Cross-reinnervation studies performed ex ovo with newly hatched chicks demonstrate that peripheral motor neurons control the phenotypic characteristics of avian muscles. The present experiments were designed to determine whether or not nerves play a similar role during the initial expression of muscle fiber types. Previous experiments indicated that differentiation of specific fiber types occurs during the first week of embryogenesis, temporally coincident with the penetration of nerves within muscle masses. These observations suggested that peripheral nerves may be associated with the initial differentiation of fiber types. To test this hypothesis directly, anterior limb buds of the chick embryo were rendered aneurogenic by deletion of the brachial segment of the neural tube. To ensure a completely aneurogenic environment for developing brachial muscles, surgery was performed at day 2 in ovo before the exit of ventral root fibers. Experimental and control embryos from Stage (St) 25 (4.5 d) through St 45 (19d) were analyzed histochemically by a silver-cholinesterase reaction to detect nerves and by the myosin ATPase reaction, following alkali and acid preincubation, to determine the fiber type composition of the muscles. In addition, the total volume of aneurogenic and control muscles was compared. Results demonstrate that the characteristic myosin ATPase profiles of individual aneurogenic and innervated (control) muscles were identical throughout the entire period analyzed. Therefore, we conclude that these enzymic profiles are endogenously expressed and are not under neuronal control during early embryogenesis. Furthermore, the entire sequence of events from the migration of myogenic cells to the anterior limb bud through the division of the primary muscle masses to form individual brachial muscles proceeded on schedule in the absence of nerves. Since the growth of aneurogenic muscles was impaired, we conclude that during embryogenesis peripheral motor nerves are necessary initially for the proper growth of muscles and ultimately, for their survival. They are not involved, however, with either the initial formation or initial differentiation of individual brachial muscles.

(1) The Ca ++, Na +, and K + contents of frog sartorius muscles were found analytically after exposure to various media including some containing labeled Ca ++. (2) During storage in media with 100 to 120 mM Na + and 1 mM Ca ++ both Na + and Ca++ are gained while K + is lost; there is a high correlation between Na + and Ca++ gains. (3) When Ca ++ gain occurs from a solution containing labeled Ca++ there is also some exchange of the original Ca++ with the labeled Ca++. The amount exchanged is considerably less (e.g. 50 per cent) than the total amount of labeled Ca ++ taken up by the tissue. (4) When the external Na + concentration is reduced to 30 m~ the amount of labeled Ca++ taken up is increased. Part of the increase is attributable to a greater net gain and part to a greater degree of exchange. (5) It is pointed out that muscles which have been loaded in vitro with labeled Ca++ will not provide a valid measure of the exchangeability of the normal Ca++ content present at the time of dissection. (6) Comparison is made between results obtained using Sr 89 and Ca 45 as labels for the Ca++. Little, if any, difference is perceptible.

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