Epigenomic Regulation of Development and Regeneration in the Mammalian Retina

Thesis (Ph.D.)--University of Washington, 2019Diseases and damage to the retina lead to broad 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, the Reh lab has shown that driving expression of the proneural transcription factor, Ascl1 in adult mouse MG stimulates neurogenesis from these cells—in vitro and in vivo—similar to that which occurs in fish. The regeneration observed in the mouse is limited however, by the types of neurons that can be derived from the MG; Ascl1-expressing MG primarily generate bipolar cells. Additionally, Ascl1-based regeneration in the mouse retina is restricted temporally as the MG develop. To better understand the limits to mammalian MG reprogramming, I used RNA-seq and ATAC-seq to compare FACS purified cells. I firstly compared reprogramming treatment combinations in adult regeneration, as well as comparing highly neurogenic newborn mouse progenitors to developing MG. Additionally, I explored the epigenomic roles on fate decisions by comparing glial type cells to retinal neurons, and by modulating the epigenome with small molecule inhibitors ex vivo. I found that while there were many similarities between MG and progenitors, E-box regulatory regions lose accessibility as NFI binding domains increase in accessibility. Analysis of young glia reveals an intermediate epigenomic and transcriptomic profile that directs intermediate reprogrammability of glia as the young retina develops. Similarly, increasing reprogramming factors increased neurogenesis-related accessibility that relates directly to adult MG regeneration outcomes. Broader modifications to the epigenome can thus redirect fate decisions as MG redifferentiate ex vivo. Fate decisions do not appear to be directed, however, by inherent epigenomic similarity to retinal neurons, but instead by pioneering factor preference for binding sites. Overall, my analysis demonstrates the roles in which epigenomic accessibility reveals glial potential for neurogenesis during Ascl1-induced regenerative responses in mammalian retinas

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

Stimulation of functional neuronal regeneration from Müller glia in adult mice

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, 2, 3, 4, 5, 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