Research Article

Gene regulatory networks controlling vertebrate retinal regeneration

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Science  20 Nov 2020:
Vol. 370, Issue 6519, eabb8598
DOI: 10.1126/science.abb8598

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Unlocking retinal regeneration in mice

Zebrafish can regenerate damaged retinal tissue, but mice cannot. Hoang et al. found that tracking changes in gene expression and chromatin accessibility upon injury revealed clues as to why retinal glial cells in zebrafish could generate new neurons but the same cell type in mice could not. In zebrafish, activated Müller glial cells shift into a proliferative phase, whereas in mice, a genetic network returns the glial cells to quiescence. A few transcription factors enforce quiescence in the mouse, and disruption of these allowed Müller glia to proliferate and generate new neurons after retinal injury.

Science, this issue p. eabb8598

Structured Abstract

INTRODUCTION

The ability to regenerate reti­nal neurons after injury varies drastically among vertebrate species. Teleost fish such as zebrafish can regenerate all major retinal cell types after injury by repro­gramming Müller glia to a progenitor-like state. In the post-hatch chick, Müller glia can generate small numbers of neurons after injury but lose regenerative ability later in life. In contrast, mammalian Müller glia do not spontaneously regenerate lost retinal neurons. Although some genes that promote retinal re­generation have been identified, the core gene regulatory networks controlling Müller glia reprogramming remain largely unknown but can be identified through cross-species tran­scriptomic and epigenomic analysis.

RATIONALE

To identify injury-induced changes in Müller glia, we performed bulk RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to separately profile gene expression and chromatin accessibility in both mouse and zebrafish. These assays included multiple time points following N-methyl-d-aspartate (NMDA) and light treatments, which damage inner retinal neurons and photoreceptors, respectively. We also conducted single-cell RNA-seq (scRNA-seq) to identify changes in gene expression after NMDA treatment and light damage, as well as after treat­ment with exogenous factors that induced in­jury-independent reprogramming, in mouse, zebrafish, and chick retinas. We then devel­oped a computational tool, which we call integrated regu­latory network analysis (IReNA), to integrate gene expression and chromatin accessibility in order to reconstruct regulatory networks of Müller glia in response to diverse stimuli. Finally, using loss-of-function approaches, we validated functions of candidate factors con­trolling Müller glia reprogramming.

RESULTS

We generated 100 RNA-seq and 40 ATAC-seq samples from zebrafish and mice, and obtained 105,666, 85,051, and 77,924 single retinal cells from zebrafish, chick, and mice, respectively. In all three species, Müller glia acquired a reactive state after treatments. In chick and zebrafish, Müller glia passed through this reactive state before becoming proliferative and neurogenic. In mice, how­ever, Müller glia reverted to a resting state after injury. By integrating these datasets, we identified changes in gene expression and chromatin accessibility after each treat­ment. Cross-species analysis identified evolu­tionarily conserved and species-specific gene regulatory networks that control the transi­tion of the quiescent, reactive, and prolifera­tive Müller glia after stimulation. In mice, a dedicated network restored Müller glia to a quiescent state. In contrast, in zebrafish and chick, genes selectively expressed in reactive Müller glia promoted the transition to a prolif­erative and neurogenic progenitor state. Loss of function of genes selectively expressed in reactive Müller glia, such as hmga1 and yap1, inhibited Müller glia reprogramming in ze­brafish. In chick, pharmacological disruption of fatty acid–binding protein 5, 7, and 8 (FABP5/7/8) activity inhibited injury-induced transition from quiescence to neurogenic com­petence. Finally, deletion of nuclear factor I factors a, b, and x (Nfia/b/x), which maintain and restore a glial quiescent state, resulted in Müller glia reprogramming into retinal bipolar and amacrine interneu­rons in adult mice after injury.

CONCLUSION

We found that transition from quiescence through the reactive state is essen­tial for Müller glia reprogramming in regen­eration-competent species such as zebrafish and chick. Furthermore, proliferative and neurogenic competence are both suppressed by a dedicated gene regulatory network in mouse Müller glia. Cross-species RNA-seq and ATAC-seq data provide a comprehen­sive resource to study cellular responses to injury and Müller glia reprogramming. Our findings indicate that treatments targeting gene regulatory networks that repress neu­rogenic competence may help to facilitate reprogramming of mammalian Müller glia to neurons.

Control of retinal Müller glia reprogramming.

RNA-seq and ATAC-seq were performed on Müller glia from zebrafish, chick, and mouse after different treatments. Integrative transcriptomic and epigenomic analysis revealed core regulatory networks controlling retinal regeneration. Loss of function of zebrafish genes expressed in reactive Müller glia blocked reprogramming, while in mouse Müller glia, disruption of Nfia/b/x, which maintain and restore quiescence, resulted in acquisition of neurogenic competence.

Abstract

Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not those of mammals, to regenerate neurons. To identify gene regulatory networks that reprogram Müller glia into progenitor cells, we profiled changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick, and mice in response to different stimuli. We identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, reactivity, and neurogenesis. In zebrafish and chick, the transition from quiescence to reactivity is essential for retinal regeneration, whereas in mice, a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice after injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

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