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.

Background Microglia and inflammation have context-specific impacts upon neuronal survival in different models of central nervous system (CNS) disease. Herein, we investigate how inflammatory mediators, including microglia, interleukin 1 beta (IL1β), and signaling through interleukin 1 receptor type 1 (IL-1R1), influence the survival of retinal neurons in response to excitotoxic damage. Methods Excitotoxic retinal damage was induced via intraocular injections of NMDA. Microglial phenotype and neuronal survival were assessed by immunohistochemistry. Single-cell RNA sequencing was performed to obtain transcriptomic profiles. Microglia were ablated by using clodronate liposome or PLX5622. Retinas were treated with IL1β prior to NMDA damage and cell death was assessed in wild type, IL-1R1 null mice, and mice expressing IL-1R1 only in astrocytes. Results NMDA-induced damage included neuronal cell death, microglial reactivity, upregulation of pro-inflammatory cytokines, and genes associated with IL1β-signaling in different types of retinal neurons and glia. Expression of the IL1β receptor, IL-1R1, was evident in astrocytes, endothelial cells, some Müller glia, and OFF bipolar cells. Ablation of microglia with clodronate liposomes or Csf1r antagonist (PLX5622) resulted in elevated cell death and diminished neuronal survival in excitotoxin-damaged retinas. Exogenous IL1β stimulated the proliferation and reactivity of microglia in the absence of damage, reduced numbers of dying cells in damaged retinas, and increased neuronal survival following an insult. IL1β failed to provide neuroprotection in the IL-1R1-null retina, but IL1β-mediated neuroprotection was rescued when expression of IL-1R1 was restored in astrocytes. Conclusions We conclude that reactive microglia provide protection to retinal neurons, since the absence of microglia is detrimental to survival. We propose that, at least in part, the survival-influencing effects of microglia may be mediated by IL1β, IL-1R1, and interactions of microglia and other macroglia. Electronic supplementary material The online version of this article (10.1186/s12974-019-1505-5) contains supplementary material, which is available to authorized users.

Müller glia-derived progenitor cells (MGPCs) have the capability to regenerate neurons in the retinas of different vertebrate orders. The formation of MGPCs is regulated by a network of cell-signaling pathways. The purpose of this study was to investigate how BMP/Smad1/5/8- and TGFβ/Smad2/3-signaling are coordinated to influence the formation of MGPCs in the chick model system. We find that pSmad1/5/8 is selectively up-regulated in the nuclei of Müller glia following treatment with BMP4, FGF2 or NMDA-induced damage, and this up-regulation is blocked by a dorsomorphin analogue DMH1. By comparison, Smad2/3 is found in the nuclei of Müller glia in untreated retinas, and becomes localized to the cytoplasm following NMDA- or FGF2-treatment. These findings suggest a decrease in TGFβ- and increase in BMP-signaling when MGPCs are known to form. In both NMDA-damaged and FGF2-treated retinas, inhibition of BMP-signaling suppressed the proliferation of MGPCs, whereas inhibition of TGFβ-signaling stimulated the proliferation of MGPCs. Consistent with these findings, TGFβ2 suppressed the formation of MGPCs in NMDA-damaged retinas. Our findings indicate that BMP/TGFβ/Smad-signaling is recruited into the network of signaling pathways that controls the formation of proliferating MGPCs. We conclude that signaling through BMP4/Smad1/5/8 promotes the formation of MGPCs, whereas signaling through TGFβ/Smad2/3 suppresses the formation of MGPCs.

We investigate the roles of mTor signaling in the formation of Müller glia-derived progenitor cells (MGPCs) in the chick retina. During embryonic development, pS6 (a readout of active mTor signaling) is present in early-stage retinal progenitors, differentiating amacrine and ganglion cells, and late-stage progenitors or maturing Müller glia. By contrast, pS6 is present at low levels in a few scattered cell types in mature, healthy retina. Following retinal damage, in which MGPCs are known to form, mTor signaling is rapidly activated in Müller glia. Inhibition of mTor in damaged retinas prevented the accumulation of pS6 in Müller glia and reduced numbers of proliferating MGPCs. Inhibition of mTor had no effect on MAPK signaling or on upregulation of the stem cell factor Klf4, whereas Pax6 upregulation was significantly reduced. Inhibition of mTor potently blocked the MGPC-promoting effects of Hedgehog, Wnt and glucocorticoid signaling in damaged retinas. In the absence of retinal damage, insulin, IGF1 and FGF2 induced pS6 in Müller glia, and this was blocked by mTor inhibitor. In FGF2-treated retinas, in which MGPCs are known to form, inhibition of mTor blocked the accumulation of pS6, the upregulation of Pax6 and the formation of proliferating MGPCs. We conclude that mTor signaling is required, but not sufficient, to stimulate Müller glia to give rise to proliferating progenitors, and the network of signaling pathways that drive the formation of MGPCs requires activation of mTor.

Recent studies suggest midkine (MDK) is involved in the development and regeneration of the zebrafish retina. We investigate the expression patterns of MDK and related factors, roles in neuronal survival, and influence upon the formation of Müller glia‐derived progenitor cells (MGPCs) in chick and mouse model systems. By using single‐cell RNA‐sequencing, we find that MDK and pleiotrophin (PTN), a MDK‐related cytokine, are upregulated by Müller glia (MG) during later stages of development in chick. While PTN is downregulated, MDK is dramatically upregulated in mature MG after retinal damage or FGF2 and insulin treatment. By comparison, MDK and PTN are downregulated by MG in damaged mouse retinas. In both chick and mouse retinas, exogenous MDK induces expression of cFos and pS6 in MG. In the chick, MDK significantly decreases numbers dying neurons, reactive microglia, and proliferating MGPCs, whereas PTN has no effect. Inhibition of MDK‐signaling with Na3VO4 blocks neuroprotective effects with an increase in the number of dying cells and negates the pro‐proliferative effects on MGPCs in damaged retinas. Inhibitors of PP2A and Pak1, which are associated with MDK‐signaling through integrin β1, suppressed the formation of MGPCs in damaged chick retinas. In mice, MDK promotes a small but significant increase in proliferating MGPCs in damaged retinas and potently decreases the number of dying cells. We conclude that MDK expression is dynamically regulated in Müller glia during embryonic maturation, following retinal injury, and during reprogramming into MGPCs. MDK mediates glial activity, neuronal survival, and the re‐programming of Müller glia into proliferating MGPCs.