Developmental changes in the accessible chromatin, transcriptome and Ascl1-binding correlate with the loss in Müller Glial regenerative potential

Abstract

Diseases and damage to the retina lead to losses in retinal neurons and eventual visual impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of Ascl1 in adult mouse MG stimulates neural regeneration. the regeneration observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-expressing MG primarily generate bipolar cells. To better understand the limits of MG-based regeneration in mouse retinas, we used ATAC-and RNA-seq to compare newborn progenitors, immature MG (P8-P12), and mature MG. Our analysis demonstrated developmental differences in gene expression and accessible chromatin between progenitors and MG, primarily in neurogenic genes. overexpression of Ascl1 is more effective in reprogramming immature MG, than mature MG, consistent with a more progenitor-like epigenetic landscape in the former. We also used ASCL1 ChIPseq to compare the differences in ASCL1 binding in progenitors and reprogrammed MG. We find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and ASCL1 binding. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming. The death of retinal neurons leads to permanent vision loss. While some species are readily capable of regenerating lost neurons, mammalian retinas are not. In mammals, neuron loss leads to reactive gliosis of the Müller glia (MG), similar to that of astrocytes in the brain 1. Teleost fish, by contrast, are capable of regenerating retinal neurons, including photoreceptors and ganglion cells, after damage. This regeneration is carried out by the MG, which respond to damage by generating progenitor-like cells, similar to those in the developing retina 2,3. Regeneration is accompanied by changes in gene expression and morphological changes to the MG, potentially regulated by epigenomic changes. The murine retina also undergoes epigenomic changes after damage, but neurogenic programs are not re-expressed, and neuronal regeneration does not occur 4. A critical difference between fish and mammalian MG in their response to damage is in their expression of the proneural transcription factor Ascl1. In fish, Ascl1 is quickly upregulated after damage, and is necessary for regeneration of new neurons 5,6. In the murine retina, Ascl1 is expressed in retinal progenitors and necessary for development of rods and bipolar cells 7 ; however it is not expressed in mature MG; moreover, after damage or in disease models, mouse MG do not spontaneously upregulate Ascl1 1,8. We recently directed Ascl1 expression to mouse MG with a inducible transgenic approach to test whether Ascl1 expression is sufficient to induce regeneration. Expression of Ascl1 in the MG of young mice (12 days post-natal (P12)) stimulated MG to generate new

miRNAs and Müller Glia Reprogramming During Retina Regeneration

“…After retinal damage in zebrafish, miR-216a is downregulated allowing increased expression of Dot1l which leads to activation of Wnt target genes, presumably by altering chromatin accessibility surrounding these genes (Kara et al, 2019 ). The idea that dedifferentiation of MG involves changes in chromatin accessibility is expected and has been experimentally supported (Jorstad et al, 2017 ; Mitra et al, 2018 ; VandenBosch et al, 2020 ). When combined with Ascl1 overexpression in an NMDA damage model in adult mice, the addition of the general histone deactylase inhibitor trichostatin A (TSA) stimulated neuronal regeneration which was otherwise only observed in developing mice <12 days old (Jorstad et al, 2017 ).…”

“…One goal for these studies is to determine the factors and pathways that allow for persistent and spontaneous regeneration. Focusing on the retina, knowledge gained from zebrafish studies (Wan and Goldman, 2016 ; Yao et al, 2018 ; Hoang et al, 2020 ; VandenBosch et al, 2020 ; Zhou et al, 2020 ) can be applied to identify common mechanisms that induce mammalian retina regeneration. Here, we will focus on the explicit role of miRNAs during MG reprogramming.…”

Inflammation Regulates the Multi-Step Process of Retinal Regeneration in Zebrafish

“…In mice the forced ectopic expression of the bHLH transcription factor, Ascl1, when combined with cell death is sufficient to stimulate a Müller glia to enter the cell cycle [ 103 ]. The efficiency of this response is enhanced when the expression of Ascl1 and retinal injury are combined with a treatment of the histone deacetylase inhibitor, trichostatin-A (TSA), indicating that chromosome structure in mammalian Müller glia may limit the regenerative capacity of these cells [ 104 , 105 ]. These results point toward common molecular mechanisms for reprogramming Müller glia in all vertebrates.…”

Meis homeobox genes control progenitor competence in the retina

“…One member of the group compensates for the loss of another and shares the same binding motif (46). bHLH transcription factors, such as Atoh7 and Ascl1, which are expressed in RPCs, bind to the same E-box motif as Atoh1 (the CAGCTG motif) (47,48), suggesting that these transcription factors may cooperate with Meis1/2. Only half of Meis1/2-bound regulatory regions in the retina contained the Meis motif, which indicated that Meis1/2 must bind DNA through noncanonical motifs in cooperation with other transcription factors.…”