Abstract
Of the 15 nicotinic ACh receptor genes identified in vertebrates, only four (alpha 1, beta 1, gamma, and delta) have been shown to be expressed in embryonic skeletal muscle at early times. In mammalian muscle a fifth gene (epsilon) replaces the gamma gene in expression at later times. The remaining 10 nicotinic receptor genes identified to date (alpha 2-alpha 8, beta 2-beta 4) are expressed in the nervous system and are considered neuronal genes. Using RNase protection assays, we show here that four of the neuronal-type genes (alpha 4, alpha 5, alpha 7, and beta 4) are expressed in developing chick skeletal muscle. Two of them (alpha 4 and alpha 7) decline substantially in transcript abundance between embryonic days 11 and 17, as does alpha 1, while the other two (alpha 5 and beta 4) show only moderate decreases over the same time period. At embryonic day 8, alpha 7 transcripts are nearly 20% as abundant as alpha 1 transcripts. In situ hybridizations confirm the presence of alpha 7 transcripts in muscle cells both in cell culture and in embryonic tissue. No evidence was found for expression of the alpha 2, alpha 3, alpha 8, or beta 3 genes in muscle. Immunoprecipitations and immunoblot analysis using subunit-specific monoclonal antibodies reveal alpha 7 protein in muscle, and the amount of protein rises and declines with the amount of alpha 7 mRNA during development. Sucrose gradient analysis demonstrates that the alpha 7 protein is present in muscle as a species of 10S, the size expected for a nicotinic receptor. The alpha 7 species in muscle binds alpha-bungarotoxin but does not contain alpha 1 subunits, indicating that the two kinds of alpha-type gene products segregate during assembly. The results suggest that neuronal AChRs may play a role in early muscle development.