Altered Dnmt1-mediated DNA Methylation Disrupts Retinal Pigment Epithelium (RPE) And Photoreceptor Differentiation

Abstract

Purpose: : DNA methylation is an essential epigenetic modification of DNA that governs chromatin remodeling and patterns of gene expression. DNA methylation is generally associated with stable silencing of gene expression and chromatin compaction. The goal of this project is to determine the role of Dnmt1 in influencing the development of neural retina (nr) and RPE.

Methods: : Dnmt12lox, Rx-Cre mice were generated to produce conditional ablation of Dnmt1 exons 4 and 5 in developing nr and RPE. We performed histological evaluation of mutant retinas by light and electron microscopy, and immunolocalization studies on retinal sections using cell-specific antibodies to photoreceptors (PRs), RPE, Muller cells, and second order neurons. Quantitation of DNA methylation was performed by bisulfite conversion followed by pyrosequencing analysis of genomic DNA of L1 elements, amplified from mouse tail and retina.

Results: : Excision of Dnmt1 exons by Cre resulted in additional exon 6 skipping (Δ4-6) and a predicted mutant DNMT1 protein lacking the PCNA binding domain. We obtained variable DNA hypomethylation and severe morphological changes in the retina and RPE of Dnmt1 mutant mice [lack of PR outer segment elongation, retinal folding and pseudorosette formation, and abnormalities in RPE development and maturation]. Notably, the general organization of the retina was unaffected and all retinal cell types (except S-cones) were produced.

Conclusions: : Our data suggest that DNA hypomethylation and altered DNMT1 function during early differentiation can lead to dramatic alterations in the morphogenesis of PRs and RPE.

Keywords: development • retina • differentiation