We do basic research on the cellular and molecular mechanisms involved in development of the mammalian neuromuscular junction, utilising a novel nerve-muscle culture system. Postsynaptic acetylcholine receptor aggregation is a critical early event in junction formation. We previously showed that the capacity to induce receptor aggregation resides primarily in the axons of ventral spinal cord neurons. This suggests that one or more of the signals for the induction of postsynaptic differentiation has a polarized distribution in these neurons. Agrin is a proteoglycan that is required for normal postsynaptic differentiation in muscle. We have used immunofluorescence to determine the distribution of secreted neuronal agrin in cultures with myotubes and in isolated motoneurons. We have found that secreted neuronal agrin is consistently concentrated at developing synaptic sites but can be less frequently found on other segments of axons as well as on dendrites or cell bodies. Further, we have found that isolated motoneurons initially secrete agrin indiscriminately but progressively target agrin to axons as they mature. We are exploring the possibility that certain physical interactions with muscle are specific to axons as opposed to dendrites, and that they may be important for the initiation of postsynaptic differentiation. We previously used scanning and transmission electron microscopy to demonstrate the formation of ruffles and microvilli on the myotube surface adjacent to sites of contact with axons that induce acetylcholine receptor aggregation, indicating a motile response to axonal contact. This response appears to occur very early after contact and, together with adhesion, may facilitate cell-surface interactions between the axon and myotube. We are now exploring the possible relationship between this response and the signal-transduction processes involved in acetylcholine receptor accumulation. Specifically, we have shown that both soluble recombinant agrin and ARIA (a growth-factor-like protein that regulates acetylcholine receptor expression by muscle) can induce a myotube surface response similar to axons. We now plan to test directly the role of these proteins in the induction of this response.
