Skeletal Muscle-specific Genes Research Articles

Beating the odds: a cardiomyocyte cell line at last.

Beating the odds: a cardiomyocyte cell line at last.

Loss of β1Integrin Function Results in a Retardation of Myogenic, but an Acceleration of Neuronal, Differentiation of Embryonic Stem Cellsin Vitro

Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell–cell and cell–matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the β1integrin gene results in embryonal death in mice around the time of implantation.In vitro,differentiation of embryonic stem (ES) cells which lack β1integrin (β1−/−) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fässleret al., J. Cell Sci.109, 2989–2999, 1996). To analyze β1integrin function during myogenesis and neurogenesis we studied differentiation of β1−/−ES cells via embryoid bodies into skeletal muscle and neuronal cellsin vitro.β1−/−cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (β1+/−) ES cells. RT–PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of β1integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from β1−/−ES cells appeared earlier than wildtype and heterozygous (β1+/−) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in β1−/−embryoid bodies. However, neuronal outgrowth was retarded in the absence of β1integrin. No functional difference between wildtype and β1−/−cells was found with respect to secretion of γ-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of β1integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in β1−/−cells, we analyzed in wildtype and β1−/−cells the regulatory role of exogenously added BMP-4 on the expression of the mesodermal and neuronal marker genes,Brachyuryandwnt-1,respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and β1−/−cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in β1−/−cells may account for the accelerated neuronal differentiation in β1−/−ES cells.

MEF-2 and Oct-1 Bind to Two Homologous Promoter Sequence Elements and Participate in the Expression of a Skeletal Muscle-specific Gene

The murine adult IIB myosin heavy chain (IIB MyHC) gene is expressed only in certain skeletal muscle fibers. Within the proximal promoter are two A + T-rich motifs, mAT1 and mAT2, which greatly enhance muscle-specific transcription; myogenic cells contain proteins that bind to these sequences. MEF-2 binds to both mAT1 and mAT2; a mutation abolishing its binding to mAT1 greatly diminishes the activity of the promoter. Both mAT motifs also form complexes with a protein requiring a target sequence typical of POU domain proteins, which migrate in electrophoretic mobility shift assays to the same position as a complex containing purified Oct-1 and which are supershifted by an antibody specific to Oct-1; this protein is therefore probably Oct-1. Footprinting experiments demonstrate that mAT1 is preferentially occupied by MEF-2 and mAT2 by Oct-1 and that these two proteins appear to bind cooperatively to their respective sites. Although the two mAT motifs have sequences that are very similar, they nonetheless exhibit distinct behaviors and perform differently in the activation of the promoter. The contribution of the IIB MyHC gene to specification of the myogenic phenotype is thus at least in part regulated by MEF-2 and Oct-1.

Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α1subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ϵ-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ϵ-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro.

Developmental modulation of a β myosin heavy chain promoter-driven transgene

The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.

Cardiomyopathy in transgenic myf5 mice.

To explore the compatibility of skeletal and cardiac programs of gene expression, transgenic mice that express a skeletal muscle myogenic regulator, bmyf5, in the heart were analyzed. These mice develop a severe cardiomyopathy and exhibit a significantly shorter life span than do their nontransgenic littermates. The transgene was expressed from day 7.5 post coitum forward, resulting in activation of skeletal muscle genes not normally seen in the myocardium. Cardiac pathology was not apparent at midgestation but was evident by day 2 of postnatal life, and by 42 days, hearts exhibited multifocal interstitial inflammation, fibrosis, cellular hypertrophy, and occasional myocyte degeneration. All four chambers of the heart were enlarged to varying degrees, with the atria demonstrating the most significant hypertrophy (>100% in 42-day-old mice). The transgene and several skeletal muscle-specific genes were expressed only in patchy areas of the heart in heterozygous mice. However, molecular markers of hypertrophy (such as alpha-skeletal actin and atrial myosin light chain- 1) were expressed with a wider distribution, suggesting that their induction was secondary to the expression of the transgene, In older (28-week-old) mice, lung weights were also significantly increased, consistent with congestive heart failure. The life span of bmyf5 mice was significantly shortened, with an average life span of 109 days, compared with at least a twofold longer life expectancy for nontransgenic littermates. Expression of the transgene was associated with an increase in Ca2+-stimulated myofibrillar ATPase in myofibrils obtained from the left ventricles of 42-day-old bmyf5 mice. Myocardial bmyf5 expression therefore induces a program of skeletal muscle gene expression that results in progressive cardiomyopathy that may be due to incompatibility of heart and skeletal muscle structural proteins.

Overexpression of Myotonic Dystrophy Kinase in BC3H1 Cells Induces the Skeletal Muscle Phenotype

Myotonic muscular dystrophy is an autosomal dominant defect that produces muscle wasting, myotonia, and cardiac conduction abnormalities. The myotonic dystrophy locus codes for a putative serine-threonine protein kinase of unknown function. We report that overexpression of human myotonic dystrophy protein kinase induces the expression of skeletal muscle-specific genes in undifferentiated BC3H1 muscle cells. BC3H1 clones expressing myotonic dystrophy kinase appear equivalent to differentiated cells with respect to expression of myogenin, retinoblastoma tumor supressor gene, M creatine kinase, beta-tropomyosin, and vimentin. In addition, differential display analysis demonstrates that the pattern of gene expression exhibited by myotonic dystrophy kinase-expressing cells is essentially identical to that of differentiated BC3H1 muscle cells. These observations suggest that myotonic dystrophy kinase may function in the myogenic pathway.

Ras Induces a General DNA Demethylation Activity in Mouse Embryonal P19 Cells

We demonstrate that expression of v-Ha-ras in mouse embryonal P19 cells results in genome-wide demethylation. Analysis of the pattern of methylation of specific genes reveals that different types of genes are demethylated in the ras transfectants: skeletal muscle specific genes, a gene specifically expressed in the adrenal cortex (c21), ubiquitous genes, and exogenously introduced sequences. Transient transfection and in vitro demethylation assays reveal that the ras transfectants express high levels of a general DNA demethylation activity. This demonstrates that the general DNA demethylation activity in mouse embryonal cells is controlled by an important cellular signal transducer and that DNA demethylase is a potential downstream effector of Ras.

Control of skeletal muscle-specific transcription: involvement of paired homeodomain and MADS domain transcription factors.

In the last few years, many aspects of skeletal muscle-specific gene regulation have been explained by the activity of the helix-loop helix (HLH) myogenic regulatory factors of the MyoD family, which are sequentially expressed during skeletal muscle formation. However, evidence is accumulating that muscle specific transcription requires functional interactions of these muscle-specific HLH factors with other regulatory proteins whose expression is not only restricted to skeletal muscle. These regulators include the SRF and MEF2 MADS domain and the MHox paired homeodomain transcription factors. Together with the aforementioned HLH factors, they build an increasingly complex network of regulatory factors. Two members of the Pax multigenic family of developmental control transcription factors, Pax-3 and 7, have been shown to be expressed not only in nervous tissue but also in skeletal muscle precursor cells. Their possible involvement in the control of muscle-specific transcription is discussed in light of known molecular properties of Pax gene products described in other biological systems.

Developmental regulation of M-cadherin in the terminal differentiation of skeletal myoblasts.

Cadherins form a large family of membrane glycoproteins which mediate homophilic calcium-dependent cell adhesion. They are thought to mediate the initial calcium-dependent cell adhesion which precedes the plasma membrane fusion of skeletal myoblasts. Two cadherin subtypes are known to be expressed in mammalian skeletal myoblasts: muscle cadherin (M-cadherin) and neural cadherin (N-cadherin). In the present study we demonstrate that 1) the expression of M- and N-cadherin is differentially regulated during myoblast differentiation in vitro, 2) the expression of M-cadherin but not N-cadherin is inhibited by 5-bromo-2'-deoxyuridine (BUdR), an agent which selectively inhibits skeletal myoblast differentiation, and 3) fusion and differentiation-competent rat L6 myoblasts do not express detectable levels of N-cadherin mRNA. In vivo, M-cadherin mRNA was detectable exclusively in skeletal muscle. M-cadherin mRNA levels peaked during the secondary myogenic wave in rat hindlimb muscle, becoming barely detectable in 1-week-old and adult rats. These observations indicate that M-cadherin is unique in two ways: It is the first cadherin to be included in the family of skeletal muscle-specific genes, and it shows peak levels of expression in developing skeletal muscle tissue. Taken together, these results suggest that M-cadherin plays an important role in skeletal myogenesis.

Intrinsic Differences in MyoD and Myogenin Expression between Primary Cultures of SJL/J and BALB/C Skeletal Muscle

The time course of expression of the skeletal muscle-specific regulatory genes MyoD and myogenin was studied in primary cultures of skeletal muscle from adult SJL/J and BALB/c mice. In situ detection of expression with MyoD and myogenin riboprobes and myogenin antibody showed that the onset of expression of these genes occurred earlier in cells from SJL/J mice. Probe-positive cells and myotubes were also more frequent in cultures from SJL/J mice than in BALB/c. The onset of expression of MyoD and myogenin was delayed in cultured cells relative to the time course seen following injury in vivo . Myogenin protein was demonstrated in replicating cells and all myogenin-positive cells expressed desmin. The observed strain-specific difference infers a greater intrinsic myogenicity of cells in SJL/J muscle in vitro and reflects the superior capacity for new muscle formation previously reported in SJL/J mice in vivo.

Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene.

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors. Finally, they demonstrate distinct transcriptional regulatory pathways in cardiac and skeletal muscle.

Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow/cardiac troponin C gene.

The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors. Finally, they demonstrate distinct transcriptional regulatory pathways in cardiac and skeletal muscle.

The transcription of MyoD1 and myogenin genes in thymic cells in vivo

The skeletal muscle specific genes MyoD1 and myogenin are closely associated with commitment of cells to the myogenic lineage and differentiation of skeletal muscle precursor cells. The transcription of these genes was studied in the thymus where mononuclear cells termed myoid cells appear to closely resemble skeletal muscle precursors. In thymus from adult SJL/J and BALB/c mice, in situ hybridization with either MyoD1 or myogenin riboprobes showed probe-positive cells concentrated in the medullary region. In neonatal thymus, mRNA for these genes was not detected. These data are the first demonstration in a higher vertebrate of MyoD1 and myogenin expression in a tissue other than skeletal muscle. The sustained expression of MyoD1 and myogenin genes in thymi of adult mice shows that myoid cells are not equivalent to quiescent stem cells of mature skeletal muscle. In addition, studies with antistriational antibodies indicate that myoid cells do not continue to differentiate within the normal murine thymic environment. This arrested differentiation process presents an unusual model for investigating conditions regulating myogenesis in vivo.

Phorbol esters selectively and reversibly inhibit a subset of myofibrillar genes responsible for the ongoing differentiation program of chick skeletal myotubes.

Phorbol esters selectively and reversibly disassemble the contractile apparatus of cultured skeletal muscle as well as inhibit the synthesis of many contractile proteins without inhibiting that of housekeeping proteins. We now demonstrate that phorbol esters reversibly decrease the mRNA levels of at least six myofibrillar genes: myosin heavy chain, myosin light chain 1/3, myosin light chain 2, cardiac and skeletal alpha-actin, and skeletal troponin T. The steady-state message levels decrease 50- to 100-fold after 48 h of exposure to phorbol esters. These decreases can be attributed at least in part to decreases in transcription rates. For at least two genes, cardiac and skeletal alpha-actin, some of the decreases are the result of increased mRNA turnover. In contrast, the cardiac troponin T steady-state message level does not change, and its transcription rate decreases only transiently upon exposure to phorbol esters. Phorbol esters do not decrease the expression of the housekeeping genes, alpha-tubulin, beta-actin, and gamma-actin. Phorbol esters do not decrease the steady-state message levels of MyoD1, a gene known to be important in the activation of many skeletal muscle-specific genes. Cycloheximide blocks the phorbol ester-induced decreases in transcription, message stability, and the resulting steady-state message level but does not block the tetradecanoyl phorbol acetate-induced rapid disassembly of the I-Z-I complexes. These results suggests a common mechanism for the regulation of many myofibrillar genes independent of MyoD1 mRNA levels, independent of housekeeping genes, but dependent on protein synthesis.

Phorbol Esters Selectively and Reversibly Inhibit a Subset of Myofibrillar Genes Responsible for the Ongoing Differentiation Program of Chick Skeletal Myotubes

Phorbol esters selectively and reversibly disassemble the contractile apparatus of cultured skeletal muscle as well as inhibit the synthesis of many contractile proteins without inhibiting that of housekeeping proteins. We now demonstrate that phorbol esters reversibly decrease the mRNA levels of at least six myofibrillar genes: myosin heavy chain, myosin light chain 1/3, myosin light chain 2, cardiac and skeletal alpha-actin, and skeletal troponin T. The steady-state message levels decrease 50- to 100-fold after 48 h of exposure to phorbol esters. These decreases can be attributed at least in part to decreases in transcription rates. For at least two genes, cardiac and skeletal alpha-actin, some of the decreases are the result of increased mRNA turnover. In contrast, the cardiac troponin T steady-state message level does not change, and its transcription rate decreases only transiently upon exposure to phorbol esters. Phorbol esters do not decrease the expression of the housekeeping genes, alpha-tubulin, beta-actin, and gamma-actin. Phorbol esters do not decrease the steady-state message levels of MyoD1, a gene known to be important in the activation of many skeletal muscle-specific genes. Cycloheximide blocks the phorbol ester-induced decreases in transcription, message stability, and the resulting steady-state message level but does not block the tetradecanoyl phorbol acetate-induced rapid disassembly of the I-Z-I complexes. These results suggests a common mechanism for the regulation of many myofibrillar genes independent of MyoD1 mRNA levels, independent of housekeeping genes, but dependent on protein synthesis.

The ryanodine receptor/junctional channel complex is regulated by growth factors in a myogenic cell line.

The ryanodine receptor/junctional channel complex (JCC) forms the calcium release channel and foot structures of the sarcoplasmic reticulum. The JCC and the dihydropyridine (DHP) receptor in the transverse tubule are two of the major components involved in excitation-contraction (E-C) coupling in skeletal muscle. The DHP receptor is believed to serve as the voltage sensor in E-C coupling. Both the JCC and DHP receptor, as well as many skeletal muscle-specific contractile protein genes, are expressed in the BC3H1 muscle cell line. In the present study, we find that during differentiation of BC3H1 cells, induced by mitogen withdrawal, induction of the JCC and DHP receptor mRNAs is temporally similar to that of the skeletal muscle contractile protein genes alpha-tropomyosin and alpha-actin. Our data suggest that there is coordinate regulation of both the contractile protein genes (which have been studied in detail previously) and the genes encoding the calcium channels involved in E-C coupling. Induction of both calcium channels is accompanied by profound changes in BC3H1 cell morphology including the development of many components of mature skeletal muscle cells, despite lack of myoblast fusion. Visualized by electron microscopy, the JCC appears as "foot structures" located in the dyad junction between the plasmalemma and the sarcoplasmic reticulum of the BC3H1 cells. Development of foot structures is concomitant with JCC mRNA expression. Expression of the JCC and DHP receptor mRNAs and formation of the foot structures are inhibited specifically by fibroblast growth factor.

A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program

MyoD1 is a nuclear phosphoprotein that is expressed in skeletal muscle in vivo and in certain muscle cell lines in vitro; it has been shown to convert fibroblasts to myoblasts through a mechanism requiring a domain with homology to the myc family of proteins. The BC3H1 muscle cell line expresses skeletal muscle-specific genes upon exposure to mitogen-deficient medium, but does not express MyoD1 at detectable levels. To determine whether BC3H1 cells may express regulatory genes functionally related to MyoD1, a cDNA library prepared from differentiated BC3H1 myocytes, was screened at reduced stringency with the region of the MyoD1 cDNA that shares homology with c-myc. From this screen, a cDNA was identified that encodes a major open reading frame with 72% homology to the myc domain and basic region of MyoD1. The mRNA encoded by this MyoD1-related gene is expressed in skeletal muscle in vivo and in differentiated skeletal myocytes in vitro and is undetectable in cardiac or smooth muscle, nonmuscle tissues, or nonmyogenic cell types. During myogenesis, the MyoD1-related mRNA accumulates several hours prior to other muscle-specific mRNAs and therefore represents an early molecular marker for entry of myoblasts into the differentiation pathway. Transient transfection of 10T1/2 or 3T3 cells with the MyoD1-related cDNA is sufficient to induce myosin heavy-chain expression and to activate a reporter gene under transcriptional control of the muscle creatine kinase 5' enhancer, which functions only in differentiated myocytes. Expression of this cDNA in stably transfected 10T1/2 cells also leads to fusion and muscle-specific gene expression upon exposure to mitogen-deficient medium. Thus, the product of this MyoD1-related gene is sufficient to activate the muscle differentiation program and may substitute for MyoD1 in certain developmental situations. Together, these results suggest the existence of a family of myogenic regulatory genes that share a conserved motif with c-myc.

A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program.

MyoD1 is a nuclear phosphoprotein that is expressed in skeletal muscle in vivo and in certain muscle cell lines in vitro; it has been shown to convert fibroblasts to myoblasts through a mechanism requiring a domain with homology to the myc family of proteins. The BC3H1 muscle cell line expresses skeletal muscle-specific genes upon exposure to mitogen-deficient medium, but does not express MyoD1 at detectable levels. To determine whether BC3H1 cells may express regulatory genes functionally related to MyoD1, a cDNA library prepared from differentiated BC3H1 myocytes, was screened at reduced stringency with the region of the MyoD1 cDNA that shares homology with c-myc. From this screen, a cDNA was identified that encodes a major open reading frame with 72% homology to the myc domain and basic region of MyoD1. The mRNA encoded by this MyoD1-related gene is expressed in skeletal muscle in vivo and in differentiated skeletal myocytes in vitro and is undetectable in cardiac or smooth muscle, nonmuscle tissues, or nonmyogenic cell types. During myogenesis, the MyoD1-related mRNA accumulates several hours prior to other muscle-specific mRNAs and therefore represents an early molecular marker for entry of myoblasts into the differentiation pathway. Transient transfection of 10T1/2 or 3T3 cells with the MyoD1-related cDNA is sufficient to induce myosin heavy-chain expression and to activate a reporter gene under transcriptional control of the muscle creatine kinase 5' enhancer, which functions only in differentiated myocytes. Expression of this cDNA in stably transfected 10T1/2 cells also leads to fusion and muscle-specific gene expression upon exposure to mitogen-deficient medium. Thus, the product of this MyoD1-related gene is sufficient to activate the muscle differentiation program and may substitute for MyoD1 in certain developmental situations. Together, these results suggest the existence of a family of myogenic regulatory genes that share a conserved motif with c-myc.