoblasts were isolated from 11-day-old embryos and adult-type myoblasts were isolated from 3-week-old chickens. We conclude that fetal myoblasts express both MyoD and myogenin within the first day in culture and rapidly transit into the differentiated myosin-expressing state. In contrast, adult myoblasts are essentially negative for MyoD and myogenin by culture Day 1 and subsequently express first MyoD and then myogenin before expressing sarcomeric myosin. The delayed MyoD-to-myogenin transition in adult myoblasts is accompanied by a lag in the fusion into myotubes, compared to fetal myoblasts. We also report on the use of a commercial antibody against the myocyte enhancer factor 2A (MEF2A) to detect terminally differentiated chicken myoblasts by their MEF2+ nuclei. Collectively, the results support the hypothesis that fetal and adult myoblasts represent different phenotypic populations. The fetal myoblasts may already be destined for terminal differentiation at the time of their isolation, and the adult myoblasts may represent progenitors that reside in an earlier compartment of the myogenic lineage.     

Identification and developmental expression of a chicken calsequestrin homolog

Calsequestrin (CAL) is a calcium-binding protein whose primary function is thought to involve sequestration of calcium in the muscle sarcoplasmic reticulum (SR). Little is known about the mechanisms regulating CAL expression, or about the role of this protein in muscle development. In addition, CAL may regulate calcium localization in some nonmuscle cells. We have identified an avian calsequestrin homolog. The predicted amino acid sequence of the avian CAL, first described as a laminin binding protein, and named aspartactin, is 70-80% identical to mammalian CAL sequences. We have used affinity-purified antibodies and cDNA probes to investigate expression in developing and adult chicken tissues. In adult chickens, the avian CAL homolog was expressed in slow and fast twitch skeletal muscle as well as in cardiac muscle. Surprisingly high levels of CAL protein were also detected in cerebellum. During development, CAL mRNA and protein were detected in Embryonic Day 5 (E-5) limb primordia, well before the initiation of myoblast fusion. In leg skeletal muscle, CAL protein and mRNA increase approximately 10-fold from E-8 to E-18 with a time course that just precedes myoblast fusion. This early expression pattern was also observed in cultured chicken pectoral myoblasts, and appears to be regulated at the level of mRNA abundance. The developmental profile of CAL expression is compared to that of other muscle proteins and possible additional functions of CAL are discussed.     

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.     

Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.     

Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen

Human skeletal muscle precursor cells (myoblasts) have significant therapeutic potential and are a valuable research tool to study muscle cell biology. Oxygen is a critical factor in the successful culture of myoblasts with low (1–6%) oxygen culture conditions enhancing the proliferation, differentiation, and/or viability of mouse, rat, and bovine myoblasts. The specific effects of low oxygen depend on the myoblast source and oxygen concentration; however, variable oxygen conditions have not been tested in the culture of human myoblasts. In this study, muscle precursor cells were isolated from vastus lateralis muscle biopsies and myoblast cultures were established in 5% oxygen, before being divided into physiological (5%) or standard (20%) oxygen conditions for experimental analysis. Five percent oxygen increased proliferating myoblast numbers, and since low oxygen had no significant effect on myoblast viability, this increase in cell number was attributed to enhanced proliferation. The proportion of cells in the S (DNA synthesis) phase of the cell cycle was increased by 50%, and p21^Cip1 gene and protein expression was decreased in 5 versus 20% oxygen. Unlike in rodent and bovine myoblasts, the increase in myoD, myogenin, creatine kinase, and myosin heavy chain IIa gene expression during differentiation was similar in 5 and 20% oxygen; as was myotube hypertrophy. These data indicate for the first time that low oxygen culture conditions stimulate proliferation, whilst maintaining (but not enhancing) the viability and the differentiation potential of human primary myoblasts and should be considered as optimum conditions for ex-vivo expansion of these cells.     

Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development

Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or intrinsic "clock." Taken together, these results suggest that, unlike embryonic and neonatal MyHCs, the expression of slow MyHC in vivo at different developmental stages during gestation is not the result of commitment to a distinct myoblast lineage, but is largely determined by the environment.     

Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca²⁺ (25 μm), Mg²⁺-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg²⁺ 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 Mg²⁺, suggesting that Mg²⁺ plays a role in sarcomere assembly. Conditions of Ca²⁺ and Mg²⁺ 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 G₀ stage of the cell cycle.     

Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts.

Patterning of fast and slow muscle fibres in limbs is regulated by signals from non-muscle cells. Myoblast lineage has, however, also been implicated in fibre type patterning. Here we test a founder cell hypothesis for the role of myoblast lineage, by implanting characterized fast and slow mouse myoblast clones into chick limb buds. In culture, late foetal mouse myoblast clones are committed to a probability (range 0-0.92) of slow myosin heavy chain (MyHC) expression. In contrast, when implanted into chick limbs, fast mouse myoblast clones express myosin characteristic of their new environment, without fusion to chick muscle cells and in the absence of innervation. Therefore, local signals exist within the chick limb bud during primary myogenesis that can override intrinsic commitment of at least some myoblasts, and induce slow MyHC.     

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.     

Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.     

Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.     

Transient induction of poly(A)-short myosin heavy chain messenger RNA during terminal differentiation of L6E9 myoblasts

The rat myoblast L₆E₉ cell line under appropriate culture conditions is a uniform population of cycling cells which can be induced to differentiate into a pure population of myotubes. The pattern and kinetics of myogenic differentiation of this cell line are similar to those of primary skeletal muscle myoblasts. We have used this cell line to investigate the controls regulating the synthesis and accumulation of myosin heavy chain during myogenic development. From pulse labeling studies of total cellular protein synthesis, we observed that activation of MHC⁴ synthesis is temporally correlated with cell fusion and myotube formation. MHC synthesis is transiently induced from <1% up to 25% of the total protein synthesized. After MHC has accumulated to the steady-state level characteristic of fully differentiated myotubes, MHC synthesis decreases very rapidly to almost basal levels. To determine whether this transient induction of MHC synthesis was due to parallel changes in MHC messenger RNA levels, the accumulation and compartmentalization of MHC mRNA during L₆E₉ cell differentiation was followed by complementary DNA/RNA hybridization using cDNA prepared against MHC mRNA purified from L₆E₉ cells. We demonstrate that the level of MHC synthesis closely parallels the level of cytoplasmic MHC mRNA. The induction of MHC mRNA accumulation is initiated at least 36 hours prior to cell fusion and at a time when all cells in the population are still uncommitted to terminal differentiation as tested by cell cloning. The level of cytoplasmic MHC mRNA is increased from ~200 molecules per cell in the growing state to ~50,000 molecules at the peak of induction (day 6 after plating). Subsequently the levels of MHC mRNA decrease very rapidly and at day 10 after plating there are only ~3000 molecules per myotube nucleus. A striking feature of this regulation is the behavior of MHC mRNA on oligo(dT) columns. Most (~90%) of the MHC mRNA transiently induced during differentiation has a very short poly(A) tail (<20 nucleotides). We conclude that the striking induction followed by deinduction of MHC synthesis is controlled primarily by the induction and deinduction of cytoplasmic MHC mRNA accumulation. The relationship of our observations to muscle physiology is discussed.     

Fibroblasts modulate expression of Thy-1 on the surface of skeletal myoblasts

Thy-1 antigen is a well-characterized cell-surface glycoprotein known to be variably expressed in many different tissues, including lymphocytes, brain, and muscle. Its function remains unknown. In skeletal muscle, both in vivo and in vitro, the antigen has been reported on immature but not on adult tissue, and its disappearance corresponds roughly to the time of myoblast fusion. Using monoclonal H36 antibody to identify myoblasts unambiguously, we demonstrate here that Thy-1 is expressed only on a small (less than 1%) fraction of rat skeletal muscle myoblasts in heterogeneous primary cultures, but the number of myoblasts that express Thy-1 rises to a steady level of about 70% when fibroblasts are removed from secondary cultures. Restitution of fibroblasts or growth of purified myoblasts in medium conditioned by fibroblasts greatly suppresses this increase in myoblast Thy-1 expression. Thus an interaction between fibroblasts and myoblasts, mediated by a soluble nondialyzable molecule, modulates expression of Thy-1 on the myoblast outer membrane.