Chick Embryonic Muscle Research Articles

Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization

We have localized myosin heavy chain (MHC) mRNAs in cells of intact embryonic chick muscle using high resolution in situ hybridization. Blocks of muscle were aldehyde-fixed prior to detergent treatment and hybridized with a biotinated cDNA probe, followed by colloidal gold-labeled antibodies, before embedment. Labeling was determined to represent MHC mRNA by extensive quantitative comparisons of electron micrographs from experimental and four different types of control samples. MHC mRNA was localized primarily to peripheral regions of 14-day chick pectoral muscle cells, where the majority of developing myofibrils were found. MHC mRNAs were consistently associated with the nonmyofibrillar cytoskeletal filaments which had diameters ranging from 4 to 10 nm. They were often oriented parallel to the longitudinal axis of the cell. The resolution of the ultrastructural approach allowed us to demonstrate that the mRNA molecules visualized were not directly associated with myofilaments, suggesting that nascent chains read from those messages do not assemble directly into myofilaments simultaneous with translation.

The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon.

Analysis of acetylcholine receptor clones isolated from a human leg muscle cDNA library, revealed that the alpha-subunit existed as two isoforms. A novel exon, coding for 25 amino acids, was located in the human genomic DNA sequence; its insertion into the alpha-subunit gives the new isoform of 462 amino acids. In addition, mRNAs for the two isoforms were found in equal proportions in poly(A)+ RNA obtained from three further sources including partially denervated and innervated human muscle and the rhabdomyosarcoma cell line TE671. Both protein isoforms can be expressed in E. coli. No evidence of a sequence related to that of the new exon was found in cDNA derived from poly(A)+ RNA isolated from fetal calf or embryonic chick muscle or Torpedo marmorata electric organ.

Cytopathic Effect of Lentogenic Strains of Newcastle Disease Virus in Cultured Chicken Muscle Cells

The ability of lentogenic strains of Newcastle disease virus (NDV) to inhibit the development of embryonic chicken muscle cells in vitro is described. In contrast to the uninfected control, which displayed a thick network of branched multinucleated muscle fibers, cultures infected with NDV exhibited a severely reduced potential to differentiate. The few differentiated muscle fibers that formed in the presence of NDV were poorly developed, displayed an attenuated morphology, and detached from the substrate 2 to 3 days before uninfected cultures showed signs of cell aging. When NDV-infected cultures were stained before muscle fiber detachment with a fluorescent-labeled NDV-specific antibody, the infected muscle fibers reacted strongly relative to neighboring myoblasts and fibroblasts. All six strains of NDV induced a similar inhibitory effect, and no strain-specific differences were detectable. These results indicate that differentiating muscle fibers are highly susceptible to infection by NDV in vitro and that this tissue exhibits an early NDV-induced cytopathic effect.

A role for the neural cell adhesion molecule, NCAM, in myoblast interaction during myogenesis

The Ca 2+-independent neural cell adhesion molecule, NCAM, is expressed by both nerve and muscle cells and has been shown to mediate both nerve-nerve and nerve-muscle cell interaction. A role for NCAM in muscle-muscle cell interaction has been proposed but not demonstrated. Here we report evidence that NCAM is expressed by embryonic chick muscle cells during in vitro development and functions together with Ca 2+-dependent adhesion molecules in mediating myoblast interaction during the formation of multinucleate cells.

Purothionin from wheat endosperm reversibly blocks myogenic differentiation of chick embryonic muscle cells in culture

Purothionin from wheat endosperm is a cysteine-rich, basic polypeptide of about 5000 Da, which modifies membrane permeability of cultured mammalian cells. This peptide was found to block fusion of chick embryonic muscle cells in culture but allows proliferation and alignment. A purothionin concentration of 6 μg/ml (1.2 μ M) was necessary for the complete prevention of myotube formation. Under similar conditions, incorporation of [ 35S]methionine occurred normally but the synthesis of muscle-specific proteins including creatine kinase and acetylcholine receptor was strongly inhibited. In addition, purothionin blocked the uptake of 86Rb +, immediately after its addition to the cultured myoblasts. No such effects were found with the purothionin chemically modified with acetic or succinic anhydride. Thus, the basic residues in purothionin appear to be associated with the inhibition of myogenic differentiation. These results suggest that purothionin exerts its regulatory effect on the transition from proliferative to differentiative myoblasts by interfering with membrane permeability or intercellular contact and recognition, which are necessary for the initiation of muscle differentiation.

Expression of nuclear lamin A and muscle-specific proteins in differentiating muscle cells in ovo and in vitro.

Primary cultures and tissue samples of chicken embryonic muscle were immunologically probed for the expression of muscle-specific proteins, such as myosin heavy chain and the tropomyosins, as well as for the nuclear lamina protein, lamin A. As determined by quantitative immunoblotting, the expression of lamin A and the muscle-specific proteins were at low levels or absent in predifferentiation myoblasts both in vitro and in ovo. During differentiation, an increase of lamin A expression preceded the induction to high levels of expression of muscle-specific proteins. Immunofluorescence staining of chicken embryonic muscle cells in culture also indicates an accumulation of lamin A before the induction of muscle-specific proteins expression. Furthermore, the accumulation of lamin A reached a plateau before the muscle-specific proteins during muscle development. In two dimensional NEPHGE gel analysis of immunoprecipitated lamin A, no detectable change in the ratio of the acidic/basic isoelectric variants of lamin A was observed during myogenesis. A potential role for lamin A in the mechanisms which underlie the differential and coordinate expression of muscle-specific genes is proposed.

Muscle-specific Regulation of a Transfected Rabbit Myosin Heavy Chain βGene Promoter

Abstract We have examined the transcriptional regulation of the rabbit myosin heavy chain (HC) beta gene by using DNA-mediated transfection experiments. To analyze the activity of the myosin HC beta promoter in a myogenic background, cultured myoblasts from 12-day-old chick embryonic breast muscle were transfected with a chimeric gene containing 781 base pairs of the promoter region fused to the gene for chloramphenicol acetyltransferase (CAT). As indicated by the transient expression of chloramphenicol acetyltransferase, the activity of the promoter in myoblast cultures increased at least 32-fold following differentiation and was selectively inhibited when myogenesis was blocked with 5-bromodeoxyuridine. Furthermore, RNase protection experiments showed that the in vivo myosin HC beta transcriptional initiation (or cap) site was utilized in the transfected skeletal muscle cells and also that the regulation of the exogenous promoter was similar to the induction of the endogenous skeletal alpha-actin gene. The results indicated that the exogenous promoter is regulated in a tissue- and stage-specific manner. By creating progressive 5' deletions of the promoter, we showed that only the region extending -294 base pairs upstream from the cap site is necessary for the muscle-specific expression. Linker-scanner mutagenesis of this region indicated that the positive regulation in differentiated skeletal muscle is mediated by at least two distinct elements within the 5'-flanking region of the myosin HC beta gene.

Identification of basic fibroblast growth factor as a cholinergic growth factor from human muscle.

Dissociated embryonic chick ciliary ganglion cells in culture were used as a bioassay to isolate a cholinergic growth-promoting protein from extracts of autopsied adult human muscle. An active protein was purified after acid and salt precipitation of extract, cation exchange, molecular sieving, heparin affinity chromatography, and in some cases, SDS-PAGE. This protein increased levels of choline acetyltransferase activity and ACh synthesis with time in culture. The protein was identified as basic FGF by several criteria. It shared the high affinity for heparin and was the same approximate molecular weight, 18 kD, as basic FGF. Activity was removed from solution by antibodies specific for basic FGF. Recombinant human basic FGF was equally effective in stimulating CAT activity, but was not additive with our purified protein at saturating concentrations. Basic FGF was also found in extracellular matrix and conditioned medium from cultured embryonic chick muscle. The activity could be released from extracellular matrix by treatment with heparinase or high salt extraction. Basic FGF stimulates neurite outgrowth as well as the capacity for transmitter synthesis. Thus, basic FGF is present in embryonic and adult muscle and capable of acting as a growth regulator for cholinergic neurons.

Enhanced survival of motoneurons in the chick lateral motor column: effects of embryonic skeletal muscle extracts and myoblast-conditioned medium.

Muscle-derived factors have shown neurotrophic effects in culture, but their possible effects on the maintenance of embryonic motoneurons have not been demonstrated in vivo. Soluble extracts derived from embryonic chick muscle, or medium conditioned by chick myoblasts, were instilled onto the chorioallantoic membrane (CAM) between the 5th and 11th days of incubation. Counts of the lumbar lateral motor column (LMC) at embryonic day 12 revealed modest but significant increases (12-15%) in motoneuron number for these experimental groups as compared with control treatments. The results suggest that sustaining effects of muscle-derived factors on motoneurons may be demonstrated on the developing LMC by the simple expedient delivery via the CAM, and that these factors can modify the normal program of cell death occurring during this critical period of development.

Distinct roles for adhesion molecules during innervation of embryonic chick muscle

In vitro studies have suggested that the cell adhesion molecules NCAM and G4/L1 contribute to a variety of events during neural development. We have directly tested the role played by these molecules in the process of initial nerve ingrowth and ramification in the embryonic chick iliofibularis muscle by in ovo injections of specific adhesion-blocking antibodies and analysis of the resultant nerve branching pattern in muscle whole mounts. Antibodies against both molecules produced axonal defasciculation, which resulted in an enhanced transverse projection to the fast region of the muscle. In the case of anti-G4/L1, we also observed a large increase in the number of side branches that form from nerve trunks in the slow region and an enhancement of nerve branching in the fast region. Conversely, anti-NCAM produced a striking decrease in both the number and length of side branches in the slow region, and a reduction in nerve branching in the fast region. A similar reduction of nerve branching was obtained following injection of an endosialidase, which removes sialic acid from NCAM, and which was observed to enhance fiber-fiber apposition, presumably by increasing cell adhesion. Based on their biochemical properties in vitro and their in vivo distribution, both NCAM and G4/L1 are in a position to contribute to axon-axon adhesive interactions, whereas NCAM would be expected to also promote axon-myotube interactions. Our observations in fact indicate that these two adhesion molecules play different but complementary roles during muscle innervation and, specifically, that axon-axon fasciculation is influenced by both NCAM and G4/L1 in an anatomically distinct manner to regulate the overall pattern of nerve branching and that NCAM-mediated axon-myotube interactions are necessary for the attainment of the normal stereotyped pattern of nerve branching in both fast and slow regions of this muscle.

Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells.

We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.

Ganglioside potentiation of NGF-independent trophic agents on sensory ganglia

The ability of gangliosides to potentiate nerve growth factor (NGF)-independent trophic agents was determined by examining the capacity of an exogenous mixture of bovine brain gangliosides (BBG) and the monosialoganglioside GM1 to enhance the neuritogenic action of conditioned media (CM). CM were prepared with cultures of C 6 glioma cells, neonatal rat astroglial cells, rat L 6 myoblasts and chick embryonic skeletal muscle. Chick embryonic (9 day) dorsal root ganglia (DRG) were cultured on collagen-coated surfaces. The nutrient media with serum added or serum-free N 1 medium were supplemented with 50% of one of the CM with or without BBG (150 μg/ml) or GM1 (150 μg/ml). The neuritogenic responses of DRG 48 h in vitro were evaluated microscopically on the basis of neurite length and number. The neurite promoting action of the factor(s) present in the various CM was potentiated by BBG or GM1 and resulted in increased neurite length and number.

CDNAs for the postsynaptic 43-kDa protein of Torpedo electric organ encode two proteins with different carboxyl termini.

Postsynaptic membranes isolated from Torpedo electric organ are highly enriched in the nicotinic acetylcholine receptor and a nonreceptor protein of 43 kDa; the distribution of the 43-kDa protein and the receptor is coextensive in the electrical membrane. As a first step in understanding the regulation of 43-kDa protein expression, we have isolated and characterized 43-kDa protein cDNAs. A lambda gt11 cDNA library was constructed from Torpedo californica electric organ mRNA and screened with a pool of 26-mer oligonucleotides encoding a short tryptic fragment of the 43-kDa synaptic protein. Positive clones were purified and sequenced; the amino acid sequences were deduced, and they matched chemically determined protein sequences of the 43-kDa protein. Two distinct classes of cDNAs were obtained; one class encoded a 43-kDa protein of 389 amino acids with a calculated molecular mass of 43,988 daltons, and another class encoded a second 43-kDa protein containing 23 additional amino acids at the C terminus. Therefore, it appears that two 43-kDa proteins with different carboxyl termini are encoded by separate mRNAs. Consistent with this idea, blot hybridization analysis revealed multiple polyadenylylated 43-kDa mRNAs in electric organ. One polyadenylylated mRNA of approximately equal to 2.0 kilobases in length was apparent in both embryonic day-11 chick muscle and the mouse muscle cell line BC3H1.

Extracts of muscle biopsies from patients with spinal muscular atrophies inhibit neurite outgrowth from spinal neurons.

Preparations derived from embryonic and neonatal chick muscle enhance neurite outgrowth when added to cultures of embryonic chick spinal neurons. In the presence of soluble extracts of biopsied muscle from 15 of 20 patients with spinal muscular atrophy (SMA), the in vitro neurite-promoting activity of neonatal chick muscle was inhibited. There was no comparable inhibition using extracts from 20 age-matched pathologic or morphologically normal controls. The neurite-promoting activity in media conditioned by embryonic myotubes was not inhibited by extracts of the SMA group.

O-267 - Chromosome condensation induced in nuclei from chick embryonic muscle in a cell-free system

O-267 - Chromosome condensation induced in nuclei from chick embryonic muscle in a cell-free system

Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures.

The expression of neonatal myosin heavy chain (MHC) was examined in developing embryonic chicken muscle cultures using a monoclonal antibody (2E9) that has been shown to be specific for that isoform (Bandman, E., 1985, Science (Wash. DC), 227: 780-782). After 1 wk in vitro some myotubes could be stained with the antibody, and the number of cells that reacted with 2E9 increased with time in culture. All myotubes always stained with a second monoclonal antibody that reacted with all MHC isoforms (AG19) or with a third monoclonal antibody that reacted with the embryonic but not the neonatal MHC (EB165). Quantitation by ELISA of an extract from 2-wk cultures demonstrated that the neonatal MHC represented between 10 and 15% of the total myosin. The appearance of the neonatal isoform was inhibited by switching young cultures to medium with a higher [K+] which has been shown to block spontaneous contractions of myotubes in culture. Furthermore, if mature cultures that reacted with the neonatal antibody were placed into high [K+] medium, neonatal MHC disappeared from virtually all myotubes within 3 d. The effect of high [K+] medium was reversible. When cultures maintained in high [K+] medium for 2 wk were placed in standard medium, which permitted the resumption of contractile activity, within 24 h cells began to react with the neonatal specific antibody, and by 72 h many myotubes were strongly positive. Since similar results were also obtained by inhibiting spontaneous contractions with tetrodotoxin, we suggest that the development of contractile activity is not only associated with the maturation of myotubes in culture, but may also be the signal that induces the expression of the neonatal MHC.

Small nuclear ribonucleoprotein antigens are absent from 10S translation inhibitory ribonucleoprotein but present in cytoplasmic messenger ribonucleoprotein and polysomes

A cytoplasmic 10S ribonucleoprotein particle (iRNP), which is isolated from chick embryonic muscle, is a potent inhibitor of mRNA translation in vitro and contains a 4S translation inhibitory RNA species (iRNA). The iRNP particle shows similarity in size to the small nuclear ribonucleoprotein (snRNP) particles. Certain autoimmune disease patients contain antibodies directed against snRNP antigenic determinants. The possibility that iRNP may be related to the small nuclear particles was tested by immunoreactivity with monospecific autoimmune antibodies to six antigenic determinants (Sm, RNP, PM-1, SS-A (Ro), SS-B (La), and Scl-70). By Ouchterlony immunodiffusion assays, the cytoplasmic 10S iRNP did not show any immunoreactivity. Also, a more sensitive hemagglutination inhibition assay for detecting Sm and RNP antigens failed to show reactivity with the 10S iRNP. Thus, the 10S iRNP particles are distinct from the similarly sized snRNP. However, free and polysomal messenger ribonucleoprotein (mRNP) particles and polysomes also isolated from chick embryonic muscle and analyzed by Ouchterlony immunodiffusion and hemagglutination inhibition for the presence of the antigenic determinants showed reactivity to Sm and RNP autoantibodies, but were not antigenic for the other four antibodies. Some of the Sm antigenic peptides of mRNP particles and polysomes were identical to those purified from calf thymus nuclear extract, as judged by Western blot analysis. The association of Sm with free and polysomal mRNP and polysomes suggests that Sm may be involved in some cytoplasmic aspects of mRNA metabolism, in addition to a nuclear function in mRNA processing.

Sialylation of lacto- N-neohexaosylceramide by sialyltransferase from embryonic chicken muscle

A sialyltransferase which catalyzes the in vitro biosynthesis of N-acetylneuraminosyllacto- N-neohexaosylceramide from lacto- N-neohexaosylceramide and CMP-NeuAc has been examined in embryonic chicken breast muscle. The maximum enzyme activity was observed in 11–12-day-old embryos. The enzyme has optimum activity at pH 6.8 in the presence of Triton CF-54 and Mg 2+. The apparent K m values for lacto- N-neohexaosylceramide and CMP-NeuAc were 0.9 and 0.67 mM, respectively. The enzymic product was characterized by TLC, neuraminidase hydrolysis and permethylation analysis. The structure was identical to authentic N-acetylneuraminosyllacto- N-neohexaosylceramide from chicken muscle. In addition, a disialo derivative has been detected that constitutes 15% of the total radioactivity incorporated. The two sialic acids connected by sialosyl-sialosyi linkage were attached to the terminal galactose residue. To our knowledge, this is the first report of biosynthesis of this disialo compound.

Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms.

A procedure was developed to determine the percentage of skeletal muscle actin and cardiac actin present in different striated muscle tissues. The method was applied to 2 mg of actin mixtures isolated from various origins. All samples show simultaneous expression of both striated muscle isoactins, with the cardiac actin being the major form (congruent to 80%) in 11-day-old chick embryonic leg muscle, decreasing to approximately 50% values in the late fetal stage of chicken, mouse, and in fused mouse muscle cell cultures and becoming the minor species (less than 5%) in adult skeletal muscle tissues. We also find a significant amount (up to 20%) of the skeletal muscle isoform in adult heart (ventricle) of porcine, bovine, and human origin and no differences in muscle actin ratios in human atrium and ventriculum cells. Similarly, no significant variation in the actin ratios was observed between a normal heart and a heart from a patient with hereditary obstructive myopathy. For those cells and tissues where comparison with levels of mRNA was possible we mostly find a good correlation between the relative ratios of expression of cardiac and skeletal actin proteins and mRNAs.

Ontogenetic study of the regulatory and coupling function of guanyl nucleotide-binding proteins in the hormone-sensitive adenylate cyclase system

1. 1. Reconstitution of catecholamine-sensitive adenylate cyclase from chick embryonic muscle membranes and guanyl nucleotide-binding proteins of mature rabbit muscle makes it possible to reveal the coupling (potentiating) effect of these nucleotides 1 week earlier than in the native condition. 2. 2. The effective insertion of guanyl-nucleotide-binding proteins into the embryonic membrane coincides with the onset of a pronounced increase in membrane lipid fluidity during the course of embryogenesis. 3. 3. The different ontogenetic time-courses for the origination of the two guanyl nucleotide effects, on catalytic adenylate cyclase activity (in early embryogenesis) and on the coupling process (in postembryonic life), suggest the existence in this system of two separate guanyl-nucleotide-binding proteins performing regulatory and coupling functions, respectively.