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.

Müller glia are capable of de-differentiating and proliferating to become Müller glia-derived progenitor cells (MGPCs) with the ability to regenerate retinal neurons. One of the cell-signaling pathways that drives the reprogramming of Müller glia into MGPCs in the zebrafish retina is the Jak/Stat-pathway. However, nothing is known about the influence of Jak/Stat-signaling during the formation of MGPCs in the retinas of warm-blooded vertebrates. Accordingly, we examined whether Jak/Stat-signaling influences the formation of MGPCs and differentiation of progeny in the avian retina. We found that Jak/Stat-signaling is activated in Müller glia in response to NMDA-induced retinal damage or by CNTF or FGF2 in the absence of retinal damage. Inhibition of gp130, Jak2, or Stat3 suppressed the formation of proliferating MGPCs in NMDA-damaged and FGF2-treated retinas. Additionally, CNTF combined with FGF2 enhanced the formation of proliferating MGPCs in the absence of retinal damage. In contrast to the zebrafish model, where activation of gp130/Jak/Stat is sufficient to drive neural regeneration from MGPCs, signaling through gp130 inhibits the neurogenic potential of MGPCs and promotes glial differentiation. We conclude that gp130/Jak/Stat-signaling plays an important role in the network of pathways that drives the formation of proliferating MGPCs; however, this pathway inhibits the neural differentiation of the progeny.

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.

No abstract

Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, Heparin-Binding EGF-like Growth Factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF stimulated not only MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas.

Retinal progenitors in the circumferential margin zone (CMZ) and Müller glia-derived progenitors have been well-described in the eyes of fish, amphibians and birds. However, there is no information regarding a CMZ and the nature of retinal glia in species phylogenetically bridging amphibians and birds. Thus, the purpose of this study was to examine the retinal glia and investigate whether a CMZ is present in the eyes of reptilian species. We used immuno-histochemical analyses to study retinal glia, neurons that could influence CMZ-progenitors, the retinal margin, and non-pigmented epithelium (NPE) of ciliary body of garter snakes, queen snakes, anole lizards, snapping turtles, and painted turtles. We compare our observations in reptile eyes to the CMZ and glia of fish, amphibians and birds. In all species, Sox9, Pax6 and the glucocorticoid receptor are expressed by Müller glia and cells at the retinal margin. However, proliferating cells were found only in the CMZ of turtles, but not in the eyes of anoles and snakes. Similar to eyes of chickens, the retinal margin in turtles contains accumulations of GLP1/glucagonergic neurites. We find that filamentous proteins, vimentin and GFAP, are expressed by Müller glia, but have different patterns of sub-cellular localization in the different species of reptiles. We provide evidence that the reptile retina may contain Non-astrocytic Inner Retinal Glial (NIRG) cells, similar to those described in the avian retina. We conclude that the retinal glia, glucagonergic neurons and CMZ of turtles appears to be the most similar to that of fish, amphibians and birds.

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...

No abstract