Multinucleation of skeletal muscle in vitro.

Healthy, mature, spontaneously contracting muscle was cultivated from explants of 13-day chick embryos for periods up to 4 months in the multipurpose chamber (Rose, 1954) using cellophane-strip technique (Rose et al., 1958) with silicone gaskets, Eagle's medium including 10 per cent horse serum reinforced with 300 mg-per cent of glucose, and the teased type of explant. This method provided optically ideal conditions for the study of muscle fibers with oil immersion, phase contrast time-lapse cinematography at 1 frame per minute without apparent damage for periods as long as 10 days. In no case was mitosis, amitosis, or nuclear "budding" observed in the course of muscle development. Multinuclear muscle fibers have been shown with cine technique to result from both myoblast fusion and polar extension of preformed (explanted) muscle tissue. Myoblast fusion was the only demonstrable way of giving rise to multinucleation. Nuclear membrane "wrinkling" was shown to be merely a temporary distortion that occurred during nuclear migration and rotation. It is suggested that this phenomenon may be responsible for numerous reports of amitosis in the genesis of muscle fibers. The histological development of new straps resulted from an orderly sequence of events. Included in these were polar extension, nuclear migration, rotation, and fixation. Following these events there was increased mitochondrial activity, myofibril formation, and cross-banding. Spontaneous contractions were seen throughout the entire course of differentiation in vitro but became more regular and stronger in the later stages.