Human iPSC-Derived Retinas Recapitulate the Fetal CRB1 CRB2 Complex Formation and Demonstrate that Photoreceptors and Muller Glia Are Targets of AAV5.
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ABSTRACT:
SUBMITTER: Quinn PM
PROVIDER: S-EPMC6522954 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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:Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration.
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Project description:Mutations in CLRN1 cause Usher syndrome type IIIA (USH3A), an autosomal recessive disorder characterized by hearing and vision loss, and often accompanied by vestibular balance issues. The identity of the cell types responsible for the pathology and mechanisms leading to vision loss in USH3A remains elusive. To address this, we employed CRISPR/Cas9 technology to delete a large region in the coding and untranslated (UTR) region of zebrafish clrn1. Retina of clrn1 mutant larvae exhibited sensitivity to cell stress, along with age-dependent loss of function and degeneration in the photoreceptor layer. Investigation revealed disorganization in the outer retina in clrn1 mutants, including actin-based structures of the Müller glia and photoreceptor cells. To assess cell-specific contributions to USH3A pathology, we specifically re-expressed clrn1 in either Müller glia or photoreceptor cells. Müller glia re-expression of clrn1 prevented the elevated cell death observed in larval clrn1 mutant zebrafish exposed to high-intensity light. Notably, the degree of phenotypic rescue correlated with the level of Clrn1 re-expression. Surprisingly, high levels of Clrn1 expression enhanced cell death in both wild-type and clrn1 mutant animals. However, rod- or cone-specific Clrn1 re-expression did not rescue the extent of cell death. Taken together, our findings underscore three crucial insights. First, clrn1 mutant zebrafish exhibit key pathological features of USH3A; second, Clrn1 within Müller glia plays a pivotal role in photoreceptor maintenance, with its expression requiring controlled regulation; third, the reliance of photoreceptors on Müller glia suggests a structural support mechanism, possibly through direct interactions between Müller glia and photoreceptors mediated in part by Clrn1 protein.
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Project description:The regenerative potential of Müller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Müller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina. Accordingly, we sought to characterize the patterns of expression of NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single-cell RNA-sequencing (scRNA-seq) and immunolabeling, we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), upregulated in E8 RPCs, further upregulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further downregulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.
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Project description:Müller glia possess stem cell characteristics that have been recognized to be responsible for the regeneration of injured retina in fish and amphibians. Although these cells are present in the adult human eye, they are not known to regenerate human retina in vivo. Human Müller glia with stem cell characteristics (hMSCs) can acquire phenotypic and genotypic characteristics of rod photoreceptors in vitro, suggesting that they may have potential for use in transplantation strategies to treat human photoreceptor degenerations. Much work has been undertaken in rodents using various sources of allogeneic stem cells to restore photoreceptor function, but the effect of human Müller glia-derived photoreceptors in the restoration of rod photoreceptor function has not been investigated. This study aimed to differentiate hMSCs into photoreceptor cells by stimulation with growth and differentiation factors in vitro to upregulate gene and protein expression of CRX, NR2E3, and rhodopsin and various phototransduction markers associated with rod photoreceptor development and function and to examine the effect of subretinal transplantation of these cells into the P23H rat, a model of primary photoreceptor degeneration. Following transplantation, hMSC-derived photoreceptor cells migrated and integrated into the outer nuclear layer of the degenerated retinas and led to significant improvement in rod photoreceptor function as shown by an increase in a-wave amplitude and slope using scotopic flash electroretinography. These observations suggest that hMSCs can be regarded as a cell source for development of cell-replacement therapies to treat human photoreceptor degenerations and may also offer potential for the development of autologous transplantation.
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Project description:PurposeThe purpose of this study was to identify the molecular basis and characterize the pathological consequences of a spontaneous mutation named cone photoreceptor function loss 8 (cpfl8) in a mouse model with a significantly reduced cone electroretinography (ERG) response.MethodsThe chromosomal position for the recessive cpfl8 mutation was determined by DNA pooling and by subsequent genotyping with simple sequence length polymorphic markers in an F2 intercross phenotyped by ERG. Genes within the candidate region of both mutants and controls were directly sequenced and compared. The effects of the mutation were examined in longitudinal studies by light microscopy, marker analysis, transmission electron microscopy, and ERG.ResultsThe cpfl8 mutation was mapped to Chromosome 12, and a premature stop codon was identified in the spectrin repeat containing nuclear envelope 2 (Syne2) gene. The reduced cone ERG response was due to a significant reduction in cone photoreceptors. Longitudinal studies of the early postnatal retina indicated that the cone photoreceptors fail to develop properly, rod photoreceptors mislocalize to the inner nuclear layer, and both rods and cones undergo apoptosis prematurely. Moreover, we observed migration defects of secondary neurons and ectopic Müller cell bodies in the outer nuclear layer in early postnatal development.ConclusionsSYNE2 is important for normal retinal development. We have determined that not only is photoreceptor nuclear migration affected, but also the positions of Müller glia and secondary neurons are disturbed early in retinal development. The cpfl8 mouse model will serve as an important resource for further examining the role of nuclear scaffolding and migration in the developing retina.
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Project description:Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1-CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1-CRB2 interactions. We observed no or a mild loss of CRB1-CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.
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Project description: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 Na3 VO4 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.
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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:Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.
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