Expression of the Acetylcholinesterase Gene in Skeletal Muscle Fibers

Abstract

In contrast to extrasynaptic compartments of skeletal muscle fibers, mRNAs encoding acetylcholinesterase (AChE) are ~ 10-fold more abundant in the postsynaptic sarcoplasm (1,2) where their expression is markedly influenced by nerve-evoked electrical activity (2,3). In order to understand the molecular events involved in the regulation of AChE expression during synapse formation in embryonic and neo-natal muscles, we have cloned a 4.7 kb DNA fragment upstream of the translation start site in the rat AChE gene and generated multiple promoter-reporter gene constructs containing LacZ and a nuclear localization signal (nlsLacZ). These constructs were directly injected into tibialis anterior muscles of mice and 14 days later, muscles were excised and frozen. Muscles were subsequently cut in a cryostat and tissue sections were histochemically stained for the demonstration of β-galactosidase. The position of blue myonuclei indicative of promoter activity, was compared to that of neuromuscular junctions identified by AChE histochemistry. Injections of promoter-reporter gene constructs containing DNA fragments ranging from 4.7 to 1.5 kb led to a strong level of expression within muscle fibers. Surprisingly, quantitative analysis further revealed that expression of nlsLacZ was not confined to synaptic areas despite the presence of an N-box motif in the promoter region shown recently to play a crucial role in directing synapse-specific expression of other genes encoding synaptic proteins (4,5). Deletion of 600 bp in intron 1 from the 1.5 kb DNA fragment completely abolished muscle expression. In addition, transfection of motoneurons with these two latter constructs showed that both were equally effective in driving expression of LacZ thereby indicating the presence of cis-acting regulatory elements essential for muscle expression in the first intron of the AChE gene.