Activity of the beta 3 nicotinic receptor promoter is a marker of neuron fate determination during retina development

Abstract

We have previously shown that transcription of the beta 3 nicotinic receptor gene within the chick CNS is regulated by a promoter 143 base pairs (bp) in length. Here, we demonstrate that in the developing visual system this promoter is active in a subset of retinal cells, the majority of which are ganglion cells. Because the beta 3 promoter is activated very early during retina development, it can provide a marker of ganglion cell induction and differentiation. Transfection of neuroretina explants enabled us to detect activity of the beta 3 promoter in premigratory cells localized on the ventricular side of the retina. Double-labeling experiments showed that activation of the beta 3 promoter takes place before the last S-phase, suggesting that this particular phenotypic trait is determined when precursor cells are still proliferating. The beta 3 phenotype is induced in about one-tenth of the total pool of retinal progenitor cells and is stable upon changing the cellular environment. Our study suggests that at the very early stages of retina neurogenesis, some lineage restrictions have already occurred in the population of retinal progenitor cells.

Functional Properties of the Neuronal Nicotinic Acetylcholine Receptor β3 Promoter in the Developing Central Nervous System

“…The widespread expression of NeuroM and NeuroD in postmitotic cells at distinct times in neural development suggests that they do not define functionally distinct neuronal phenotypes but rather successive stages of a cell's life course (21,33). Transcription of the ␤3 gene is activated in a small subset of proliferating retinal cells, and then it is continuously expressed during cell differentiation and in the mature ganglion and amacrine cells (11). Although ASH-1, NeuroM, and NeuroD are expressed in subsets of retinal cells and instances of overlapping expression between these factors and ␤3 have been detected, we found that the ␤3 promoter was also active in cells that do not express these bHLH genes.…”

“…Chick embryos were staged according to Hamburger and Hamilton (25). Cells from different regions of the CNS were prepared as described previously (11,24). Neuroretina, pigment epithelium, optic tectum, telencephalon, and cerebellum were dissected from E4 -E13 embryos, collected in Ca 2ϩ -and Mg 2ϩ -free Hanks' balanced salt solution and incubated with 0.05% trypsin for 10 min (E4 -E8) or with 0.1% trypsin for 20 min (E9 -E13).…”

“…We have previously shown that essential neuron-specific promoter elements are located in a short EcoRISphI DNA fragment, 143 bp in length and located just upstream of the transcription start site (11,15). This ␤3RS fragment contains several putative binding sites for transcription factors: CACCC and CAGCTG (E-box) motifs, a CAAT box, and two TATA-like motifs located at Ϫ56 bp and Ϫ30 bp relative to the transcription initiation site (Fig.…”

“…Transcription of ␤3 in neuroretina is first detected on E4, whereupon activity of the promoter rapidly increases and peaks on E5, decreasing later to relatively low levels (11). We wanted to determine whether ectopic MyoD could activate the ␤3 promoter earlier in development, increase the peak value at E5, or maintain a high level of activity at later stages.…”

“…Forsayeth and Kobrin (16) have shown that the ␤3 subunit co-assembles in vivo with the ␣4, ␤2, and ␤4 subunits to form a functional nicotinic receptor endowed with distinctive properties. We have isolated a short 5Ј-sequence of the ␤3 gene containing promoter elements that are sufficient to target reporter gene expression to those retinal neurons that normally express ␤3 in vivo (11,15). The stringent neuronal specificity of the ␤3 promoter and its activation during the period of neuronal fate determination make it an attractive system in which to study the functional interactions between transcription factors and cis-acting regulatory elements that help establish the diverse neuronal phenotypes.…”