The pattern of organogenesis of the soleus muscle of the 129 ReJ mouse was evaluated quantitatively using spaced, serial, ultrathin sections and computer-assisted morphometric analysis. Muscles from 14-, 16-, and 18-day in utero mice and muscles of 1- and 5-day-old mice were analyzed to determine age-related alterations in the maximal girth and length of the muscle, number of myotubes, cluster frequency, and the lengths and diameters of myotubes. Primary myotubes are found in the muscle at 14 days in utero. There is little de novo myotube formation between 14 and 16 days in utero, this interval being principally one of primary myotube growth and maturation. The interval between 16 and 18 days in utero is marked by extensive secondary myotube formation, with more myotubes being formed during this period than in any period studied. Morphometric data support the hypothesis that secondary generation myotubes use primary myotubes as a scaffold on which they are formed. Morphometric data also confirm the hypothesis that cluster formation and cluster dispersal occur concurrently during the prenatal period. Secondary myotubes continue to form until birth. At birth, the soleus muscle contains the adult number of myofibers. The first 5 days postnatally are marked by myofiber growth and maturation.
The organogenesis of the soleus muscle of the 129 ReJ mouse (a mixed muscle, which in the adult contains approximately equal numbers of slow-twitch oxidative and fast-twitch oxidative-glycolytic myofibers) was studied in spaced, serial transverse, and longitudinal sections of muscles of 14-, 16-, and 18-day in utero and 1- and 5-day postnatal mice. A discrete soleus muscle was distinguished by 14 days in utero. It consisted of groups of closely apposed primary myotubes displaying junctional complexes and a pleomorphic population of mononucleated cells. Between 14 and 16 days in utero there was little de novo myotube formation. At 16 days in utero, basal lamina surrounded groups of primary myotubes; and primitive motor endplates were found on these myotubes. At 18 days in utero, the basal-lamina-enclosed groups of primary myotubes were no longer present. At this stage, basal lamina surrounded clusters (consisting of one primary myotube and one or more secondary myotubes) or independent myotubes (single myotubes surrounded by their own basal lamina). Cluster formation and cluster dispersal occurred concurrently, beginning at 18 days in utero and extending until birth. At birth, there was still a substantial population of immature, secondary myotubes that interdigitated with larger, more mature primary myofibers. At this stage, intermuscular axons had begun to myelinate, and postsynaptic specialization of the motor endplates had begun. Cluster dispersal and myonuclear migration was completed during the first 5 days postnatally with the muscle taking on adult characteristics. Beginning at 16 days in utero and extending into the neonatal period, there was evidence of myotube death in the soleus muscle.
The extensor digitorum longus muscles of 2-, 4-, and 12-week-old 129-ReJ mice were subjected to homotopic, whole-muscle transplantation. Subsequent to myofiber necrosis and phagocytosis, a new population of myotubes was produced. The three-dimensional cytoarchitecture of these newly formed myotubes was determined in spaced, serial, ultrathin sections. Myotubes, which for long distances along their length appeared to be separate and discrete, were found to branch and recombine, forming a complex syncytium.
A technique is described whereby it is possible to surgically ablate the lumbosacral spinal cord of a developing mouse fetus without interfering with fetal viability. The lumbosacral spinal cords of 14-day in utero, 129ReJ mice were ablated with a Cooper Nd-YAG laser, and the fetuses, enclosed in their membranes and attached to the uterus by their placentae, were allowed to develop in the abdominal cavity of the dam. The cytoarchitecture and the temporal pattern of organogenesis of aneural soleus muscles were studied in spaced, serial, transverse, ultrathin sections of muscles of 16-and 18-day gestation and newborn (20-day gestation) mice. At the time of surgery, the soleus muscle was a discrete mass consisting of primary myotubes and a pleomorphic population of mononucleated cells. Axon bundles and blood vessels were found at the muscle's periphery, but had not penetrated throughout the muscle mass. The organogenesis of the aneural muscle was remarkably similar to that of the innervated soleus muscle (Ontell et al., Am J Anat 181: [267][268][269][270][271][272][273][274][275][276][277][278]1988). In the aneural muscle, as in the innervated muscle, significant numbers of secondary myotubes formed all along the lengths of primary myotubes. Moreover, the time course of myotube formation, the dynamics of cluster formation and cluster dispersal, and the ultrastructural appearance of the myotubes mimicked that observed in innervated muscle. The frequency of necrotic myotubes was no greater in the aneural muscle than in the innervated soleus muscle. Myotube maturation was similar in aneural and innervated soleus muscles until 18 days gestation. However, at birth, aneural myotubes appeared to be slightly less mature than innervated myotubes. Thus, the major morphogenic phenomena that characterize the development of the soleus muscle appear to be independent of innervation.
It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration-regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJdy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) per milligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability.
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