Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons1–6. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage1,7 in zebrafish and is necessary for regeneration8. Although Ascl1 is not expressed in mammalian MG after injury9, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro10 and in vivo after NMDA (N-methyl-D-aspartate) damage in young mice11. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression11. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC–seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.
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
Damage to neuronal tissues in mammals leads to permanent loss of tissue function that can have major health consequences. While mammals have no inherent regenerative capacity to functionally repair neuronal tissue, other species such as amphibians and teleost fish readily replace damaged tissue. The exploration of development and native regeneration can thus inform the process of inducing regeneration in non-regenerative systems, which can be used to develop new therapeutics. Increasing evidence points to an epigenetic component in the regulation of the changes in cellular gene expression necessary for regeneration. In this review, we compare evidence of epigenetic roles in development and regeneration of neuronal tissue. We have focused on three key systems of important clinical significance: the neural retina, the inner ear, and the spinal cord in regenerative and non-regenerative species. While evidence for epigenetic regulation of regeneration is still limited, changes in DNA accessibility, histone acetylation and DNA methylation have all emerged as key elements in this process. To date, most studies have used broadly acting experimental manipulations to establish a role for epigenetics in regeneration, but the advent of more targeted approaches to modify the epigenome will be critical to dissecting the relative contributions of these regulatory factors in this process and the development of methods to stimulate the regeneration in those organisms like ourselves where only limited regeneration occurs in these neural systems.
Hearing loss is often due to the absence or the degeneration of hair cells in the cochlea. Understanding the mechanisms regulating the generation of hair cells may therefore lead to better treatments for hearing disorders. To elucidate the transcriptional control mechanisms specifying the progenitor cells (i.e. prosensory cells) that generate the hair cells and support cells critical for hearing function, we compared chromatin accessibility using ATAC-seq in sorted prosensory cells (Sox2-EGFP + ) and surrounding cells (Sox2-EGFP − ) from E12, E14.5 and E16 cochlear ducts. In Sox2-EGFP + , we find greater accessibility in and near genes restricted in expression to the prosensory region of the cochlear duct including Sox2 , Isl1 , Eya1 and Pou4f3 . Furthermore, we find significant enrichment for the consensus binding sites of Sox2, Six1 and Gata3—transcription factors required for prosensory development—in the open chromatin regions. Over 2,200 regions displayed differential accessibility with developmental time in Sox2-EGFP + cells, with most changes in the E12-14.5 window. Open chromatin regions detected in Sox2-EGFP + cells map to over 48,000 orthologous regions in the human genome that include regions in genes linked to deafness. Our results reveal a dynamic landscape of open chromatin in prosensory cells with potential implications for cochlear development and disease.
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