Cis-Regulatory Accessibility Directs Muller Glial Development and Regenerative Capacity

Abstract

19Diseases and damage to the retina lead to losses in retinal neurons and eventual visual 20 impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have 21 robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of 22 Ascl1 in adult mouse MG stimulates neurogenesis similar to fish regeneration. The regeneration 23 observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-24 expressing MG primarily generate bipolar cells. To better understand the limits of MG-based 25 regeneration in mouse retinas, we used ATAC-and RNA-seq to compare newborn progenitors 26 with MG. Our analysis demonstrated striking similarities between MG and progenitors, with 27 losses in regulatory motifs for neurogenesis genes. Young MG were found to have intermediate 28 expression profiles and accessible DNA, which is mirrored in the ability of Ascl1 to direct 29 bipolar neurogenesis in young MG. When comparing what makes bipolar and photoreceptor 30 cells distinct from glial cells, we find that bipolar-specific accessible regions are more frequently 31 linked to bHLH motifs and Ascl1 binding, indicating that Ascl1 preferentially binds to bipolar 32 regions. Overall, our analysis indicates a loss of neurogenic gene expression and motif 33 accessibility during glial maturation that may prevent efficient reprogramming. 34 35 36 Neuron loss caused by disease and damage to the mammalian retina can lead to 37 permanent vision loss. While some species are readily capable of regenerating lost neurons, 38 mammalian retinas are not regenerative. In the mammalian retina, neuron loss caused by direct 39 damage to the retina leads to reactive gliosis of the Müller glia (MG), similar to that of astrocytes 40 in the brain (Bringmann et al. 2009).41 Teleost fish, by contrast, are capable of regenerating retinal neurons, including 42 photoreceptors and ganglion cells, after damage. This regeneration is carried out by the MG, 43 which respond to damage by generating progenitor-like cells, similar to those in the developing 44 retina (Goldman 2014; Gemberling et al. 2013). This regeneration is accompanied by waves of 45 gene expression and morphological changes to the MG, regulated by epigenomic changes 46 directing regeneration (for review see VandenBosch and Reh 2019). The murine retina also 47 undergoes epigenomic changes after damage, but neurogenic programs are not re-expressed, and 48 neuronal regeneration does not occur (VandenBosch and Reh 2019). 49A critical difference between the fish MG and the mammalian MG in their response to 50 damage is in their expression of the proneural transcription factor Ascl1. In fish, Ascl1 is quickly 51 upregulated after damage, and is necessary for regeneration of new neurons (Ramachandran et 52 al. 2010; Fausett et al. 2008). In the murine retina, Ascl1 is expressed in retinal progenitors and 53 necessary for development of rods and bipolar cells (Ohsawa and Kageyama 2008); however it is 54 not expressed in...