Interferon-gamma promotes proliferation of rat skeletal muscle cells in vitro and alters their AChR distribution
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Bromodeoxyuridine
Sarcolemma
Caveolin 3
Colocalization
Multinucleate
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Abstract Developing muscles contain at least two types of myoblasts. Early myoblasts are the first myoblast to form and are the only myoblasts present during primary myotube formation. By the time secondary myotube formation begins, early myoblasts are rare and late myoblasts are common. The late myoblasts have been postulated to give rise to secondary myotubes. While this is generally accepted, it is unclear whether late myoblasts also contribute to the growth of primary myotubes. One study has produced evidence that myoblasts present during secondary myogenesis selectively fuse with each other or with secondary myotubes, but not with primary myotubes (Harris et al. [1989a] Development 107:771–784). However, the sizes of primary myotubes increase during secondary myotube formation. We have therefore re‐examined the question of whether primary myotubes absorb new nuclei during secondary myotube formation. Pregnant rats were given a single intraperitoneal injection of 5 mg of 5‐bromodeoxyuridine (BrdU) on one embryonic day (from E13 to E19) and their embryos removed on E20. The brominated‐nuclei were labelled with an antibody to BrdU and the myotubes were marked with anti‐myosin antibodies. Double labelled sections from the soleus, tibialis anterior, and extensor digitorum longus muscles were examined with a confocal microscope. The numbers and locations of labelled nuclear profiles in primary and secondary myotubes were counted and recorded. The results show: (1) that primary myotubes absorb nuclei at all stages of development, including the period of secondary myotube formation; (2) that in the early stages of secondary myotube formation, more myoblasts fuse with primary than secondary myotubes whereas this situation is reversed by the end of secondary myotube formation; and (3) that the nuclei added to primary and secondary myotubes during the early stages of their formation are located within the middle of E20 muscles. The nuclei added to growing myotubes are preferentially located at the ends of the muscles. © 1995 wiley‐Liss, Inc.
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alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.
C2C12
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Previous studies suggested that all myoblasts are present in the head and limb prior to the commencement of primary myotube formation. As a consequence, these myoblasts must be in various developmental states during myogenesis, i.e. proliferating, differentiating or terminally differentiated. There are few in vivo studies investigating dynamic quantitative changes of subgroups of these myoblasts during myogenesis. In this report, using anti-Pax7 and anti-myosin heavy chain antibodies, we examined the quantitative change of proliferating (Pax7(+ve)) and terminally differentiated (MF20(+ve)) myoblasts during primary and secondary myogenesis in the chick head and limb. Our results show that during primary myogenesis, less than 30% of myoblasts are in the proliferating phase, but as soon as secondary myogenesis begins, over 95% of myoblasts start to proliferate. Moreover, we have found that the proportion of terminally differentiated myoblasts is maintained at a low level (less than 3%) during primary and secondary myogenesis.
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Multinucleate
Cell fusion
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Limb bud
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Repair and regeneration are mutually exclusive responses to injury. Previous studies have shown that wound fluids promote proliferation, but not differentiation, of myoblasts in vitro. This study explored the ability of the repair environment within polyvinyl alcohol sponges to support cellular events of skeletal muscle regeneration in vivo. Neonatal rat L8 myoblasts were modified to express beta-galactosidase then inoculated into plain sponges or sponges containing minced muscle. Labeled myoblasts were found in myotubes within minced muscle. In contrast, myoblasts inoculated into sponges lacking muscle remained mononucleate. Occurrence of labeled myoblasts within myotubes, which required fusion, represents differentiation of inoculated myoblasts to participate in regeneration. Failure of myoblasts to form myotubes in sponges lacking muscle suggests that this wound repair environment cannot support morphologic differentiation of myoblasts. Although this repair environment can support the survival of myoblasts, it did not support myogenesis, an event necessary to complete skeletal muscle regeneration. Data from this study reinforce earlier studies in vitro and suggest that the properties attributed to wound fluids are inherent in the wound environment. Whether the inability of this environment to support myogenesis is the consequence of the absence of essential factors or the presence of inhibitors remains to be determined.
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ABSTRACT The distribution of secondary myotubes and undifferentiated mononucleated cells (presumed to be myoblasts) within foetal IVth lumbrical muscles of the rat was analyzed with serial section electron microscopy. In all myotube clusters for which the innervation zone was located, every secondary myotube overlapped the endplate region of the primary myotube. No secondary myotubes were ever demonstrated to occur at a distance from the primary myotube innervation zone. This indicates that new secondary myotubes begin to form only in the innervation zone of the muscle. Some young secondary myotubes made direct contact with a nerve terminal, but we cannot say if this is true for all developing secondary myotubes. Myoblasts were not clustered near the innervation zone, but were uniformly distributed throughout the muscle. Myoblasts were frequently interposed between a primary and a secondary myotube, in equally close proximity to both cell membranes. We conclude that specificity in myoblast-myotube fusion does not depend on restrictions in the physical distribution of myoblasts within the muscle, and therefore must reflect more subtle mechanisms for intercellular recognition.
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Bromodeoxyuridine
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Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.
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