Abstract
Purpose :
The mammalian retina does not regenerate, while retinas of cold-blooded animals generally have great regenerative capacity. The chick retina falls in between, generating Muller glia-derived progenitor cells (MGPCs) with limited neurogenic capacity. We investigated epigenetic mechanisms underlying MGPC production in the chick retina. We investigated the role of the Polycomb Repressor Complex 2 (PRC2), which catalyzes histone methylation, in damage-induced MGPC production.
Methods :
Retinal damage was induced in chicks by intraocular injection of NMDA. PRC2 component activity was inhibited by injection of pharmacological inhibitors into one eye; the other eye (injected with vehicle) served as a control. PRC2 components were inhibited using DZNexp4 (inhibitor of EZH2), Jib04 (inhibitor of JARID2), and EEDi (inhibitor of EED). Eyes were isolated, hemisected, fixed, and processed for immunohistochemistry or single cell RNA or ATAC seq. MGPCs were identified by double labelling for SOX2 and incorporated EdU and counted. Statistical comparisons were performed using Student’s t-test.
Results :
The PRC2 subunits EZH2, JARID2, and EED displayed increased levels of expression in damaged retinas. Inhibition of EZH2 resulted in decreased MGPC production [control: 121 ± 21.21 MGPCs per retinal section, treated: 12.25 ± 11.40 MGPCs per section, p < 3.3E-06 (n = 8)]. Similarly, inhibition of JARID2 and EED resulted in significant reductions in MGPCs [66.56 ± 29.64% reduction for JARID2, p < 1E-07 (n = 7); 51.98 ± 31% reduction for EED, p < 0.003 (n = 15)]. Analysis of DZNexp4-treated retinas by scRNA-seq revealed changes in differential expression of genes involved in glial and neuronal development and differentiation. Many of these differentially-expressed genes were found to exhibit differential chromatin accessibility by scATAC-seq.
Conclusions :
We conclude that the activity of PRC2, in general, and EZH2, in particular, are required for the reprogramming of Müller glia into MGPCs, and that the PRC2 complex and EZH2 normally influence chromatin access to a network of genes associated with glial differentiation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.