Role of Epigenetics (DNA Methylation) in RPE and Photoreceptor Development

Abstract

Purpose: : Differential DNA methylation takes place during organogenesis in cells acquiring different cell fates, and is conducted in all mammalian species (including humans and mice) by three DNA methyltransferases. Dnmt3a and Dnmt3b are responsible for de novo methylation of DNA, while Dnmt1 maintains the newly established methylation patterns in differentiating cells. DNA methylation mostly leads to stable silencing of gene expression. We hypothesize that differentiation and/or maturation of neural retina (nr) and retinal pigment epithelium (RPE) are influenced by DNA methylation.

Methods: : The Dnmt1-floxed mice were mated to Rx-Cre mice to generate conditional loss of Dnmt1 expression in developing nr and RPE. Histological changes were evaluated by light and electron microscopy. Immunolocalization studies were performed on retinal sections using cell-specific antibodies to photoreceptors, RPE and Muller cells, and second order neurons.

Results: : Severe morphological changes were observed in the retina and RPE of Dnmt1 knockout animals. These pathologic changes included lack of photoreceptor outer segment elongation, retinal folding and pseudorosette formation, and abnormalities in RPE development and maturation. Inner segments and rhodopsin and recoverin were present in Dnmt1 2lox, Rx-Cre mutant retinas. Retina-RPE adhesion in mutants was affected during early postnatal development. Multiple localized RPE defects such as lack of organized regular hexagonal cell structure were observed in flatmount RPE preparations of newborn mutant pups (P0.5) and even in e16.5 embryos stained with phalloidin. PNA and cone arrestin staining of P10.5 and P15.5 mutant retinas demonstrated significant underrepresentation of cones.

Conclusions: : Our studies provide the first evidence of Dnmt-mediated epigenetic mechanisms guiding the development of RPE and photoreceptors.

Keywords: retinal development • retinal pigment epithelium • photoreceptors