Evaluation Of P53-otx1 Gene Expression Pathway In Progenitor Cell Differentiation In The Retina

Purpose:Wild type p53 has been recently demonstrated to play developmental and differentiation functions. Recently we have established that the p53 protein directly induces OTX1 expression by acting on its promoter. Otx1 with Otx2 are critical molecules for eye morphogenesis and may have a central role in progenitor cell proliferation and in photoreceptor differentiation. Furthermore, recent studies have suggested that Müller cells display characteristics of progenitor and stem cells, producing new neurons following specific types of retinal injury. The aim of our study is to evaluate p53, p63, 73, Otx1 and Otx2 gene expression and their interaction and regulation during Muller cells differentiation. Methods:Mice eyes were enucleated, cut-open at the ora serrata and retina was placed in PBS + PEN/STREP + 0,025% Trypsin for 10 minutes at 4C and then for 10 minutes at 37C. Retina was transferred to PBS + FBS 2% + PEN/STREP and mechanically dissociated with a Pasteur pipette. Dissociated cell were placed in a Petri dish and let settle in incubator for 2h, then the dish was rinsed with complete medium. Adherents cell were then cultured in complete medium. Real-time PCR was performed following manifacturer's instructions. Results:We demonstrated that p53 directly control Otx1 gene expression during cell differentiation. The immunostain positivity of cell component in the retina demonstrated the possible biological function of Otx1/Otx2 in adult retina. In detail Otx1/Otx2 antibody stained the nuclei of both cones, rods and bipolar cells. We show faint positivity also in the cytoplasm of photoreceptors. Conclusions:Primary Müller Cell and Müller Cell line will be used to evaluate the role of the pathway of Otx1, Otx2, p53, p63 and p73 in Muller cells differentiation. The understanding of the p53-Otx1 molecular interaction in Muller cells could be critical to intervene in their differentiation