Chick muscle expresses various ARIA isoforms: Regulation during development, denervation, and regeneration

Acetylcholine receptor inducing activity (ARIA) is a glycoprotein released from the motor neuron to stimulate the synthesis of acetylcholine receptors (AChRs) on the postsynaptic muscle fiber. Transcripts encoding ARIA were detected not only in brain but also in muscle, and immunohistochemical staining showed that muscle-derived ARIA was restricted to the neuromuscular junctions. RT-PCR analysis revealed three biological active isoforms of ARIA in chick muscle, namely ARIA(beta 1), ARIA(alpha 1), and ARIA(beta 2), that were classified based on their variation in the carboxyl-terminus of the EGF-like domain. The expression of these ARIA isoforms in muscle changed during development, denervation, and nerve regeneration. ARIA(beta 1), ARIA(alpha 2), and ARIA(beta 2) were expressed in embryonic and young chick muscles, while ARIA(beta 1) was the major isoform expressed in adult chicken. The embryonic-like expression of ARIA(alpha 2) and ARIA(beta 2) was induced after nerve injury in adult chicken. However, the prominent expression of ARIA(beta 1) in adult-like profile was restored after nerve regeneration. A splicing variation in the region between Ig-like and EGF-like domains of ARIA was also revealed; a zero-amino acid insertion (ARIA(SP0)), a 17-amino acid insertion (ARIA(SP17)), or a 34-amino acid insertion (ARIA(SP34)) were identified. Unlike ARIA(SP0), the expression of ARIA(SP17) and ARIA(SP34) was found in muscle and sciatic nerve only. The expression of ARIA(SP0), ARIA(SP17), and ARIA(SP34) in chick muscle remained unchanged during development and after nerve injury. Moreover, the specific expression of these ARIA isoforms in cultured myotubes was not affected by drug treatments or by coculturing with neurons. Our findings provide strong evidence that muscle ARIA may play an important role in the formation of neuromuscular junctions.