Coexpression of multiple acetylcholine receptor genes in neurons: Quantification of transcripts during development

Abstract

A large family of genes encoding subunits of nicotinic ACh receptors (AChRs) has been identified in vertebrates and shown to be expressed in the nervous system. The multiplicity of genes raises questions about which gene products coassemble to produce native receptor subtypes and how the expression of receptor genes is regulated in neurons. We report here that five neuronal AChR genes are expressed in the chick ciliary ganglion at both early and late times in development. Quantitative RNase protection experiments demonstrated that at embryonic day 18 (E18) the ganglion contains about 1800 copies of α7 transcript per neuron, 900 copies of α3 transcript per neuron, and 200-300 copies each of α5, β2, and β4 transcripts per neuron. The same five genes are expressed at significantly lower levels at E8 but show the same rank order of abundance in transcripts per neuron. Few, if any, transcripts were found for the α2, α4, α8, and β3 AChR genes in ciliary ganglion RNA at either E8 or E18. The 6- and 13-fold increases previously reported for two classes of AChRs on the neurons between E8 and E18 approximate the 4-14-fold increases observed here in AChR gene mRNA levels per neuron over the same time period. The α3, α5, α7, and β4 genes have previously been correlated with subunits of ciliary ganglion AChRs, but the β2 gene has not. The abundance of β2 transcripts raises the possibility either that the known AChRs in the ganglion have a more complex subunit composition than previously described or that additional receptor subtypes remain to be discovered. Northern blot analysis revealed no changes in transcript pattern for the α3, α5, and β4 genes between E8 and E18; a small change may occur in the transcript pattern for the α7 gene. In situ hybridizations demonstrated that α5 and β4 transcripts are expressed in essentially all ciliary ganglion neurons as has been shown previously for the more abundant α3 transcript and inferred for the α7 transcript. The results indicate that neurons can stably coexpress multiple AChR genes, including three of the α type, and that transcript levels may be rate limiting for accumulation of AChRs during development.