Corriveau R A, Berg D K
Department of Biology, University of California, San Diego, La Jolla 92093-0322.
J Neurosci. 1993 Jun;13(6):2662-71. doi: 10.1523/JNEUROSCI.13-06-02662.1993.
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 alpha 7 transcript per neuron, 900 copies of alpha 3 transcript per neuron, and 200-300 copies each of alpha 5, beta 2, and beta 4 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 alpha 2, alpha 4, alpha 8, and beta 3 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 alpha 3, alpha 5, alpha 7, and beta 4 genes have previously been correlated with subunits of ciliary ganglion AChRs, but the beta 2 gene has not. The abundance of beta 2 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 alpha 3, alpha 5, and beta 4 genes between E8 and E18; a small change may occur in the transcript pattern for the alpha 7 gene. In situ hybridizations demonstrated that alpha 5 and beta 4 transcripts are expressed in essentially all ciliary ganglion neurons as has been shown previously for the more abundant alpha 3 transcript and inferred for the alpha 7 transcript. The results indicate that neurons can stably coexpress multiple AChR genes, including three of the alpha type, and that transcript levels may be rate limiting for accumulation of AChRs during development.
在脊椎动物中已鉴定出一大类编码烟碱型乙酰胆碱受体（AChRs）亚基的基因，并表明其在神经系统中表达。基因的多样性引发了一些问题，即哪些基因产物共同组装以产生天然受体亚型，以及受体基因在神经元中的表达是如何调控的。我们在此报告，五个神经元AChR基因在鸡睫状神经节发育的早期和晚期均有表达。定量核糖核酸酶保护实验表明，在胚胎第18天（E18），神经节中每个神经元含有约1800个α7转录本拷贝、900个α3转录本拷贝，以及每个神经元200 - 300个α5、β2和β4转录本拷贝。相同的五个基因在E8时表达水平显著较低，但在每个神经元的转录本丰度上显示出相同的排序。在E8或E18时，在睫状神经节RNA中几乎未发现α2、α4、α8和β3 AChR基因的转录本（如果有的话）。先前报道的E8和E18之间两类AChRs在神经元上6倍和13倍的增加，与在此观察到的同一时期每个神经元AChR基因mRNA水平4 - 14倍的增加相近。α3、α5、α7和β4基因先前已与睫状神经节AChRs的亚基相关联，但β2基因尚未关联。β2转录本的丰度增加了这样一种可能性，即神经节中已知的AChRs具有比先前描述的更复杂的亚基组成，或者仍有待发现其他受体亚型。Northern印迹分析显示，E8和E18之间α3、α5和β4基因的转录本模式没有变化；α7基因的转录本模式可能会有小的变化。原位杂交表明，α5和β4转录本在基本上所有睫状神经节神经元中表达，正如先前对更丰富的α3转录本所显示的以及对α7转录本所推断的那样。结果表明，神经元可以稳定地共表达多个AChR基因，包括三个α型基因，并且转录水平可能是发育过程中AChRs积累的限速因素。
