No abstract

A transient transfection assay has been developed to analyse promoter activity in neuronal cells freshly dissociated from the chick central nervous system. The assay enabled us to identify cis-acting regulatory elements within the 5'-flanking region of the a7 nicotinic acetylcholine receptor gene. In differentiated retina, regulatory elements direct reporter gene expression to a small subset of neurons which has been identified as ganglion cells, i.e. to the population of neurons in which C7 transcripts were localized by in situ hybridization. However, these promoter elements exhibit ubiquitous activity in undifferentiated neural cells and in mesodermal stem cells. Our study supports the idea that c7 regulatory elements acquire their neuronal specificity in the course of embryogenesis.

Genomic and cDNA clones encoding the chicken neuronal nicotinic acetylcholine receptor beta 3 subunit were isolated and sequenced. The beta 3 gene consists of six protein-encoding exons and the deduced protein has the structural features found in all other members of the neuronal nicotinic acetylcholine receptor subunit family. Although they are undetectable in most brain compartments, beta 3 mRNAs are relatively abundant in the developing retina and in the trigeminal ganglion. In situ hybridization and immunohistochemical analysis demonstrated that in retina, beta 3 transcripts and protein are confined to subpopulations of cells in the inner nuclear and ganglion cell layers. Beta 3 is expressed in the proximal and distal regions of the developing trigeminal ganglion, i.e. in both placode- and neural crest-derived neurons. Transient transfection assays in cells freshly dissociated from selected regions of the central nervous system at different developmental stages allowed the identification of genetic elements involved in the neuronal-selective expression of the beta 3 gene. A promoter fragment 143 base pairs in length and containing TATA, CAAT, and other consensus sequences is sufficient to restrict reporter gene expression to a subpopulation of retinal neurons. This promoter is totally inactive upon transfection into neuronal and non-neuronal cells from other regions of the central nervous system.

Within the chick central nervous system, expression of the ␤3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the ␤3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of ␤3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the ␤3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of ␤3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of ␤3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3 of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.In vertebrates, both negative and positive regulations play an important role in determining neuronal gene expression. Negative regulation (reviewed in Refs. 1 and 2) is best exemplified by REST/NRSF, a factor that represses transcription of the SCG-10 and type II Na ϩ channel genes in non-neuronal cell-types, whereas most neurons lack REST/NRSF and thus express these two genes (3, 4). Conversely, the Olf-1 transcription factor positively regulates several genes (e.g. the olfactory neuron-specific G protein, the type III adenylyl cyclase) specifically expressed in olfactory neurons (5). Vertebrate homologs of the basic helix-loop-helix (bHLH) 1 factors involved in Drosophila neurogenesis (6) act as positive or negative regulators in the acquisition of neuronal identity. For instance, the atonaland achaete-scute-related activators are transiently expressed in parts of the central and peripheral nervous system during early development, and their null mutation or ectopic expression profoundly influences neurogenesis (reviewed by Lee et al. (7)). However, the direct regulation by bHLH proteins of genes that define neuronal identity has never been documented.Several neuronal nicotinic acetylcholine receptor (nAChR) genes are expressed early in neural development (8 -12), and, since they encode transmembrane sensors capable of fluxing Ca 2ϩ and other cations upon stimulation (reviewed in Ref. 13), an understanding of their regulation should help explain how the genetic program puts together the mechanisms needed for epigenetic environmental cues to participate in development. This is illustrated in a recent report by Shatz and associates (14) showing that, in the developing retina, cholinergic synaptic transmission between newly generated amacrine and ganglion cells is respo...

Genes encoding transcription factors of the helix-loop-helix family are essential for the development of the nervous system in Drosophila and vertebrates. Screens of an embryonic chick neural cDNA library have yielded NeuroM, a novel neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. In situ hybridization studies have been conducted, in conjunction with pulse-labeling of S-phase nuclei, to compare NeuroM to NeuroD expression in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. We propose that availability of the NeuroM helix-loop-helix factor defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors.