Early stages of myogenesis in a large mammal: Formation of successive generations of myotubes in sheep tibialis cranialis muscle

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Abstract
The generation of myotubes was studied in the tibialis cranialis muscle in the sheep hindlimb from the earliest stage of primary myotube formation until a stage shortly before muscle fascicles began to segregate. Primary myotubes were first seen on embryonic day 32 (E32) and reached their maximum number by E38. Small numbers of secondary myotubes were first identified at E38, and secondary myotube numbers continued to increase during the period of study. The ratio of adult muscle fibre to primary myotube numbers was approximately 70∶1, making it seem unlikely that every later generation myotube used a primary myotube as scaffold for its formation, as described in small mammals. By E62, some secondary myotubes were supporting the formation of a third generation of myotubes. Experiments with diffusible dye markers showed that primary myotubes extended from tendon to tendon of the muscle, whereas most adult fibres ran for only part of the muscle length, terminating with myo-myonal attachments to other muscle fibres in a series arrangement. Acetylcholinesterase (AChE) and acetylcholine receptor (AChR) aggregations appeared in multiple bands across the muscle shortly after formation of the primary generation of myotubes was complete. The number of bands and their pattern of distribution across the muscle as they were first formed was the same as in the adult. Primary myotubes teased from early muscles had multiple focal AChE and AChR deposits regularly spaced along their lengths. We suggest that the secondary generation of myotubes forms at endplate sites in a series arrangement along the length of single primary myotubes, and that tertiary and possibly later generations of myotubes in their turn use the earlier generation myofibres as a scaffold. Although the fundamental cellular mechanisms appear to be similar, the process of muscle fibre generation in large mammalian muscles is more complex than that described from previous studies in small laboratory rodents.