| During the development of the neuromuscular junction, it is generally accepted that the nerve exerts complex control over the differentiation of the postsynaptic apparatus. In contrast, although muscle is widely believed to influence nerve terminal differentiation in some manner, little evidence exists as to a specific role for target in presynaptic differentiation. To test the hypothesis that target supplies retrograde signals to the developing nerve, and to establish the effects of these signals on nerve terminal differentiation, a study of neuromuscular differentiation both in vitro and in vivo was undertaken. Chinese-hamster-ovary (CHO) cells expressing the basal lamina heparan sulfate proteoglycan agrin on their surfaces were cocultured with cholinergic motor neurons of the chick ciliary ganglia (CG). In such a system, agrin is sufficient to induce clustering of the synaptic vesicle protein synaptotagmin (syt) and displays the additional properties of a general motorneuronal stop signal. In CG-muscle cocultures, antibodies to agrin in high concentrations specifically disrupt both pre- and postsynaptic differentiation. In the presence of intermediate concentrations of antibody, postsynaptic differentiation still occurs, while presynaptic differentiation is eliminated. These experiments suggest a role for agrin in the induction of differentiation of nerve terminals in vitro. To ascertain the role of target in the regulation of presynaptic gene expression, syt mRNA expression was examined in the lumbar lateral motor columns of the chick embryo by in situ hybridization. Contact with target is associated with an increase in syt I gene expression which is prevented by unilateral removal of the target limb prior to the time of motoraxonal outgrowth. Further, there appear to be two stages of presynaptic gene regulation, an early increase in syt I expression, with a later switch to predominantly syt II expression. These data suggest that target induces the formation of the presynaptic terminal and that similar to postsynaptic differentiation, presynaptic differentiation includes both relocalization of protein and new gene expression. |
