Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not 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, transition from the quiescence to reactivity is essential for retinal regeneration, while in mice a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I (NFI) transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice following injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

Damage to the zebrafish retina stimulates resident Müller glia to reprogram, reenter the cell cycle, divide asymmetrically, and produce neuronal progenitor cells that amplify and differentiate into the lost neurons. The transition from quiescent to proliferative Müller glia involves both positive and negative regulators. We previously demonstrated that the Notch signaling pathway represses retinal regeneration by maintaining Müller glia quiescence in zebrafish. Here we examine which Notch receptor is necessary to maintain quiescence. Quantitative RT‐PCR and RNA‐Seq analyses reveal that notch3 is expressed in the undamaged retina and is downregulated in response to light damage. Additionally, Notch3 protein is expressed in quiescent Müller glia of the undamaged retina, is downregulated as Müller glia proliferate, and is reestablished in the Müller glia. Knockdown of Notch3 is sufficient to induce Müller glia proliferation in undamaged retinas and enhances proliferation during light damage. Alternatively, knockdown of Notch1a, Notch1b, or Notch2 decreases the number of proliferating cells during light damage, suggesting that Notch signaling is also required for proliferation during retinal regeneration. We also knockdown the zebrafish Delta and Delta‐like proteins, ligands for the Notch receptors, and find that the deltaB morphant possesses an increased number of proliferating cells in the light‐damaged retina. As with Notch3, knockdown of DeltaB is sufficient to induce Müller glia proliferation in the absence of light damage. Taken together, the negative regulation of Müller glia proliferation in zebrafish retinal regeneration is mediated by Notch3 and DeltaB.

sentence: This study identifies gene regulatory networks controlling proliferative and neurogenic competence in retinal Müller glia. AbstractInjury induces retinal Müller glia of cold-blooded, but not mammalian, vertebrates to regenerate neurons. To identify gene regulatory networks that control neuronal reprogramming in retinal glia, we comprehensively profiled injury-dependent changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick and mice using bulk RNA-Seq and ATAC-Seq, as well as single-cell RNA-Seq. Crossspecies integrative analysis of these data, together with functional validation, identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, gliosis and neurogenesis. In zebrafish and chick, transition from the resting state to gliosis is essential for initiation of retinal regeneration, while in mice a dedicated network suppresses neurogenic competence and restores quiescence. Selective disruption of NFI family transcription factors, which maintain and restore quiescence, enables Müller glia to proliferate and generate neurons in adult mice following retinal injury. These findings may aid in the design of cell-based therapies aimed at restoring retinal neurons lost to degenerative disease. previously shown to induce MG reprogramming independent of injury (20). In the mouse, we also tested FGF2 and insulin treatment, which induces limited MG proliferation, but not neurogenic competence (21).For bulk RNA-Seq and ATAC-Seq, in zebrafish, we used the reporter line Tg[gfap:EGFP]nt11 (22) and cell sorting to enrich MG cells, purifying 9.8% of total input retinal cells (Fig. S1A). In mouse, we performed intraperitoneal injection of tamoxifen at postnatal day (P) 21 to induce MG-specific Sun1-GFP expression in GlastCreERT2;CAG-lsl-Sun1-GFP mice (23), purifying 3.5% of total input cells ( Fig. S1A). Based on RT-qPCR, we had an overall enrichment of 30-fold of canonical MG marker genes such as rlbp1a/Rlbp1 and Glul in the GFP-positive (GFP+) fraction in both species (Fig. S1B). Bulk RNA-Seq samples were generated from both the GFP+ MG and GFP-neuronal fractions in zebrafish and mouse, while ATAC-Seq samples were generated from the GFP+ cells. Each sample had a minimum of two biological replicates. In both species, we analyzed retinas with a time course following either inner retinal injury induced by NMDA excitotoxicity or outer retinal injury induced by light damage. In total, we generated 100 bulk RNA-Seq libraries and 40 bulk ATAC-Seq libraries ( Fig. 1A, Table S1, Supplementary Data 1).In parallel, we conducted scRNA-Seq analysis of whole retinas at the same time points as used for the bulk RNA-Seq. We profiled retinas following either NMDA or light damage in zebrafish and mouse, as well as following NMDA damage in P10 chick. To distinguish injury-responsive genes from injury-independent reprogramming genes, we profiled zebrafish retinas at multiple time points following T+R treatment, and a single time point in chick and mouse retinas...

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as Retinitis Pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Müller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, MG in zebrafish models of photoreceptor degeneration fail to re-enter the cell cycle and regenerate lost cells. Here, we used the zebrafish cep290 mutant to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to pre-lesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.

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Zebrafish possess the innate ability to fully regenerate any neurons lost following a retinal injury. This response is mediated by Müller glia that reprogram and divide asymmetrically to produce neuronal precursor cells that differentiate into the lost neurons. However, little is understood about the early signals that induce this response. Ciliary neurotrophic factor (CNTF) was previously shown to be both neuroprotective and pro-proliferative within the zebrafish retina, however CNTF is not expressed following injury. Here we demonstrate that alternative ligands of the Ciliary neurotrophic factor receptor (CNTFR), such as Cardiotrophin-like cytokine factor 1 (Clcf1) and Cytokine receptor-like factor 1a (Crlf1a), are expressed within Müller glia of the light-damaged retina. We found that CNTFR, Clcf1, and Crlf1a are required for Müller glia proliferation in the light-damaged retina. Furthermore, intravitreal injection of CLCF1/CRLF1 protected against rod photoreceptor cell death in the light-damaged retina and induced proliferation of rod precursor cells in the undamaged retina, but not Müller glia. While rod precursor cell proliferation was previously shown to be Insulin-like growth factor 1 receptor (IGF-1R)-dependent, co-injection of IGF-1 with CLCF1/CRLF1 failed to induce further proliferation of either Müller glia or rod precursor cells. Together, these findings demonstrate that CNTFR ligands have a neuroprotective effect and are required for induction of Müller glia proliferation in the light-damaged zebrafish retina.

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as Retinitis Pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Muller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, MG in zebrafish models of photoreceptor degeneration fail to re-enter the cell cycle and regenerate lost cells. Here, we used the zebrafish cep290 mutant to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to pre-lesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.

Damage to light-sensing photoreceptors (PRs) occurs in highly prevalent retinal diseases. As humans cannot regenerate new PRs, these diseases often lead to irreversible blindness. Intriguingly, animals, such as the zebrafish, have the ability to regenerate PRs efficiently and restore functional vision. Upon injury, mature Müller glia (MG) undergo reprogramming to adopt a stem cell-like state. This process is similar to cellular dedifferentiation, and results in the generation of progenitor cells, which, in turn, proliferate and differentiate to replace lost retinal neurons. In this study, we tested whether factors involved in dedifferentiation of Drosophila CNS are implicated in the regenerative response in the zebrafish retina. We found that hairy-related 6 (her6) negatively regulates of PR production by regulating the rate of cell divisions in the MG-derived progenitors. prospero homeobox 1 (prox1) is expressed in differentiated PRs, and likely promotes PR differentiation through phase separation. Interestingly, upon Her6 downregulation, Prox1 is precociously upregulated in the PRs, to promote PR differentiation; conversely, loss of Prox1 also induces a downregulation of Her6. Together, we identified two novel candidates of PR regeneration that cross regulate each other, and may be exploited to promote human retinal regeneration and vision recovery.