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.

The retina of warm-blooded vertebrates is believed to be incapable of neural regeneration. Here we provide evidence that the retina of postnatal chickens has the potential to generate new neurons. In response to acute damage, numerous Müller glia re-entered the cell cycle, and shortly thereafter, expressed CASH-1, Pax6 and Chx10, transcription factors expressed by embryonic retinal progenitors. These progenitor-like cells transiently expressed neurofilament. Newly formed cells became distributed throughout the inner and outer nuclear layers of the retina, and remained for at least three weeks after damage. Some of these newly formed cells differentiated into retinal neurons, a few formed Müller glia, and most remained undifferentiated, with continued expression of Pax6 and Chx10. These cells continued to proliferate when grown in culture, with some differentiating into retinal neurons or Müller glia. We propose that, in response to damage, Müller glia in the retina are a potential source of neural regeneration.

In warm-blooded vertebrates it is generally accepted that after early stages of development new neurons are not added to the retina. Contrary to this belief, we show here that hatched chickens have a zone of proliferating cells at the peripheral margin of the retina, similar to that of fish and amphibians. We found that cells at the peripheral edge of the retina incorporated the thymidine analog BrdU and expressed the cell cycle regulator proliferating cell nuclear antigen (PCNA). Furthermore, cells in the ciliary epithelium and retinal margin coexpressed the homeodomain transcription factors Pax6 and Chx-10, similar to multipotent progenitors of embryonic retina. Expression of PCNA, Pax6, and Chx-10 in cells at the retinal margin was maintained in adult birds. Double-labeling studies showed that BrdU-labeled cells that were integrated into the retina expressed proteins found only in differentiated neurons. Increased rates of ocular growth, induced by visual deprivation, resulted in increased numbers of BrdU-labeled cells at the retinal margin. Unlike the progenitors in the retinal marginal zone of fish and amphibians, the progenitors of the chick retina do not increase their rate of proliferation in response to acute damage. Furthermore, insulin, insulin-like growth factor-I, and epidermal growth factor increased proliferation of progenitors at the retinal margin, while basic fibroblast growth factor had no effect. These results indicate that the avian retina has a marginal growth zone containing proliferating cells that share similarities with multipotent embryonic retinal progenitors and the retinal stem cells of cold-blooded vertebrates.

The purpose of this study was to investigate whether signaling through insulin, Fibroblast Growth Factor (FGF) receptors, and Mitogen-Activated Protein Kinase (MAPK) pathways protect retinal neurons against excitotoxicity and regulate the proliferation of Müller glia. We found that intraocular injections of insulin or FGF2 had variable effects upon the phosphorylation of ERK1/2, p38 MAPK and CREB, and the expression of immediate early genes, cFos and Egr1. Accumulations of pERK1/2, p38 MAPK, pCREB, cFos and Egr1 in response to insulin or FGF2 were confined to Müller glia, whereas retinal neurons did not appear to respond to growth factors. Unlike FGF2, insulin stimulated microglia-like cells to up-regulate the intermediate filament transitin and lysosomal membrane glycoprotein (LMG). The affects of insulin and FGF2 upon microglia-like cells and/or MAPKsignaling in Müller glia had profound effects upon numbers of dying neurons in response to excitotoxic damage. Although FGF2 significantly reduced numbers of dying neurons, insulin significantly increased numbers of dying neurons. In addition to neuroprotective affects, FGF2 also "primed" the Müller glia to proliferate following retinal damage, whereas insulin had no effect upon glial proliferation. Further, we found that FGF receptor isoform 1 (FGFR1) and FGFR3 are prominently expressed in the retina, whereas the insulin receptor and FGFR2 are not expressed, or are expressed at very low levels. We conclude that MAPK-signaling through FGF receptors stimulates Müller glia to become more neuroprotective and progenitor-like, whereas insulin acting on Müller and microglia-like cells through unidentified receptors had the opposite effect.

We have reported previously that neurotoxic damage to the chicken retina causes Müller glia to dedifferentiate, proliferate, express transcription factors common to retinal progenitors, and generate new neurons and glia, whereas the majority of newly produced cells remain undifferentiated (Fischer and Reh, 2001). Because damaged retinal cells have been shown to produce increased levels of insulin-related factors and FGFs, in the current study we tested whether intraocular injections of growth factors stimulate Müller glia to proliferate and produce new neurons. We injected growth factors and bromodeoxyuridine into the vitreous chamber of the eyes of chickens and assayed for changes in glial phenotype and proliferation within the retina. Although insulin or FGF2 alone had no effect, the combination of insulin and FGF2 caused Müller glia to coexpress transcription factors common to retinal progenitors (Pax6 and Chx10) and initiated a wave of proliferation in Müller cells that began at the retinal margin and spread into peripheral regions of the retina. Most of the newly formed cells remain undifferentiated, expressing Pax6 and Chx10, whereas some differentiate into Müller glia, and a few differentiate into neurons that express the neuronal markers Hu or calretinin. There was no evidence of retinal damage in eyes treated with insulin and FGF2. We conclude that the combination of insulin and FGF2 stimulated Müller glia to dedifferentiate, proliferate, and generate new neurons. These findings imply that exogenous growth factors might be used to stimulate endogenous glial cells to regenerate neurons in the CNS.