Identification of Pleiotrophin as a CNTF–Regulated Gene Involved in Rod Differentiation

Abstract

Abstract: : Purpose: In order to identify genes regulated by Ciliary Neurotrophic Factor (CNTF) in the developing retina, we carried out a subtractive screen for differentially CNTF–regulated genes in retinal explants from postnatal day 0 (P0) rats. Our approach has revealed a candidate in the form of Pleiotrophin (Ptn), an heparin–binding growth factor involved in neural development. The complete sequencing, the spatio–temporal expression of Ptn and its function during retinal development in relationship with CNTF effects has been addressed. Methods: Quantitative real–time PCR experiments and western blot analyses were used to determine the expression of Ptn during retinal development and to evaluate its regulation by CNTF in retinal explants of P0 rats after different times of culture. In vitro electroporation has been used for introducing Ptn cDNA into retinal explants to examine the role of Ptn during post natal retinal development. Retinal explants were maintained for 3 to 7 days in vitro before dissociation and counting rhodopsin–positive cells by immunocytochemistry. Results: Out of the 10 clones obtained from the "CNTF treated – Control untreated", one clone corresponded to the gene of rat pleiotrophin. Quantitative real–time PCR experiments with specific primers demonstrate the differential expression of Ptn between retinal explants untreated or treated with CNTF during 1, 3 and 6 days. Analysis at different developmental stages revealed that Ptn increased at P0 to reach a maximum at P10 and decreased thereafter. Western blot analyses confirmed the upregulation of Ptn in CNTF–treated retinal explants. Ectopic expression of Ptn in retinal explants from P0 rats induced a large reduction of rhodopsin–positive cells (rods) compared to the control explants. Conclusions: Our results demonstrate that Ptn mRNA and protein are upregulated by CNTF in the retina during the postnatal development, and that surexpression of Ptn prevented the photoreceptor differentiation, suggesting that Ptn may partially mediate the effect of CNTF on retinal development.

Keywords: photoreceptors • retinal development • growth factors/growth factor receptors