Neuronal specificity of the alpha 7 nicotinic acetylcholine receptor promoter develops during morphogenesis of the central nervous system.

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.

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

“…100 -150 g of cytosolic proteins were used in CAT assays such that the proportion of acetylated [ 14 C]chloramphenicol in cells transfected with pSV-CAT did not exceed 70%. The activity of the SV40 promoter was similar in different neuronal and non-neuronal cell-types at different embryonic stages (24). The means and S.D.…”

“…Isolation of Nuclear Proteins and Gel Mobility Shift Analysis-Retinae and optic tecta were dissected in cold phosphate-buffered saline, and nuclei were isolated as described by Matter-Sadzinski et al (24). Nuclear proteins were obtained by adding NaCl to the suspension of nuclei to a concentration of 1 M, in the presence of the protease inhibitors leupeptin, phenylmethylsulfonyl fluoride (both at 0.7 g/ml), and Trasylol (1%), incubating for 15 min on ice, and centrifuging for 45 min at 50,000 rpm (Beckman TLA 100).…”

“…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).…”

“…In all instances, the ratio of DNA to Lipofectin was 4/1. Glial cells and primary or secondary cultures of CEFs were prepared and transfected as described by Matter-Sadzinski et al (24). 24 or 48 h after transfection, cells were collected and processed for CAT assay.…”

“…bHLH Proteins Regulate the ␤3 nAChR Genefreshly dissociated from E5 chick neuroretina (24), and 48 h later the transfected cells were processed for CAT activity. Mutations in the E-box and in the CAAT box produced spectacular effects, completely abolishing promoter activity in retinal cells.…”