Characterization of genes regulated by CNTF during retinal development

Abstract

Abstract: : Purpose: During development, progenitor cells give birth to the different subtypes of neurons and glial cells of the neural retina. Cell division and differentiation of these progenitors are under the control of endogenous factors and exogenous factors such as ciliary neurotrophic factor (CNTF), an inhibitor of the photoreceptor differentiation in rat. Therefore the identification of CNTF target genes is a key step to understand the molecular events underlying photoreceptor differentiation and will be a challenging issue for cell therapy. Methods: In order to characterized such candidates, retinal explants of Post Natal day 0 (PN0) rats were cultured in vitro and remained untreated or were treated with CNTF. After 6 days, mRNA were extracted from "CNTF–treated" and "control untreated" retinal explants and subjected to the Suppressive Subtractive Hybridization (SSH) technique. The subtraction of "control untreated" mRNA from "CNTF–treated" mRNA by SSH should enriched the final cDNA pool with genes involved in the photoreceptor lineage. Results: A "Control untreated – CNTF treated" library was constructed from these subtracted cDNA and 192 clones were selected randomly and screened for differential expression by dot blot analysis using subtracted and control probes, and by reverse Northern blot analysis. After these successive selection steps, 15 candidates were sequenced and compared to public (the human genome, cDNA, and EST) databases. Out of these 15 clones, 11 correspond to orthologue of mouse genes which functions are known, 4 clones present no identity with known proteins and/or sequences. Primers were designed to specifically amplified each of these 15 clones in quantitative real–time PCR experiments. These clones present a CNTF–regulated expression in retinal explants after different times of culture. Conclusions: This library allowed the characterization of several genes known to be involved in neural differentiation as well as new genes expressed during retinal differentiation. In vivo and in vitro overexpression experiments will address the function of these genes.

Keywords: retinal development • photoreceptors • gene/expression