NEURONS AND ECDYSTEROIDS PROMOTE THE PROLIFERATION OF MYOGENIC CELLS CULTURED FROM THE DEVELOPING ADULT LEGS OF MANDUCA-SEXTA

During metamorphosis in the hawkmoth Manduca sexta, larval leg motoneu rons survive the degeneration of their target muscles to innervate new muscles that form during the development of the adult legs. Observati ons of muscle development in vivo suggest that there are close interac tions between motor terminals and the muscle precursor cells at the ea rliest stages of muscle formation and surgical denervation compromises further development of adult muscles. Here we describe a nerve/muscle coculture system that allows further exploration of this critical dev elopmental interaction. Muscle precursor cells derived from the develo ping thoracic legs of early pupae and cultured in the presence of neur ons assumed a spindlelike morphology and fused to form multinucleate c ontractile myotubes. Contractile fibers did not form in cultures of mu scle precursor cells alone. In the presence of neurons the rate of bro modeoxyuridine (BrdU) incorporation into myonuclei was significantly e nhanced, suggesting that neurons promote the proliferation of myogenic cells. This effect was not unique to thoracic leg motoneurons of the early pupal stage, in that larval thoracic neurons as well as neurons from the pupal brain or abdominal ganglia were also effective at enhan cing BrdU incorporation and the formation of contractile muscle fibers . Medium conditioned by neurons was ineffective at promoting BrdU inco rporation, and in cocultures BrdU incorporation was enhanced only in r egions of physical overlap between neurons and muscle precursor cells, suggesting that a very close-range interaction was involved. Tetrodot oxin-sensitive neuronal activity was not required for the effect on mu scle development, but fixed neurons were ineffective. The insect stero id hormone 20-hydroxyecdysone enhanced BrdU incorporation into the nuc lei of myogenic cells in both the presence and the absence of neurons. The results suggest that both neurons and ecdysteroids play an import ant regulatory role in adult muscle development, at least in part by e nhancing the proliferation of myogenic cells. (C) 1996 Academic Press, Inc.