Glia Dedifferentiation Research Articles

Organoid-derived human retinal progenitor cells promote early dedifferentiation of Müller glia in Royal College of Surgeons rats.

To explore whether the subretinal transplantation of retinal progenitor cells from human embryonic stem cell-derived retinal organoid (hERO-RPCs) could promote Müller glia dedifferentiation and transdifferentiation, thus improving visual function and delaying retinal degenerative progression. hERO-RPCs were subretinally transplanted into Royal College of Surgeons (RCS) rats. Electroretinography (ERG) recording was performed at 4 and 8wk postoperation to assess retinal function. Using immunofluorescence, the changes in outer nuclear layer (ONL) thickness and retinal Müller glia were explored at 2, 4, and 8wk postoperation. To verify the effect of hERO-RPCs on Müller glia in vitro, we cocultured hERO-RPCs with Müller glia with a Transwell system. After coculture, Ki67 staining and quantitative polymerase chain reaction (qPCR) were performed to measure the proliferation and mRNA levels of Müller glia respectively. Cell migration experiment was used to detect the effect of hERO-RPCs on Müller glial migration. Comparisons between two groups were performed by the unpaired Student's t-test, and comparisons among multiple groups were made with one-way ANOVA followed by Tukey's multiple comparison test. The visual function and ONL thickness of RCS rats were significantly improved by transplantation of hERO-RPCs at 4 and 8wk postoperation. In addition to inhibiting gliosis at 4 and 8wk postoperation, hERO-RPCs significantly increased the expression of dedifferentiation-associated transcriptional factor in Müller glia and promoted the migration at 2, 4 and 8wk postoperation, but not the transdifferentiation of these cells in RCS rats. In vitro, using the Transwell system, we found that hERO-RPCs promoted the proliferation and migration of primary rat Müller glia and induced their dedifferentiation at the mRNA level. These results show that hERO-RPCs might promote early dedifferentiation of Müller glia, which may provide novel insights into the mechanisms of stem cell therapy and Müller glial reprogramming, contributing to the development of novel therapies for retinal degeneration disorders.

Involvement of HB-EGF/Ascl1/Lin28a Genes in Dedifferentiation of Adult Mammalian Müller Glia.

Previous studies from this lab have determined that dedifferentiation of Müller glia occurs after eye drop application of an α7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, to the adult rodent eye. PNU-282987 acts on α7 nAChRs on retinal pigment epithelial cells to stimulate production of Müller-derived progenitor cells (MDPCs) and ultimately lead to neurogenesis. This current study was designed to test the hypothesis that the activation of genes involved in the HB-EGF/Ascl1/Lin28a signaling pathway in Müller glia leads to the genesis of MDPCs. RNA-seq was performed on a Müller glial cell line (rMC-1) following contact with supernatant collected from a retinal pigment epithelial (RPE) cell line treated with PNU-282987. Differentially regulated genes were compared with published literature of Müller glia dedifferentiation that occurs in lower vertebrate regeneration and early mammalian development. HB-EGF was significantly up-regulated by 8 h and expression increased through 12 h. By 48 h, up-regulation of Ascl1 and Lin28a was observed, two genes known to be rapidly induced in dedifferentiating zebrafish Müller glia. Up-regulation of other genes known to be involved in mammalian development and zebrafish regeneration were also observed, as well as down-regulation of some factors necessary for Müller glia cell identity. RNA-seq results were verified using qRT-PCR. Using immunocytochemistry, the presence of markers associated with MDCP identity, Otx2, Nestin, and Vsx2, were found to be expressed in the 48 h treatment group cultures. This study is novel in its demonstration that Müller glia in adult rodents can be induced into regenerative activity by stimulating genes involved in the HB-EGF/Ascl1/Lin28a pathway that leads to MDPCs after introducing conditioned media from PNU-282987 treated RPE. This study furthers our understanding of the mechanism by which Müller glia dedifferentiate in response to PNU-282987 in the adult mammalian retina.

Inflammation-induced mammalian target of rapamycin signaling is essential for retina regeneration.

Upon retina injury, Müller glia in the zebrafish retina respond by generating multipotent progenitors to repair the retina. However, the complete mechanisms underlying retina regeneration remain elusive. Here we report inflammation-induced mammalian target of rapamycin (mTOR) signaling in the Müller glia is essential for retina regeneration in adult zebrafish. We show after a stab injury, mTOR is rapidly activated in Müller glia and later Müller glia-derived progenitor cells (MGPCs). Importantly, mTOR is required for Müller glia dedifferentiation, as well as the proliferation of Müller glia and MGPCs. Interestingly, transient mTOR inhibition by rapamycin only reversibly suppresses MGPC proliferation, while its longer suppression by knocking down Raptor significantly inhibits the regeneration of retinal neurons. We further show mTOR promotes retina regeneration by regulating the mRNA expression of key reprogramming factors ascl1a and lin-28a, cell cycle-related genes and critical cytokines. Surprisingly, we identify microglia/macrophage-mediated inflammation as an important upstream regulator of mTOR in the Müller glia and it promotes retina regeneration through mTOR. Our study not only demonstrates the important functions of mTOR but also reveals an interesting link between inflammation and the mTOR signaling during retina regeneration.

Neurotransmitter-Regulated Regeneration in the Zebrafish Retina.

SummaryCurrent efforts to repair damaged or diseased mammalian retinas are inefficient and largely incapable of fully restoring vision. Conversely, the zebrafish retina is capable of spontaneous regeneration upon damage using Müller glia (MG)-derived progenitors. Understanding how zebrafish MG initiate regeneration may help develop new treatments that prompt mammalian retinas to regenerate. We show that inhibition of γ-aminobutyric acid (GABA) signaling facilitates initiation of MG proliferation. GABA levels decrease following damage, and MG are positioned to detect decreased ambient levels and undergo dedifferentiation. Using pharmacological and genetic approaches, we demonstrate that GABAA receptor inhibition stimulates regeneration in undamaged retinas while activation inhibits regeneration in damaged retinas.

Oct4 Methylation-Mediated Silencing As an Epigenetic Barrier Preventing Müller Glia Dedifferentiation in a Murine Model of Retinal Injury.

Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog, and Lin28, which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition.

Lin28B promotes Müller glial cell de-differentiation and proliferation in the regenerative rat retinas.

Retinal regeneration and repair are severely impeded in higher mammalian animals. Although Müller cells can be activated and show some characteristics of progenitor cells when injured or under pathological conditions, they quickly form gliosis scars. Unfortunately, the basic mechanisms that impede retinal regeneration remain unknown. We studied retinas from Royal College of Surgeon (RCS) rats and found that let-7 family molecules, let-7e and let-7i, were significantly overexpressed in Müller cells of degenerative retinas. It demonstrated that down-regulation of the RNA binding protein Lin28B was one of the key factors leading to the overexpression of let-7e and let-7i. Lin28B ectopic expression in the Müller cells suppressed overexpression of let-7e and let-7i, stimulated and mobilized Müller glia de-differentiation, proliferation, promoted neuronal commitment, and inhibited glial fate acquisition of de-differentiated Müller cells. ERG recordings revealed that the amplitudes of a-wave and b-wave were improved significantly after Lin28B was delivered into the subretinal space of RCS rats. In summary, down-regulation of Lin28B as well as up-regulation of let-7e and let-7i may be the main factors that impede Müller cell de-differentiation and proliferation in the retina of RCS rats.

Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia-Derived Progenitors During Zebrafish Retinal Regeneration.

PurposeThe purpose of this study was to investigate the effect of the antiviral drug ganciclovir (GCV) on Müller glia dedifferentiation and proliferation and the underlying cellular and molecular mechanisms in adult zebrafish.MethodsA Tg(1016tuba1a:GFP) transgenic line was generated to identify injury-induced dedifferentiation of Müller glia. Mechanical retinal damage was induced by a needle-poke injury on the back of the eyes in adult zebrafish. Phosphate-buffered saline or GCV was injected into the vitreous of the eye at the time of injury or through the cornea. The GCV clearance rate from the eye was determined by a reversed-phase HPLC method. Green fluorescent protein (GFP) and bromodeoxyuridine (BrdU) immunofluorescence were used to determine the effect of GCV on retinal regeneration. Cell apoptosis was evaluated by TUNEL staining. Microglia were labeled by vitreous injection of isolectin IB4 conjugates. Quantitative (q)PCR and Western blot analysis were used to determine gene expression in the retina.ResultsGanciclovir treatment significantly reduced the number of BrdU+ Müller glia–derived progenitor cells (MGPCs) at 4 days post injury. Further analysis showed that GCV had no impact on Müller glia dedifferentiation and the initial formation of MGPCs. Our data indicate that GCV irreversibly inhibited MGPC proliferation likely through a p53-p21cip1–dependent pathway. Interestingly, unlike control cells, GCV-treated Müller glia cells were “locked” in a prolonged dedifferentiated state.ConclusionsOur study uncovered a novel inhibitory effect of GCV on MGPC proliferation and suggests its potential use as a tool to uncover molecular mechanisms underlying retinal regeneration in zebrafish.

MicroRNA changes through Müller glia dedifferentiation and early/late rod photoreceptor differentiation

Cell-type determination is a complex process driven by the combinatorial effect of extrinsic signals and the expression of transcription factors and regulatory genes.MicroRNAs (miRNAs) are non-coding RNAs that, generally, inhibit the expression of target genes and have been involved, among other processes, in cell identity acquisition. To search for candidate miRNAs putatively involved in mice rod photoreceptor and Müller glia (MG) identity, we compared miRNA expression profiles between late-stage retinal progenitor cells (RPCs), CD73-immunopositive (CD73+) rods and postnatal MG. We found a close similarity between RPCs and CD73+ miRNA expression profiles but a divergence between CD73+ and MG miRNA signatures. We validated preferentially expressed miRNAs in the CD73+ subpopulation (miR-182, 183, 124a, 9∗, 181c and 301b∗) or MG (miR-143, 145, 214, 199a-5p, 199b∗, and 29a). Taking advantage of the unique capacity of MG to dedifferentiate into progenitor-like cells that can be differentiated to a rod phenotype in response to external cues, we evaluated changes of selected miRNAs in MG-derived progenitors (MGDP) during neuronal differentiation. We found decreased levels of miR-143 and 145, but increased levels of miR-29a in MGDP. In MGDPs committed to early neuronal lineages we found increased levels of miR-124a and upregulation of miR-124a, 9∗ and 181c during MGDP acquisition of rod phenotypes. Furthermore, we demonstrated that ectopic miR-124 expression is sufficient to enhance early neuronal commitment of MGDP.Our data reveal a dynamic regulation of miRNAs in MGDP through early and late neuronal commitment and miRNAs that could be potential targets to exploit the silent neuronal differentiation capacity of MG in mammals.

MiR-203 regulates progenitor cell proliferation during adult zebrafish retina regeneration

Damage of the zebrafish retina triggers a spontaneous regeneration response that is initiated by Müller Glia (MG) dedifferentiation and asymmetric cell division to produce multipotent progenitor cells. Subsequent expansion of the progenitor pool by proliferation is critical for retina regeneration. Pax6b expression in the progenitor cells is necessary for their proliferation, but exact regulation of its expression is unclear. Here, we show that miR-203 is downregulated during regeneration in proliferating progenitor cells. Elevated miR-203 levels inhibit progenitor cell expansion without affecting MG dedifferentiation or progenitor cell generation. Using GFP-reporter assays and gain and loss of function experiments in the retina, we show that miR-203 expression must be suppressed to allow pax6b expression and subsequent progenitor cell proliferation.

HB-EGF Is Necessary and Sufficient for Müller Glia Dedifferentiation and Retina Regeneration

Müller glia (MG) dedifferentiation into a cycling population of multipotent progenitors is crucial to zebrafish retina regeneration. The mechanisms underlying MG dedifferentiation are unknown. Here we report that heparin-binding epidermal-like growth factor (HB-EGF) is rapidly induced in MG residing at the injury site and that pro-HB-EGF ectodomain shedding is necessary for retina regeneration. Remarkably, HB-EGF stimulates the formation of multipotent MG-derived progenitors in the uninjured retina. We show that HB-EGF mediates its effects via an EGFR/MAPK signal transduction cascade that regulates the expression of regeneration-associated genes, like ascl1a and pax6(b). We also uncover an HB-EGF/Ascl1a/Notch/hb-egf(a)-signaling loop that helps define the zone of injury-responsive MG. Finally, we show that HB-EGF acts upstream of the Wnt/β-catenin-signaling cascade that controls progenitor proliferation. These data provide a link between extracellular signaling and regeneration-associated gene expression in the injured retina and suggest strategies for stimulating retina regeneration in mammals.

Injury-Dependent Müller Glia and Ganglion Cell Reprogramming during Tissue Regeneration Requires Apobec2a and Apobec2b

Unlike mammals, adult zebrafish are able to regenerate multiple tissues including those of the CNS. In the zebrafish retina, injury stimulates Müller glia dedifferentiation into a multipotent retinal progenitor that is capable of regenerating all lost cell types. This dedifferentiation is driven by the reactivation of gene expression programs that share many characteristics with those that operate during early development. Although the mechanisms underlying the reactivation of these programs remain unknown, it is likely that changes in DNA methylation play a significant role. To begin investigating whether DNA demethylation may contribute to retina regeneration, we characterized the expression of genes associated with DNA demethylation in the uninjured and injured retina. We found that two cytidine deaminases (apobec2a and apobec2b) were expressed basally in the uninjured retina and that they were induced in proliferating, dedifferentiated Müller glia. The maximal induction of apobec2b required Ascl1a, but was independent of Lin28, and therefore defines an independent signaling pathway stemming from Ascl1a. Strikingly, when Apobec2a or Apobec2b was knocked down by antisense morpholino oligonucleotides, the proliferative response of Müller glia following injury was significantly reduced and injury-dependent induction of ascl1a and its target genes were inhibited, suggesting the presence of a regulatory feedback loop between Apobec proteins and ascl1a. Finally, Ascl1a, Apobec2a and Apobec2b were found to be essential for optic nerve regeneration. These data identify an essential role for Apobec proteins during retina and optic nerve regeneration and suggest DNA demethylation may underlie the reprogramming of cells to mount a regenerative response.

Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7 microRNA signalling pathway

Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function1, 2. This regenerative response relies on Müller glia (MG) dedifferentiation into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. Here we report that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor Ascl1a and the pluripotency factor Lin-28 are induced in MG within 6 hrs following retinal injury and are necessary for MG dedifferentiation. We demonstrate that Ascl1a is necessary for lin-28 expression and that Lin-28 suppresses let-7 miRNA expression. Furthermore we show that let-7 represses expression of regeneration-associated genes like, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. Interestingly, hspd1, oct4 and c-myca exhibit basal expression in the uninjured retina and let-7 may inhibit this expression to prevent premature MG dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on MG differentiation/dedifferentiation are similar to that of embryonic stem cells3 and suggest novel targets for stimulating MG dedifferentiation and retina regeneration in mammals.