Transient DNA methyltransferase 3a (DNMT3a) Inhibition Unlocks dedifferentiation and Neurogenic Potential in Mouse Retinal Müller Glia

Abstract

The regenerative response of retinal cells to injury and aging depends on the epigenomic plasticity that enables the dedifferentiation and neuronal differentiation capacities of Muller glial cells (MG). In mammals, this regenerative ability is extremely limited, and disruptions in epigenetic mechanisms, particularly those involving DNA methylation and demethylation, may underlie this restricted potential. To explore this possibility, we aimed to develop DNA methylation-targeting molecular tools to enhance the dedifferentiation and neurogenic capacity of primary MG cultures derived from mouse retina. Using CRISPR/dCas9-based gene regulation technology, we selectively and transiently inhibited Dnmt3a, a de novo DNA methyltransferase previously implicated in maintaining transcriptional repression. Our results show that Dnmt3a knockdown leads to sustained upregulation of pluripotency-associated genes, including Ascl1, Lin28, and Nestin, as measured by RT-qPCR and immunofluorescence. This epigenetic modulation also promoted increased cell proliferation and migration, both hallmarks of a regenerative response. Furthermore, Dnmt3a knockdown, either alone or in combination with neurogenic stimuli, induced MG to acquire neuronal-like morphologies and express the early neuronal marker {beta}III-tubulin. These findings suggest that Dnmt3a acts as a repressive regulator of MG plasticity, likely serving as an epigenetic barrier that counteracts injury-induced demethylation events. Overall, our study identifies Dnmt3a as a critical modulator of MG fate and highlights the potential of its targeted downregulation to facilitate reprogramming. By prolonging the transient progenitor-like state of MG, DNMT3a inhibition may serve as a complementary approach to unlock the neurogenic and regenerative potential of the mammalian retina, offering promising avenues for future therapeutic strategies. Author SummaryRetinal damage caused by injury, disease, or aging can lead to vision loss and ultimately blindness. Unlike mammals, the small freshwater zebrafish possesses a remarkable ability to regenerate its retina and restore vision after injury. Extensive research has focused on uncovering the molecular mechanisms behind this regenerative process in zebrafish, with the goal of understanding what is absent or suppressed in the mammalian retina. It is now well established that this regenerative capacity depends on a specific type of retinal cell: Muller glia. These cells can undergo dedifferentiation, a process in which they lose their specialized function, morphology, and gene expression profile. This is followed by neuronal differentiation, allowing them to replace lost neurons with newly generated ones. In recent years, numerous molecules and molecular pathways have been identified that may limit regenerative potential in mammals. In this study, we developed a molecular tool to specifically block one of these inhibitory factors, DNMT3a, a DNA methyltransferase involved in epigenetic repression. We demonstrate that in the absence of DNMT3a, mouse Muller glia can more efficiently dedifferentiate and subsequently adopt neuronal-like characteristics. These findings suggest that DNMT3a acts as a barrier to retinal regeneration and may represent a promising target for future therapeutic strategies aimed at promoting neural repair in the mammalian retina.