The growth of muscle nerves in relation to the formation of primary myotubes in the developing chick forelimb.

A study has been made of the development of muscle nerves to primary myotube clusters destined to become the flexor carpi ulnaris (fcu) and flexor digitorum profundus (fdp) muscles in the avian forelimb. Myotubes and nerves were identified by immunofluorescent techniques using antibodies to the heavy and light subunits of myosin and neurofilament, respectively. At stage 24 the main ventral nerve trunk (the brachialis longus inferior nerve; bli n) had entered the limb before the appearance of myotubes in the limb. At stage 25/26 the bli n within the ventral compartment of the forearm had given rise to the interosseus nerve (in n) and the medial-ulnar nerve (m-u n) at the junction of the stylopodium and zeugopodium. The first few myotubes of the fdp and fcu muscles were observed at this level within the ventral premuscle cell mass; however, no nerves projected toward these myotubes from either the in n or the m-u n at this time. At stage 26/27 the fcu and the fdp muscles each consisted of clusters of 20-40 myotubes; each cluster was clearly delineated within the ventral premuscle cell mass. By this time small groups of axons had left the in n and the m-u n to grow into the fdp and fcu myotube clusters, respectively; these axons formed the muscle nerves. At stage 28/29 the number of primary myotubes in the clusters composing the fdp and fcu muscles had greatly increased, as did the size of the muscle nerves; each muscle was still clearly identifiable within the ventral muscle mass. By stage 32 the fdp and fcu muscles had clearly separated and the muscle nerves had divided into several well-spaced branches within each muscle. The present observations show that the main nerve trunks grow into the limb before the formation of myotubes expressing myosin isozymes. When myotubes do form they appear in small clusters at specific sites within the premuscle mass, before muscle nerves appear; a distinct muscle is destined to form from each of these clusters. Muscle nerves first branch from the main limb nerves when the myotube cluster contains more than about ten myotubes.