Zebrafish Muller glia-derived progenitors are multipotent, exhibit proliferative biases and regenerate excess neurons.
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ABSTRACT:
SUBMITTER: Powell C
PROVIDER: S-EPMC4837407 | biostudies-other | 2016 Apr
REPOSITORIES: biostudies-other
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Project description:The adult zebrafish retina exhibits a robust regenerative response following light-induced photoreceptor cell death. This response is initiated by the Müller glia proliferating in the inner nuclear layer (INL), which gives rise to neuronal progenitor cells that continue to divide and migrate to the outer nuclear layer (ONL), where they differentiate into rod and cone photoreceptors. We previously conducted a microarray analysis of retinal gene expression at 16, 31, 51, 68, and 96 h of constant intense-light treatment to identify genes and their corresponding proteins that may be involved in the generation and proliferation of the neuronal progenitor cells. We examined the expression of two candidate transcription factors, Pax6 and Ngn1, and one candidate transgene, olig2:EGFP, in the regenerating light-damaged retina. We compared the temporal and spatial expression patterns of these markers relative to PCNA (proliferating cell nuclear antigen), an established marker for proliferating cells in the zebrafish retina, and the Tg(gfap:EGFP) nt11 transgenic line that specifically labels Müller glial cells. We found that Müller glial cells dedifferentiate during regeneration, based on the loss of cell-specific markers such as GFAP (glial fibrillary acidic protein) and glutamine synthetase following their reentry into the cell cycle to produce neuronal progenitors. Pax6 expression was first detected in the proliferating neuronal progenitors by 51 h of constant light treatment, which is significantly after the Müller glia first reenter the cell cycle after 31h of light. This suggests that Pax6 expression increases in neuronal progenitors, rather than in the proliferating Müller glia. EGFP expression from the olig2 promoter was first detected by 68 h of constant light treatment in the dedifferentiated Müller glia, with Pax6 expressed in the closely associated proliferating neuronal progenitors migrating to the ONL. Both Pax6 and olig2 expression persisted until 3 days post-light treatment, when the neuronal progenitors begin differentiating into new rod and cone photoreceptors. Ngn1 protein expression was initially detected in proliferating neuronal progenitors at 68 h of light treatment. However, Ngn1 expression persisted in a subset of the INL nuclei until 17 days post-light treatment. Using the Tg(gfap:EGFP) nt11 transgenic line, Ngn1 was localized to the Müller glial nuclei that were reestablished following the regenerative response. These markers, therefore, can be used to identify different cell types at particular stages of retinal regeneration: neuronal progenitor formation, proliferation, and the reestablishment of the Müller glia cells. These markers will be important to further characterize the regeneration response in other retinal damage models and to elucidate the defects associated with mutants and morphants that disrupt the regeneration response.
| S-EPMC2586672 | biostudies-literature
Project description:Identification of the signaling pathways that influence the reprogramming of Müller glia into neurogenic retinal progenitors is key to harnessing the potential of these cells to regenerate the retina. Glucocorticoid receptor (GCR) signaling is commonly associated with anti-inflammatory responses and GCR agonists are widely used to treat inflammatory diseases of the eye, even though the cellular targets and mechanisms of action in the retina are not well understood. We find that signaling through GCR has a significant impact upon the ability of Müller glia to become proliferating Müller glia-derived progenitor cells (MGPCs). The primary amino acid sequence and pattern of GCR expression in the retina is highly conserved across vertebrate species, including chickens, mice, guinea pigs, dogs and humans. In all of these species we find GCR expressed by the Müller glia. In the chick retina, we find that GCR is expressed by progenitors in the circumferential marginal zone (CMZ) and is upregulated by Müller glia in acutely damaged retinas. Activation of GCR signaling inhibits the formation of MGPCs and antagonizes FGF2/MAPK signaling in the Müller glia. By contrast, we find that inhibition of GCR signaling stimulates the formation of proliferating MGPCs in damaged retinas, and enhances the neuronal differentiation while diminishing glial differentiation. Given the conserved expression pattern of GCR in different vertebrate retinas, we propose that the functions and mechanisms of GCR signaling are highly conserved and are mediated through the Müller glia. We conclude that GCR signaling directly inhibits the formation of MGPCs, at least in part, by interfering with FGF2/MAPK signaling.
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Project description:Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Müller glia (MG) that activate the gene encoding the proneural factor Achaete-scute homolog 1 (Ascl1; also known as Mash1 in mammals) and de-differentiate into progenitor cells. By contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether ASCL1 could restore neurogenic potential to mammalian MG, we overexpressed ASCL1 in dissociated mouse MG cultures and intact retinal explants. ASCL1-infected MG upregulated retinal progenitor-specific genes and downregulated glial genes. Furthermore, ASCL1 remodeled the chromatin at its targets from a repressive to an active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers and displayed neuron-like physiological responses. These results indicate that a single transcription factor, ASCL1, can induce a neurogenic state in mature MG.
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Project description:The transcription factor Sox11 plays important roles in retinal neurogenesis during vertebrate eye development. However, its function in retina regeneration remains elusive. Here we report that Sox11b, a zebrafish Sox11 homolog, regulates the migration and fate determination of Müller glia-derived progenitors (MGPCs) in an adult zebrafish model of mechanical retinal injury. Following a stab injury, the expression of Sox11b was induced in proliferating MGPCs in the retina. Sox11b knockdown did not affect MGPC formation at 4 days post-injury, although the nuclear morphology and subsequent radial migration of MGPCs were altered. At 7 days post-injury, Sox11b knockdown resulted in an increased proportion of MGPCs in the inner retina and a decreased proportion of MGPCs in the outer nuclear layer, compared with controls. Furthermore, Sox11b knockdown led to reduced photoreceptor regeneration, while it increased the numbers of newborn amacrines and retinal ganglion cells. Finally, quantitative polymerase chain reaction analysis revealed that Sox11b regulated the expression of Notch signaling components in the retina, and Notch inhibition partially recapitulated the Sox11b knockdown phenotype, indicating that Notch signaling functions downstream of Sox11b. Our findings imply that Sox11b plays key roles in MGPC migration and fate determination during retina regeneration in zebrafish, which may have critical implications for future explorations of retinal repair in mammals.
| S-EPMC9396499 | biostudies-literature
Project description:Müller glia function as retinal stem cells in adult zebrafish. In response to loss of retinal neurons, Müller glia partially dedifferentiate, re-express neuroepithelial markers and re-enter the cell cycle. We show that the immunoglobulin superfamily adhesion molecule Alcama is a novel marker of multipotent retinal stem cells, including injury-induced Müller glia, and that each Müller glial cell divides asymmetrically only once to produce an Alcama-negative, proliferating retinal progenitor. The initial mitotic division of Müller glia involves interkinetic nuclear migration, but mitosis of retinal progenitors occurs in situ. Rapidly dividing retinal progenitors form neurogenic clusters tightly associated with Alcama/N-cadherin-labeled Müller glial radial processes. Genetic suppression of N-cadherin function interferes with basal migration of retinal progenitors and subsequent regeneration of HuC/D(+) inner retinal neurons.
| S-EPMC3817940 | biostudies-literature
Project description:The 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.A 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.Ganciclovir 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-p21(cip1)-dependent pathway. Interestingly, unlike control cells, GCV-treated Müller glia cells were "locked" in a prolonged dedifferentiated state.Our 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.
| S-EPMC4849886 | biostudies-other
Project description:Notch signaling is known to play important roles during retinal development. Recently, Notch signaling has been shown to be active in proliferating Müller glia in acutely damaged chick retina (Hayes et al., 2007). However, the roles of Notch in mature, undamaged retina remain unknown. Thus, the purpose of this study was to determine the role of the Notch-signaling pathway in the postnatal retina. Here we show that components of the Notch-signaling pathway are expressed in most Müller glia at low levels in undamaged retina. The expression of Notch-related genes varies during early postnatal development and across regions, with higher expression in peripheral versus central retina. Blockade of Notch activity with a small molecule inhibitor before damage was protective to retinal interneurons (amacrine and bipolar cells) and projection neurons (ganglion cells). In the absence of damage, Notch is upregulated in retinas treated with insulin and FGF2; the combination of these factors is known to stimulate the proliferation and dedifferentiation of Müller glia (Fischer et al., 2002b). Inhibition of Notch signaling during FGF2 treatment reduces levels of the downstream effectors of the MAPK-signaling pathway-p38 MAPK and pCREB in Müller glia. Further, inhibition of Notch activity potently inhibits FGF2-induced proliferation of Müller glia. Together, our data indicate that Notch signaling is downstream of, and is required for, FGF2/MAPK signaling to drive the proliferation of Müller glia. In addition, our data suggest that low levels of Notch signaling in Müller glia diminish the neuroprotective activities of these glial cells.
| S-EPMC2834965 | biostudies-literature
Project description:Vertebrates such as zebrafish have the outstanding ability to fully regenerate their retina upon injury, while mammals, including humans, do not. In zebrafish, upon light-induced injury, photoreceptor regeneration is achieved through reprogramming of Müller glia cells, which proliferate and give rise to a self-renewing population of progenitors that migrate to the lesion site to differentiate into the new photoreceptors. The Hippo pathway effector YAP was recently implicated in the response to damage in the retina, but how this transcription coactivator is integrated into the signaling network regulating Müller glia reprogramming has not yet been explored. Here, we show that Yap is required in Müller glia to engage their response to a lesion by regulating their cell cycle reentry and progenitor cell formation, contributing to the differentiation of new photoreceptors. We propose that this regulation is accomplished through a lin28a-ascl1a-dependent mechanism, bona fide Müller glia-reprogramming factors. Overall, this study presents Yap as a key regulator of zebrafish Müller glia reprogramming and consequently retina regeneration upon injury.
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Project description:Unlike mammals, zebrafish have the capacity to regenerate neurons in response to damage. Most zebrafish retinal injury models employ acute damage, which is unlike the chronic, gradual damage that occurs in human retinal diseases. Here, we studied the regenerative response in the zebrafish aipl1b mutant, gold rush (gosh). In gosh mutants, both cones and rods degenerate by 3 weeks post-fertilization (wpf). Müller glia do not exhibit a regenerative response by 3 wpf; however, they do present non-proliferative gliosis. Only at 5 wpf, is proliferation of Müller cells and rod precursor cells activated. Rods start to recover at 5 wpf and by 12 wpf they reach a level of recovery comparable to wild type, but cones remain absent in the adult stage. TNF? was detected in degenerating cones at 5-7 wpf and in Müller glia at 7 wpf in gosh mutants. At 5 wpf, proliferating Müller glia express Sox2, followed by Pax6 expression in neuronal progenitor cells (NPCs), confirming that the neuronal regeneration program is activated in gosh mutants after 5 wpf. Although acute light-induced damage did not activate proliferation of Müller glia, TNF? injection caused Müller glia to commence a proliferative response at 3 wpf in gosh mutants. These results suggest that Müller glia transition from non-proliferative gliosis to a regenerative state in gosh mutants, and that ectopic introduction of TNF? promotes this Müller cell transition even at 3 wpf. Thus, zebrafish gosh mutants provide a useful model to investigate mechanisms underlying retinal regeneration in a chronic photoreceptor degeneration model.
| S-EPMC6962764 | biostudies-literature
Project description:We analyzed the role of Stat3, Ascl1a, and Lin28a in Müller glia reentry into the cell cycle following damage to the zebrafish retina. Immunohistochemical analysis was employed to determine the temporal and spatial expression of Stat3 and Ascl1a proteins following rod and cone photoreceptor cell apoptosis. Stat3 expression was observed in all Müller glia, whereas Ascl1a expression was restricted to only the mitotic Müller glia. Knockdown of Stat3 protein expression did not affect photoreceptor apoptosis, but significantly reduced, without abolishing, the number of proliferating Ascl1a-positive Müller glia. Knockdown of Ascl1a protein also did not change the extent of photoreceptor apoptosis, but did yield significantly fewer Müller glia that reentered the cell cycle relative to the stat3 morphant and significantly decreased the number and intensity of Stat3-expressing Müller glia. Finally, introduction of lin28a morpholinos resulted in decreased Müller glia expression of Stat3 and Ascl1a, significantly reducing the number of proliferating Müller glia. Thus, there are three populations of Müller glia in the light-damaged zebrafish retina: 1) Stat3-expressing Ascl1a-nonexpressing nonproliferating (quiescent) Müller glia; 2) Stat3-dependent Ascl1a-dependent proliferating Müller glia; and 3) Stat3-independent Ascl1a-dependent proliferating Müller glia. Whereas Ascl1a and Lin28a are required for Müller glia proliferation, Stat3 is necessary for the maximal number of Müller glia to proliferate during regeneration of the damaged zebrafish retina.
| S-EPMC3478445 | biostudies-literature