Marilyn J. Duxson

Background: Vertebrate muscles are defined and patterned at the stage of primary myotube formation, but there is no clear description of how these cells form in vivo. Of particular interest is whether primary myotubes are “seeded” by a unique myoblast population that differentiates as mononucleated myocytes, similar to the founder myoblasts of insects. Results: We analyzed the cell populations and processes leading to initiation of primary myogenesis in limb buds of rats and mice. Pax3^+ve myogenic precursors migrate into the limb bud and initially consolidate into dorsal and ventral muscle masses in the absence of Pax7 expression. Approximately a day later, Pax7^+ve cells appear in the central aspect of the limb base and subsequently throughout the limb muscle masses. Primary myogenesis is initiated within each muscle mass at a time when only Pax3, and not Pax7, protein can be detected. Primary myotubes form initially as elongate mononucleated myocytes, well before cleavage of the muscle masses has occurred. Multinucleate myotubes appear approximately a day later. A similar process is seen during initiation of chick limb primary myogenesis. Conclusions: Primary myotubes of vertebrate limb muscles are initiated by mononucleated myocytes, that appear structurally analogous to the founder myoblasts of insects. Developmental Dynamics 242:1043–1055, 2013.

The myotome is a segmented paraxial muscle present in all early vertebrate embryos, which in amniotes disappears in mid-embryogenesis, and is replaced by complex epaxial and hypaxial musculature. Little is known about how this transition occurs. Here, we describe the detailed morphogenesis of the epaxial muscles from the epaxial myotome, in rodent embryos. The results show there is no apoptosis of myotomal fibres during the transition, and that the epaxial muscles arise by translocation, re-orientation, and elongation of the myotomal myocytes followed by cleavage of the myotomal masses. Myotomal myocytes transit from a mononucleated to a multinucleated state just before onset of this transformation. Each newly-formed epaxial muscle anlagen includes populations of Pax3- and Pax7-positive muscle progenitors, with different distributions. Using transgenic mouse embryos bearing a GFP marker for Scleraxis, we show that tendon progenitors are tightly associated with the sides and ends of myotomal myocytes as they re-orient and elongate. Developmental Dynamics 239:2898–2910, 2010.

The segmented muscular myotome is the first muscle to form in all vertebrates. In fish and amphibian embryos, the myotome becomes innervated very early and is essential for larval swimming. Its role in birds and mammals, however, is not clear. Using immunohistochemistry on sections and whole mounts of rat embryos, we demonstrate that the mammalian myotome differentiates and develops over a period of 3 days without being invaded by the outgrowing spinal nerves. In contrast, the limb muscle masses become filled with fine nerve branches from the first time that myocyte differentiation can be detected. Additionally, we show that the mammalian myotome does not express clustered acetylcholine receptors until after embryonic day 13.5, which corresponds to the beginning of its transformation into the adult epaxial muscles, showing that there are no functional myotomal neuromuscular junctions before this age. We suggest that the mammalian myotome has entirely lost the function of neurally controlled muscular contraction: its remaining functions are likely to be as a signaling tissue, as a structural scaffold, and as an incubator for myogenic precursors of the deep back, abdominal, and intercostal muscles.

This work investigates the role of NT‐3 in peripheral myelination. Recent articles, based in vitro, propose that NT‐3 acting through its high‐affinity receptor TrkC may act to inhibit myelin formation by enhancing Schwann cell motility and/or migration. Here, we investigate this hypothesis in vivo by examining myelination formation in NT‐3 mutant mice. On the day of birth, soon after the onset of myelination, axons showed normal ensheathment by Schwann cells, no change in the proportion of axons which had begun to myelinate, and no change in either myelin thickness or number of myelin lamellae. However in postnatal day 21 mice, when myelination is substantially complete, we observed an unexpected reduction in mRNA and protein levels for MAG and P₀, and in myelin thickness. This is the opposite result to that predicted from previous in vitro studies, where removal of an inhibitory NT‐3 signal would have been expected to enhance myelination. These results suggest that, in vivo, the importance of NT‐3 as a major support factor for Schwann cells (Meier et al., (1999) J Neurosci 19:3847‐3859) over‐rides its potential role as an myelin inhibitor, with the net effect that loss of NT‐3 results in degradation of Schwann cell functions, including myelination. In support of this idea, Schwann cells of NT‐3 null mutants showed increased expression of activated caspase‐3. Finally, we observed significant reduction in width of the Schwann cell periaxonal collar in NT‐3 mutant animals suggesting that loss of NT‐3 and resulting reduction in MAG levels may alter signaling at the axon‐glial interface.

This paper examines early postnatal development of the neuromuscular system in mice with a null mutation in the gene for neurotrophin‐3. We report that alpha‐motoneurons at first develop substantially normally, despite a known 15% deficit in their somal size [Woolley et al. (1999)Neurosci. Lett., 272, 107–110.] and the absence of proprioceptive input [Ernfors et al. (1994)Cell, 77, 503–512]. At birth, motor axons have extended into the muscle, forming normal‐looking neuromuscular junctions with focal accumulations of acetylcholine receptors. Detailed ultrastructural analysis does however, reveal subtle abnormalities at this time, particularly a decrease in the extent of occupancy of the postsynaptic site by nerve terminals, and a small but significant deficit in myofibre number. After the relative normality of this early neuromuscular development, there then occurs a catastrophic postnatal loss of motor nerve terminals, resulting in complete denervation of hindlimb muscles by P7. In systematic semi‐serial samples through the entire muscle endplate zones, no neuromuscular junctions can be found. Intramuscular axons are fragmented, as shown by both electron microscopic observations and neurofilament immunohistochemistry, and alpha‐bungarotoxin detection of acetylcholine receptors indicates dispersal of the junctional accumulation. At earlier times (postnatal days three and four) the terminal Schwann cells show ultrastructural abnormalities, and preliminary observations suggest marked disturbance of myelination. Based on comparison with other literature, the peripheral nerve degeneration seems unlikely to have arisen as a secondary effect of de‐afferentation. We discuss whether the neural degeneration is secondary to the disturbance of Schwann cell function, or due directly to a loss of neurotrophin‐3 based support of the motoneuron.

This study examines the processes by which multiply innervated, serially fibered mammalian muscles are constructed during development. We previously reported that primary myotubes of such a muscle, the guinea pig sternomastoid muscle, span from tendon to tendon and are innervated at each of the muscle's four innervation zones. Secondary myotubes form later, in association with each point of innervation (Duxson and Sheard, Dev. Dyn., 1995; 204:391–405). We now describe the further growth and development of the muscle. Secondary myotubes initially insert onto and grow along the primary myotube. However, as they reach a critical length, they encounter other secondary myotubes growing from serially adjacent innervation zones and may transfer their attachment(s) to these serially positioned secondary myotubes. Other secondary myotubes maintain attachment at one or both ends to their primary myotube. Thus, an interconnected network of primary and secondary myotubes is formed. Patterns of reactivity for cell adhesion molecules suggest that early attachment points between myotubes are the embryonic precursors of adult myomyonal junctions, characterized by the expression of α7Bβ1 integrin. Finally, the results show that secondary myotubes positioned near a tendon are generally longer than those lying in the mid belly of the muscle, and we suggest that the environment surrounding the tendinous zone may somehow stimulate myotube growth. Anat Rec Part A 278A:571–578, 2004.