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.

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.

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.

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 …