Formation of terminal fields by primary motoneurons in the zebrafish

I have studied the formation of motoneuronal terminal fields in live zebrafish embryos to elucidate mechanisms underlying the specification of synaptic connections. My investigations have been aimed at determining the extent to which the specification of synaptic connections depends on interactions between the motoneuron and other cells. Zebrafish primary motoneurons are identifiable as individuals and innervate neighboring but mutually exclusive muscle territories. I studied the first week of their development by labeling individual motoneurons with fluorescent dyes which facilitated sequential observations of axonal branches. The results show that primary motoneurons establish their cell-specific terminal fields primarily by directed outgrowth of branches and formation of neuromuscular junctions almost exclusively on appropriate muscle fibers, rather than by overproduction and selective elimination of inappropriate branches. Retraction of the few branches that were inappropriately placed, although correlated in time with the ingrowth of branches from appropriate motoneurons, occurred independently of the presence of these other cells and when neuromuscular transmission was blocked. To monitor neuron-target interactions I looked at the distribution of acetylcholine receptor clusters in the muscles of living zebrafish embryos using a fluorescent ligand. Receptor clusters were not detected in the muscle early in development, before the first axons had grown into the muscle segment. This situation changed rapidly as the first growth cones reached the muscle and dense clusters of receptors formed. Concurrent labeling of acetylcholine receptors and motoneurons revealed that all receptor clusters were closely associated with motoneuronal branches. Are the receptor clusters present in embryonic muscles induced by neuronal contact? I looked at the distribution of receptor clusters in muscle segments deprived of motor innervation. Segments completely lacking motor innervation, as a result of spinal ablation, failed to cluster acetylcholine receptors for the duration of the observations. These results differ from those obtained in cell culture and in other vertebrates where clusters of acetylcholine receptors have been found in the absence of nerve contact. I attribute this disparity to a difference between tissue culture and embryonic conditions because cultured zebrafish muscles, too, produce clusters of acetylcholine receptors in the absence of nerve contact.