Early Stages Of Myogenesis Research Articles

α-actin proteins and gene transcripts are colocalized in embryonic mouse muscle

The alpha-actins are among the earliest muscle-specific mRNAs to appear in developing cardiac and skeletal muscle. To determine if there is coexpression of the alpha-actin proteins at early stages of myogenesis, we have used an alpha-actin-specific polyclonal antibody and in situ hybridization with specific cRNA probes to cardiac and skeletal alpha-actin transcripts on serial slides of mouse embryo sections. As soon as we can detect alpha-actin mRNAs in embryonic striated muscle, we also detect the protein suggesting that alpha-actin transcripts are translated very rapidly after transcription during myogenesis. In skeletal muscle, this colocalization of alpha-actin mRNA and protein was observed both in the myotomes of somites and in developing muscles in the limbs. In cardiac muscle, alpha-actin transcripts and proteins are abundantly expressed as soon as a cardiac tube forms.

Development of muscle fiber types in the prenatal rat hindlimb

Immunohistochemistry was used to examine the expression of embryonic, slow, and neonatal isoforms of myosin heavy chain in muscle fibers of the embryonic rat hindlimb. While the embryonic isoform is present in every fiber throughout prenatal development, by the time of birth the expression of the slow and neonatal isoforms occurs, for the most part, in separate, complementary populations of fibers. The pattern of slow and neonatal expression is highly stereotyped in individual muscles and mirrors the distribution of slow and fast fibers found in the adult. This pattern is not present at the early stages of myogenesis but unfolds gradually as different generations of fibers are added. As has been noted by previous investigators (e.g., Narusawa et al., 1987, J. Cell Biol. 104, 447–459) , all of the earliest generation (primary) muscle fibers initially express the slow isoform but some of these primary fibers later lose this expression. In this study we show that loss of slow myosin in these fibers is accompanied by the expression of neonatal myosin. This switch in isoform expression occurs in all primary fibers located in specific regions of particular muscles. However, in other muscles primary fibers which retain their slow expression are extensively intermixed with those that switch to neonatal expression. Later generated (secondary) muscle fibers, which are interspersed among the primary fibers, express neonatal myosin, although a few of them in stereotyped locations later switch from neonatal to slow myosin expression. Many of the observed changes in myosin expression occur coincidentally with the arrival of axons in the limb or the invasion of axons into individual muscles. Thus, although both fiber birth date and intramuscular position are grossly predictive of fiber fate, neither factor is sufficient to account for the final pattern of fiber types seen in the rat hindlimb. The possibility that fiber diversification is dependent upon innervation is tested in the accompanying paper (K. Condon, L. Silberstein, H. M. Blau, and W. J. Thompson, 1990, Dev. Biol. 138, 275–295) .

Changes in the distribution of slow skeletal myosin heavy chain SM1 in developing avian muscle fibres

The use of monoclonal antibodies against fast skeletal and slow skeletal myosin heavy chains (MHC) has shown the presence of significant amounts of slow skeletal type MHC in embryonic skeletal muscles of white leghorn chickens. The presence of this slow skeletal myosin heavy chain (SMHC) was not restricted to presumptive slow muscles only, as it was also observed in presumptive fast skeletal muscles. As was the case for embryonic MHC reactive with the antibody against fast skeletal myosin heavy chain (FMHC), the presence of SMHC could be detected at the earliest stages of myogenesis. It appeared to be present in most muscle cells during early embryonic development. The changes in its cellular distribution during subsequent embryonic and post-hatch period indicated its suppression in a certain proportion of the cells in both presumptive fast and slow skeletal muscles. Its time course of suppression, however, was much prolonged, not synchronized, and varied in fast and slow skeletal muscles during both embryonic and post-hatch development.

Binding of Ricinus communis I lectin to developing dystrophic muscle in human fetus

In previous studies it was shown that a d-galactose-specific lectin, Ricinus communis I (RCA I), does not bind to the plasma membrane of muscle fibres from patients with Duchenne muscular dystrophy (DMD) in contrast to normal muscle. We have now studied RCA I binding to the membranes of developing human fetal muscle in fetuses at 95% risk of DMD (n = 6) and normal controls (n = 5) with a developmental range of 12–20 weeks of gestation. The results were compared to the membrane appearance with conventional ultrastructure. Binding of RCA I to the muscle basement membrane was consistently strong from the early stages of myogenesis, such as in fusing myoblasts/myocytes. RCA I binding to the plasma membrane was weak but detectable in both DMD and normal fetuses at 12–14 weeks of gestation. Both the normal and diseased condition showed an increase of RCA I labelling of the muscle plasma membrane at 15–17 weeks and strong labelling at 18–20 weeks of gestation. No difference was observed in the RCA I localization of normal and diseased human fetal muscle plasma membrane. It is concluded that (a) the plasma membrane in developing fetal muscle undergoes a maturation process between 12 and 20 weeks gestational age leading to an increase in expression of RCA I binding carbohydrate moieties; and (b) that the absence of RCA I binding glycoprotein in mature DMD muscle plasma membrane reflects a change acquired during the course of disease.

Characterization of a prostaglandin dysfunction myopathy.

Inhibition of prostaglandin synthesis in chicken embryos generated a myopathy characterized by ringbinden, loss of Z disks and thick and thin filaments from the myofibrillar apparatus. During the early stages of myogenesis there was also abnormal clustering and contacts between the developing myotubes. This disruption of myofibrillar apparatus occurred in both primary and secondary myotubes. There was also a slight increase in the proportion of type 1 fibers, which resulted from a selective absence of type 2 fibers. The number of motoneurons in the lumbar spinal cord was normal. This, along with the nature of the disruption of the myofibrillar apparatus, was taken as an indication that the generation of this myopathy was not secondary to abnormal motoneuron development.

Ultrastructural Localisation Of Ricinus Communis-I Lectin To Skeletal Muscle From Foetuses At High Risk For Duchenne Muscular Dystrophy

Our previous studies have shown that a D-galactose-specific lectin. Ricinus communis-I (RCA-I), does not bind to the plasma membrane of skeletal muscle fibres In DMD whereas it does in normal muscle. RCA-I was subsequently found to recognize a Mr 370000 glycoproteln which is absent or altered in DMD (1. 2). We have now used RCA-I to characterize the Ultrastructural glycoprotein pattern of developing human skeletal muscle membranes. Five foetuses at high risk of DMD (95% by DNA probe analysis), ranging from 12 to 20 weeks gestation, were compared with five age-matched normal foetuses. The results were compared to the membrane appearance in conventional ultrastructure. Binding of RCA-I to the muscle fibre basement membrane was consistently strong from the early stages of myogenesis, such as in fusing myoblasts. In both the normal and DMD foetuses RCA-I binding to the plasma membrane was weak but detectable at 12 weeks, more marked by 15 weeks gestation and showed strong labelling at 20 weeks gestation. No difference was observed in RCA-Z binding to the plasma membrane 1n normal and diseased human foetal muscle. It is concluded that a) the human foetal muscle plasma membrane is undergoing a maturation process between 12 and 20 weeks gestational age leading to an increase 1n expression of RCA-I binding glycoproteins; and b) that the absence of RCA-I binding glycoprotein in mature DMD muscle represents a secondary change developing in the course of the disease. 1) J Neurol Sci 63: 129-142. 1984 2) J Neurol Sci 68: 225-231. 1985

Possible role of c- fos, c-N- ras and c- mos proto-oncogenes in muscular development

Time course analyses of various proto-oncogene transcripts compared with cytoskeleton-specific and muscle-specific messenger RNAs (mRNAs) were carried out during growth and differentiation of a clonal line of rat myoblasts that retain the capacity to form non-contractile fibres in vitro. Throughout their growth phase, these cells express consistent levels of c- fos, c- myc, c-Ki- ras and c-N- ras RNA and no c- mos RNA. When the cultures approach confluency the level of c- fos RNA rises sharply 3–4-fold, peaks, and rapidly declines when muscle-specific transcripts start accumulating, to become negligible in myotube-forming cells. These changes occur whatever the concentration in seric factors. By contrast, the level of c-N- ras RNA rises up to 3-fold and both c- myc and c-Ki- ras RNAs are slowly eliminated during the myogenic process, whereas no c- mos RNA is detectable. However, skeletal muscles from prenatal fetuses and adult animals were reproducibly found to contain both low and high levels of c- mos RNA respectively. These data and the demonstration that inactivation of the c- fos gene correlates with the loss of myogenic capability in six lines of neoplastic myoblasts, including four lines transformed by the v- fos oncogene, suggest a physiological function for this proto-oncogene during early stages of myogenesis and for the c-N- ras and c- mos genes in later stages of muscular development.

Calcium channel components of action potential in chick skeletal muscle cells developing in culture

The action potential was recorded from cultured chick skeletal muscle cells in Na-, Ca-, Cl-free saline containing Ba and tetraethylammonium ions (Ba saline). The action potential consisted of two components: (1) a low-threshold, fast inactivating component and (2) a high-threshold, long-lasting component. Both components of the action potential were dependent on external Ba ions and eliminated by Co ions. It is concluded that both components are generated by inward currents carried by Ba ions through Ca channels. The two Ca channel components of the action potential differed with regard to activation and inactivation potential, presence or absence of fast inactivation, sensitivity to an organic Ca channel blocker, and developmental profile. In addition, the failure of occurrence of one or the other components was observed in some cells. These results could be explained by assuming that two components of the action potential in Ba saline were mediated by the two different Ca channels. Furthermore, there was a tendency for younger cells to have more prominent Ca channel components. This may suggest that Ca channels have some function in the early stages of myogenesis.

Monoclonal antibodies to desmin: evidence for stage-dependent intermediate filament immunoreactivity during cardiac and skeletal muscle development.

Monoclonal antibodies reactive with desmin (D3 and D76) have been generated and their specificities validated by immunoblots, RIAs, and immunocytochemistry. No cross-reaction with other IFPs has been observed. The McAbs recognized different epitopes but both reside in the amino-terminal rod domain of desmin. Whereas McAb D3 produces a staining pattern characteristic of desmin throughout the development of cardiac and skeletal muscles, McAb D76 was selectively unreactive with certain regions of early (three days in ovo) embryonic cardiac anlage, with cultured cardiac myocytes derived from 7-day-old embryos, and with skeletal myotubes in early stages of myogenesis in vitro. Positive reactivity of D76 was seen at stages of myofibrillogenesis when the sarcomeres assume lateral alignment. Evidence was presented that differential reactivity of D76 did not result from the biosynthesis of a new desmin isoform or the post-translational modification of an existing protein. We suggest that the appearance of D76 immunoreactivity during striated muscle development represents an unmasking of the epitope by some IF-associated protein. Since this transition during skeletal muscle differentiation occurs during lateral alignment of the myofibrils, this antibody may serve as a useful probe for exploring this reorganization of the contractile apparatus during myogenesis and muscle regeneration.

Neural dependence and independence of myotube production in chicken hindlimb muscles

Chicken embryos were paralyzed by repeated injections of the neuromuscular blocking agent d-tubocurarine, from the fourth day in ovo until sacrificed. This treatment affected the late but not early stages of myogenesis. The production of the initial generation (primary) of myotubes was unaffected whereas, in contrast, the acquisition of subsequent generations (secondary) of myotubes was totally inhibited. The elongation and growth of primary myotubes proceeded normally although their differentiation was retarded. An atrophy of these myotubes was not observed even in Day 17 embryos. It is concluded that the factors regulating myogenesis change as the muscle matures.

CAMP and cGMP changes associated with the differentiation of cultured chick embryo muscle cells

Changes in the intracellular concentration of cAMP and cGMP were examined in primary cultures of chick embryo skeletal muscle cells. The reciprocal changes, increase in cAMP and decrease in cGMP level, were observed during the early stage of myogenesis. Consequently, the ratio of cAMP to cGMP increased significantly during that period reaching the maximum level on day 3. The most marked change in the nucleotide ratio, an approx. 2-fold increase, was observed between the 2nd and 3rd day of culture which coincided with the period of cell fusion. After the 3rd day, the value of nucleotide ratio remained constant throughout the period examined. These findings pose the possibility that the intracellular levels of both cyclic nucleotides are involved in the regulation in myoblast differentiation.