Agrin-related molecules are concentrated at acetylcholine receptor clusters in normal and aneural developing muscle

Agrin induces the clustering of acetylcholine receptors (AchRs) and other postsynaptic components on the surface of cultured muscle cells. Molecules closely related if not identical to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a key part in orchestrating synapse regeneration. Agrin or agrin-related molecules are thus likely to play a role in directing the differentiation of the postsynaptic apparatus at the regenerating neuromuscular junction. The present studies are aimed at understanding the role of agrin at developing synapses. We have used anti-agrin monoclonal antibodies combined with alpha-bungarotoxin labeling to establish the localization and time of appearance of agrin-related molecules in muscles of the chick hindlimb. Agrinlike immunoreactivity was observed in premuscle masses from as early as stage 23. AchR clusters were first detected late in stage 25, coincident with the entry of axons into the limb. At this and all subsequent stages examined, greater than 95% of the AchR clusters colocalized with agrin-related molecules. This colocalization was also observed in unpermeabilized whole mount preparations, indicating that the agrin-related molecules were disposed on the external surface of the cells. Agrin-related molecules were also detected in regions of low AchR density on the muscle cell surface. To examine the role of innervation in the expression of agrin-related molecules, aneural limbs were generated by two methods. Examination of these limbs revealed that agrin-related molecules were expressed in the aneural muscle and they colocalized with AchR clusters. Thus, in developing muscle, agrin or a closely related molecule (a) is expressed before AchR clusters are detected; (b) is colocalized with the earliest AchR clusters formed; and (c) can be expressed in muscle and at sites of high AchR density independently of innervation. These results indicate that agrin or a related molecule is likely to play a role in synapse development and suggest that the muscle cell may be at least one source of this molecule.