Differential distribution of agrin isoforms in the developing and adult avian retina

At the developing and regenerating neuromuscular junction, agrin is responsible for the formation of aggregates containing the acetylcholine receptor (AChR) and other molecules. Multiple isoforms of agrin are generated by alternative splicing, and the presence of an 8, 11, or 19 (8 + 11) amino acid insert at splice site B is required for agrin's AChR aggregation activity. An antiserum was generated against the 19 amino acid peptide which reacted specifically with the B11 and B19 agrin isoforms. The antiserum blocked the formation of agrin-induced AChR aggregates on myotubes, but the peptide itself had no aggregation activity, suggesting that agrin's active site is close to the splice site, but not the peptide itself. In the embryonic and adult retina anti-peptide immunoreactivity was concentrated in the synapse-containing layers. In contrast, the inner limiting membrane and radial cells, which express strong immunoreactivity with a pan-specific anti-agrin antiserum, were not stained by the anti-peptide antiserum, showing that agrin isoforms are differentially distributed in the retina. In addition, agrin B11 and B19 isoforms were associated with ganglion cell axons, particular at early developmental stages before synapse formation, indicating additional functions for these isoforms in the developing CNS.