The influence of innervation on the initial differentiation of muscle fibre types was investigated by using the trochlear nucleus—superior oblique muscle system of duck. The adult muscle is composed of three types of fibres (designated as type I, II, III) as identified with the histochemical techniques for ATPase pH sensitivity. Type I fibre ATPase activity was acidstable, alkali-labile; type II alkali-stable, acid-labile; and type III both acid- and alkali-stable. These types showed variable mitochondrial α-glycerophosphatase dehydrogenase, nicotinamide adenine dinucleotide tetrazolium reductase, and phosphorylase activity. Type I and II fibres are primarily located in the portion of the muscle adjacent to the orbit whereas the rest of the muscle is primarily composed of type III fibres. In the normally developing muscle, type II and III fibres are present as early as embryonic day 9; one day prior to the arrival of nerve fibres in the muscle. Type I fibres are first observed on embryonic day 17. On day 22 the percentages of type I, II and III fibres are 29, 53 and 18, respectively. As the development progressed the percentages of type I and II fibres decrease and after hatching 76 % of the fibres belong to type III, 17 % to type II and only 7 % to type I. In embryos paralysed with daily application of 3 mg d-tubocurarine (d-TC) from day 9 onwards the differentiation of type II and III fibres occurs, but type I fibres were never observed in the paralysed muscles. These muscles also contained significantly fewer myotubes than the normal muscle. By contrast, when the muscle was made aneural by permanent destruction of motor neurones on embryonic day 7 all three types of fibres differentiated. When embryos with aneural muscles were also subjected to d-TC treatment the type I fibres failed to differentiate. It is concluded that the initial differentiation of fibre types is independent of innervation and that primary myotubes are capable of differentiating into all three types of fibres. The absence of type I fibres in curarized muscles may be due to some unique effect of d-TC on the muscle itself.

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