ABSTRACT
Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.
Subject(s)
Activins , Cell Differentiation , Hedgehog Proteins , Organoids , Retina , Signal Transduction , Tretinoin , Humans , Activins/pharmacology , Activins/metabolism , Organoids/drug effects , Organoids/metabolism , Organoids/cytology , Hedgehog Proteins/metabolism , Tretinoin/pharmacology , Retina/metabolism , Retina/cytology , Retina/drug effects , Signal Transduction/drug effects , Cell Differentiation/drug effects , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolismABSTRACT
PURPOSE: To identify the genotypic and phenotypic characteristics of rhodopsin (RHO)-associated retinitis pigmentosa (RP) in the Japanese population. STUDY DESIGN: Cross-sectional, single-center study METHODS: The medical records of 1336 patients with RP who underwent genetic testing at our clinic between November 2008 and September 2021 were reviewed, and patients with RHO variants were included. The patients were divided into class A and class B to assess genotype-phenotype correlations based on previous reports. The clinical findings, including best-corrected visual acuity (BCVA), OCT parameters (ellipsoid zone [EZ] width and central retinal thickness [CRT]), and presence of macular degeneration, of the 2 groups were compared. RESULTS: The study included 28 patients diagnosed with RHO-associated RP (class A, 19; class B, 9). The BCVA was significantly worse in class A patients than in class B patients (P = 0.045). Superior EZ width was significantly shorter in class A than in class B patients (P = 0.016). Class A patients tended to have thinner CRT and shorter inferior EZ width than those of class B patients, although this difference was not significant. Macular degeneration was observed in 61.5% of class A and 12.5% of class B patients, demonstrating that macular degeneration can be a common complication in class A variants. CONCLUSION: Patients with class A variants presented with a severer form of RP than that of patients with class B variants in the Japanese population. These results suggest that the phenotype of RHO-associated RP is linked to the location of the variants and that such a genotype-phenotype correlation is less affected by ethnicities with different genetic backgrounds.
Subject(s)
Macular Degeneration , Retinitis Pigmentosa , Humans , Rhodopsin/genetics , Cross-Sectional Studies , East Asian People , Tomography, Optical Coherence/methods , Retinitis Pigmentosa/diagnosis , Retinitis Pigmentosa/genetics , Phenotype , GenotypeABSTRACT
Several clinical studies have been conducted into the practicality and safety of regenerative therapy using hESC/iPSC-retinal pigment epithelium (RPE) as a treatment for the diseases including age-related macular degeneration. These studies used either suspensions of RPE cells or an RPE cell sheet. The cells can be injected using a minimally invasive procedure but the delivery of an intended number of cells at an exact target location is difficult; cell sheets take a longer time to prepare, and the surgical procedure is invasive but can be placed at the target area. In the research reported here, we combined the advantages of the two approaches by producing a quickly formed hiPSC-RPE strip in as short as 2 days. The strip readily expanded into a monolayer sheet on the plate, and after transplantation in nude rats, it showed a potency to partly expand with the correct apical/basal polarity in vivo, although limited in expansion area in the presence of healthy host RPE. The strip could be injected into a target area in animal eyes using a 24G canula tip.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Minimally Invasive Surgical Procedures/methods , Retinal Degeneration/surgery , Retinal Pigment Epithelium/transplantation , Animals , Male , Rabbits , Rats , Rats, Inbred F344 , Rats, Nude , Retinal Degeneration/pathology , Retinal Degeneration/therapyABSTRACT
ESC/iPSC-retinal sheet transplantation, which supplies photoreceptors as well as other retinal cells, has been shown to be able to restore visual function in mice with end-stage retinal degeneration. Here, by introducing a novel type of genetically engineered mouse ESC/iPSC-retinal sheet with reduced numbers of secondary retinal neurons but intact photoreceptor cell layer structure, we reinforced the evidence that ESC/iPSC-retinal sheet transplantation can establish synaptic connections with the host, restore light responsiveness, and reduce aberrant retinal ganglion cell spiking in mice. Furthermore, we show that genetically engineered grafts can substantially improve the outcome of the treatment by improving neural integration. We speculate that this leads to reduced spontaneous activity in the host which in turn contributes to a better visual recovery.
ABSTRACT
Retinal ganglion cells (RGCs) are impaired in patients such as those with glaucoma and optic neuritis, resulting in permanent vision loss. To restore visual function, development of RGC transplantation therapy is now underway. Induced pluripotent stem cells (iPSCs) are an important source of RGCs for human allogeneic transplantation. We therefore analyzed the immunological characteristics of iPSC-derived RGCs (iPSC-RGCs) to evaluate the possibility of rejection after RGC transplantation. We first assessed the expression of human leukocyte antigen (HLA) molecules on iPSC-RGCs using immunostaining, and then evaluated the effects of iPSC-RGCs to activate lymphocytes using the mixed lymphocyte reaction (MLR) and iPSC-RGC co-cultures. We observed low expression of HLA class I and no expression of HLA class II molecules on iPSC-RGCs. We also found that iPSC-RGCs strongly suppressed various inflammatory immune cells including activated T-cells in the MLR assay and that transforming growth factor-ß2 produced by iPSC-RGCs played a critical role in suppression of inflammatory cells in vitro. Our data suggest that iPSC-RGCs have low immunogenicity, and immunosuppressive capacity on lymphocytes. Our study will contribute to predicting immune attacks after RGC transplantation.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/immunology , T-Lymphocytes/immunology , Cell Differentiation , Coculture Techniques , Graft Rejection , HLA Antigens/genetics , HLA Antigens/immunology , HLA Antigens/metabolism , Humans , Immune Tolerance , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Retinal Ganglion Cells/transplantation , Transforming Growth Factor beta/metabolismABSTRACT
VEGF secreted from retinal pigment epithelial (RPE) cells is responsible for the choroidal vascular development; however, the molecular regulatory mechanism is unclear. We found that Aldh1a1-/- mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region, although Aldh1a1, an enzyme that synthesizes retinoic acids (RAs), is expressed in the dorsal neural retina, not in the RPE/choroid complex. The level of VEGF in the RPE/choroid was significantly decreased in Aldh1a1-/- mice, and RA-dependent enhancement of VEGF was observed in primary RPE cells. An RA-deficient diet resulted in dorsal choroidal hypoplasia, and simple RA treatment of Aldh1a1-/- pregnant females suppressed choroid hypoplasia in their offspring. We also found downregulation of Sox9 in the dorsal neural retina and RPE of Aldh1a1-/- mice and RPE-specific disruption of Sox9 phenocopied Aldh1a1-/- choroidal development. These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion.
Subject(s)
Aldehyde Dehydrogenase/metabolism , Choroid/physiology , Gene Expression Regulation , Neovascularization, Physiologic , Retinal Pigment Epithelium/physiology , SOX9 Transcription Factor/metabolism , Vascular Endothelial Growth Factor A/metabolism , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Animals , Epithelial Cells/physiology , Mice, Inbred C57BL , Mice, Knockout , Retinal Dehydrogenase , Tretinoin/metabolismABSTRACT
Purpose: We aimed to establish purification and culture systems for retinal ganglion cells (RGCs) differentiated from mouse and human pluripotent stem cells (PSC) for in vitro and regenerative medicine studies. Methods: We used a two-step immunopanning method to purify RGCs from mouse and human PSC-derived three-dimensional (3D) retinal organoids. To assess the method, we purified RGCs from 3D retinal organoids derived from embryonic stem cells (ESCs) generated from Thy1-EGFP transgenic (TG) mice. In addition, 3D retinal organoids differentiated from human induced PSCs (iPSCs) were cultured for up to differentiation day (DD) 120, and RGCs were purified by immunopanning. RGC marker expressions were confirmed by immunostaining and reverse transcription-quantitative PCR. The purified RGCs were cultured, and neurite outgrowth was measured and analyzed using an IncuCyte Zoom system. Results: Mouse RGCs purified from Thy1-EGFP TG mouse retinas and the ESC-derived 3D retinas could be maintained for approximately 2 to 3 weeks, expressing the markers BRN3B and SMI-312. Purified RGCs from human iPSC-derived retinal organoids expressed RGC markers and could be maintained for up to 4 weeks. The RGCs collected at DD 90 to 110 extended longer neurites than those collected at younger stages. Conclusions: We successfully purified RGCs from mouse and human PSC-derived 3D retinal organoids cultured for approximately 120 days. RGCs from older retinal organoids would be useful for neurite tracking. This method would be effective not only for studying the pathology of human RGC diseases but also for therapeutic drug studies and RGC transplantation.
Subject(s)
Cell Culture Techniques/methods , Cell Separation/methods , Pluripotent Stem Cells/cytology , Retinal Ganglion Cells/cytology , Animals , Biomarkers/metabolism , Blotting, Western , Cell Differentiation , Cells, Cultured , Humans , Immunohistochemistry , Mice , Mice, Transgenic , Neuronal Outgrowth , Organoids , Regenerative Medicine , Retinal Ganglion Cells/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
PURPOSE: Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins. METHODS: Retinal organoids differentiated from mouse Nrl-eGFP iPS cells were cultured in various mediums during photoreceptor development. To promote rod photoreceptor development, organoids were maintained in media containing 9-cis retinoic acids (9cRA). To obtain retinal organoids with M-opsin expression, we cultured in medium with 1% fetal bovine serum (FBS) supplemented with T3, BMP4, and DAPT. Section immunohistochemistry was performed to visualize the expression of photoreceptor markers. RESULTS: In three-dimensional (3D) retinas exposed to 9cRA, rhodopsin was expressed earlier and S-cone opsins were suppressed. We could maintain 3D retinas up to DD 35 in culture media with 1% FBS. The 3D retinas expressed rhodopsin, S- and M-opsins, but most cone photoreceptors expressed either S- or M-opsins. CONCLUSION: By modifying culture conditions in the SFEBq protocol, we obtained rod-dominated 3D retinas and S- and M-opsin expressing 3D retinas.
Subject(s)
Bioartificial Organs , Cone Opsins/metabolism , Retina/cytology , Retina/growth & development , Rhodopsin/metabolism , Stem Cells/cytology , Tissue Engineering/methods , Animals , Cell Differentiation/physiology , Cells, Cultured , Mice , Organogenesis/physiology , Printing, Three-Dimensional , Retinal Cone Photoreceptor Cells/metabolism , Stem Cells/physiologyABSTRACT
In the developing retina, as in other regions of the CNS, neural progenitors give rise to individual cell types during discrete temporal windows. Pax6 is expressed in retinal progenitor cells (RPCs) throughout the course of retinogenesis, and has been shown to be required during early retinogenesis for generation of most early-born cell types. In this study, we examined the function of Pax6 in postnatal mouse retinal development. We found that Pax6 is essential for the generation of late-born interneurons, while inhibiting photoreceptor differentiation. Generation of bipolar interneurons requires Pax6 expression in RPCs, while Pax6 is required for the generation of glycinergic, but not for GABAergic or non-GABAergic-non-glycinergic (nGnG) amacrine cell subtypes. In contrast, overexpression of either full-length Pax6 or its 5a isoform in RPCs induces formation of cells with nGnG amacrine features, and suppresses generation of other inner retinal cell types. Moreover, overexpression of both Pax6 variants prevents photoreceptor differentiation, most likely by inhibiting Crx expression. Taken together, these data show that Pax6 acts in RPCs to control differentiation of multiple late-born neuronal cell types.
Subject(s)
Neurons/physiology , PAX6 Transcription Factor/physiology , Photoreceptor Cells, Vertebrate/physiology , Retina/physiology , Amacrine Cells/cytology , Amacrine Cells/metabolism , Amacrine Cells/physiology , Animals , Cell Differentiation/physiology , Female , Interneurons/cytology , Interneurons/metabolism , Interneurons/physiology , Male , Mice , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/physiology , Neurogenesis/physiology , Neurons/cytology , Neurons/metabolism , PAX6 Transcription Factor/metabolism , Photoreceptor Cells, Vertebrate/cytology , Photoreceptor Cells, Vertebrate/metabolism , Retina/cytology , Retina/metabolism , Retinal Neurons/cytology , Retinal Neurons/metabolism , Retinal Neurons/physiologyABSTRACT
Induced pluripotent stem cells (iPSCs), which can be differentiated into various tissues and cell types, have been used for clinical research and disease modeling. Self-organizing three-dimensional (3D) tissue engineering has been established within the past decade and enables researchers to obtain tissues and cells that almost mimic in vivo development. However, there are no reports of practical experimental procedures that reproduce photoreceptor degeneration. In this study, we induced photoreceptor cell death in mouse iPSC-derived 3D retinal organoids (3D-retinas) by 4-hydroxytamoxifen (4-OHT), which induces photoreceptor degeneration in mouse retinal explants, and then established a live-cell imaging system to measure degeneration-related properties. Furthermore, we quantified the protective effects of representative ophthalmic supplements for treating the photoreceptor degeneration. This drug evaluation system enables us to monitor drug effects in photoreceptor cells and could be useful for drug screening.
Subject(s)
Cytoprotection , Induced Pluripotent Stem Cells/metabolism , Organoids/pathology , Photoreceptor Cells, Vertebrate/pathology , Retina/pathology , Retinal Degeneration/pathology , Tamoxifen/analogs & derivatives , Animals , Apoptosis/drug effects , Cell Differentiation/drug effects , Cytoprotection/drug effects , Green Fluorescent Proteins/metabolism , Induced Pluripotent Stem Cells/drug effects , Mice, Inbred C57BL , Organoids/drug effects , Photoreceptor Cells, Vertebrate/drug effects , Tamoxifen/toxicityABSTRACT
We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).
Subject(s)
Induced Pluripotent Stem Cells/cytology , Macular Degeneration/therapy , Retinal Pigment Epithelium/cytology , Aged , Cell Culture Techniques , Cell Differentiation , Feasibility Studies , Female , Fibroblasts , Humans , Male , Retinal Pigment Epithelium/transplantation , Transplantation, AutologousABSTRACT
PURPOSE: To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC). METHODS: Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated. RESULTS: We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation. CONCLUSION: We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.
Subject(s)
Cell Culture Techniques , Cell Differentiation , Induced Pluripotent Stem Cells/cytology , Retinal Pigment Epithelium/metabolism , Animals , Cell Communication , Cell Proliferation , Cells, Cultured , Collagenases/chemistry , Embryonic Stem Cells/cytology , Lymphocytes/cytology , Mice , Peptide Hydrolases/chemistry , Phagocytosis , Rod Cell Outer Segment/metabolism , Signal Transduction , rho-Associated Kinases/metabolismABSTRACT
PURPOSE: To establish a practical research tool for studying the pathogenesis of retinal ganglion cell (RGC) diseases, we optimized culture procedures to induce neurite outgrowth from three-dimensional self-organizing optic vesicles (3D-retinas) differentiated in vitro from mouse and human embryonic stem cells (ESCs). MATERIALS AND METHODS: The developing 3D-retinas isolated at various time points were placed on Matrigel-coated plates and cultured in media on the basis of the 3D-retinal culture or the retinal organotypic culture protocol. The number, length, and morphology of the neurites in each culture condition were compared. RESULTS: First, we confirmed that Venus-positive cells were double-labeled with a RGC marker, Brn3a, in the 3D-retina differentiated from Fstl4::Venus mouse ESCs, indicating specific RGC-subtype differentiation. Second, Venus-positive neurites grown from these RGC subsets were positive for beta-III tubulin and SMI312 by immunohistochemistry. Enhanced neurite outgrowth was observed in the B27-supplemented Neurobasal-A medium on Matrigel-coated plates from the optic vesicles isolated after 14 days of differentiation from mouse ESCs. For the differentiated RGCs from human ESCs, we obtained neurite extension of >4 mm by modifying Matrigel coating and the culture medium from the mouse RGC culture. CONCLUSION: We successfully optimized the culture conditions to enhance lengthy and high-frequency neurite outgrowth in mouse and human models. The procedure would be useful for not only developmental studies of RGCs, including maintenance and projection, but also clinical, pathological, and pharmacological studies of human RGC diseases.
Subject(s)
Human Embryonic Stem Cells/cytology , Nerve Degeneration/therapy , Neurites/pathology , Neuronal Outgrowth/physiology , Retinal Ganglion Cells/pathology , Animals , Cell Differentiation , Cells, Cultured , Disease Models, Animal , Embryo, Mammalian , Humans , Imaging, Three-Dimensional , Immunohistochemistry , Mice , Mice, Knockout , Nerve Degeneration/pathologyABSTRACT
In vertebrate retinal development, various transcription factors are known to execute essential activities in gene regulation. Although epigenetic modification is considered to play a pivotal role in retinal development, the exact in vivo role of epigenetic regulation is still poorly understood. We observed that G9a histone methyltransferase, which methylates histone H3 at lysine 9 (H3K9), is substantially expressed in the mouse retina throughout development. To address in vivo G9a function in the mouse retina, we ablated G9a in retinal progenitor cells by conditional gene knock-out (G9a Dkk3 CKO). The G9a Dkk3 CKO retina exhibited severe morphological defects, including photoreceptor rosette formation, a partial loss of the outer nuclear layer, elevated cell death, and persistent cell proliferation. Progenitor cell-related genes, including several cyclins, Hes1, Chx10, and Lhx2, are methylated on histone H3K9 in the wild-type retina, but they were defective in H3K9 methylation and improperly upregulated at late developmental stages in the G9a Dkk3 CKO retina. Notably, conditional depletion of G9a in postmitotic photoreceptor precursors (G9a Crx CKO) led to the development of an almost normal retina, indicating that G9a activity mainly in retinal progenitor cells, but not in photoreceptor precursors, is essential for normal terminal differentiation of and survival of the retina. Our results suggest that proper epigenetic marks in progenitor cells are important for subsequent appropriate terminal differentiation and survival of retinal cells by repressing progenitor cell-related genes in differentiating retinal cells.
Subject(s)
Cell Death/physiology , Cell Differentiation/physiology , Gene Expression Regulation, Developmental/physiology , Histone-Lysine N-Methyltransferase/physiology , Retina/growth & development , Stem Cells/enzymology , Animals , Cell Death/genetics , Cell Differentiation/genetics , Epigenesis, Genetic/genetics , Epigenesis, Genetic/physiology , Gene Expression Regulation, Developmental/genetics , Histone-Lysine N-Methyltransferase/genetics , Methylation , Mice , Mice, Knockout , Mice, Transgenic , Retina/anatomy & histology , Retina/enzymology , Stem Cells/physiologyABSTRACT
MicroRNA-124a (miR-124a) is the most abundant microRNA expressed in the vertebrate CNS. Despite past investigations into the role of miR-124a, inconsistent results have left the in vivo function of miR-124a unclear. We examined the in vivo function of miR-124a by targeted disruption of Rncr3 (retinal non-coding RNA 3), the dominant source of miR-124a. Rncr3(-/-) mice exhibited abnormalities in the CNS, including small brain size, axonal mis-sprouting of dentate gyrus granule cells and retinal cone cell death. We found that Lhx2 is an in vivo target mRNA of miR-124a. We also observed that LHX2 downregulation by miR-124a is required for the prevention of apoptosis in the developing retina and proper axonal development of hippocampal neurons. These results suggest that miR-124a is essential for the maturation and survival of dentate gyrus neurons and retinal cones, as it represses Lhx2 translation.
Subject(s)
Axons/physiology , Hippocampus/cytology , Homeodomain Proteins/metabolism , MicroRNAs/metabolism , Neurons/cytology , Retinal Cone Photoreceptor Cells/physiology , Transcription Factors/metabolism , Analysis of Variance , Animals , Animals, Newborn , Brain/abnormalities , Cell Differentiation/genetics , Cell Survival/genetics , Electroporation/methods , Electroretinography/methods , Embryo, Mammalian , Evoked Potentials, Visual/genetics , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , In Situ Nick-End Labeling/methods , LIM-Homeodomain Proteins , Mice , Mice, Transgenic , MicroRNAs/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Untranslated/genetics , Retina/cytology , Retina/physiology , Tissue Culture Techniques , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
In the vertebrate retina, the Otx2 transcription factor plays a crucial role in the cell fate determination of both rod and cone photoreceptors. We previously reported that Otx2 conditional knockout (CKO) mice exhibited a total absence of rods and cones in the retina due to their cell fate conversion to amacrine-like cells. In order to investigate the entire transcriptome of the Otx2 CKO retina, we compared expression profile of Otx2 CKO and wild-type retinas at P1 and P12 using microarray. We observed that expression of 101- and 1049-probe sets significantly decreased in the Otx2 CKO retina at P1 and P12, respectively, whereas, expression of 3- and 4149-probe sets increased at P1 and P12, respectively. We found that expression of genes encoding transcription factors involved in photoreceptor development, including Crx, Nrl, Nr2e3, Esrrb, and NeuroD, was markedly down-regulated in the Otx2 CKO at both P1 and P12. Furthermore, we identified three human retinal disease loci mapped in close proximity to certain down-regulated genes in the Otx2 CKO retina including Ccdc126, Tnfsf13 and Pitpnm1, suggesting that these genes are possibly responsible for these diseases. These transcriptome data sets of the Otx2 CKO retina provide a resource on developing rods and cones to further understand the molecular mechanisms underlying photoreceptor development, function and disease.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Developmental , Otx Transcription Factors/genetics , Photoreceptor Cells/pathology , Retina/pathology , Animals , Humans , Mice , Mice, Knockout , Otx Transcription Factors/deficiency , Photoreceptor Cells/metabolism , Retinal Diseases/genetics , Transcription FactorsABSTRACT
Selective expression of retinal cone opsin genes is essential for color vision, but the mechanism mediating this process is poorly understood. Both vertebrate rod and medium wavelength-sensitive (M) cone photoreceptors differentiate by repression of a short wavelength-sensitive (S) cone differentiation program. We found that Pias3 acts in mouse cone photoreceptors to activate expression of M opsin and repress expression of S opsin, with the transcription factors Trbeta2 and Rxrgamma mediating preferential expression of Pias3 in M cones. Finally, we observed that Pias3 directly regulated M and S cone opsin expression by modulating the cone-enriched transcription factors Rxrgamma, Roralpha and Trbeta1. Our results indicate that Pias3-dependent SUMOylation of photoreceptor-specific transcription factors is a common mechanism that controls both rod and cone photoreceptor subtype specification, regulating distinct molecular targets in the two cell types.
Subject(s)
Cell Differentiation/physiology , Neurogenesis/physiology , Protein Inhibitors of Activated STAT/metabolism , Retinal Cone Photoreceptor Cells/cytology , Retinal Cone Photoreceptor Cells/physiology , Small Ubiquitin-Related Modifier Proteins/metabolism , Animals , Base Sequence , Cone Opsins/genetics , Cone Opsins/metabolism , Gene Expression Regulation/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Inhibitors of Activated STAT/genetics , Retinal Rod Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/physiology , Sequence Homology, Nucleic Acid , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Mutation of rod photoreceptor-enriched transcription factors is a major cause of inherited blindness. We identified the orphan nuclear hormone receptor estrogen-related receptor beta (ERRbeta) as selectively expressed in rod photoreceptors. Overexpression of ERRbeta induces expression of rod-specific genes in retinas of wild-type as well as Nrl(-/-) mice, which lack rod photoreceptors. Mutation of ERRbeta results in dysfunction and degeneration of rods, whereas inverse agonists of ERRbeta trigger rapid rod degeneration, which is rescued by constitutively active mutants of ERRbeta. ERRbeta coordinates expression of multiple genes that are rate-limiting regulators of ATP generation and consumption in photoreceptors. Furthermore, enhancing ERRbeta activity rescues photoreceptor defects that result from loss of the photoreceptor-specific transcription factor Crx. Our findings demonstrate that ERRbeta is a critical regulator of rod photoreceptor function and survival, and suggest that ERRbeta agonists may be useful in the treatment of certain retinal dystrophies.
Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/physiology , Receptors, Estrogen/metabolism , Retina/embryology , Retinal Rod Photoreceptor Cells/metabolism , Trans-Activators/physiology , Animals , Cell Survival , Electroretinography/methods , Homeodomain Proteins/metabolism , Ligands , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Retina/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Rhodopsin/metabolism , Trans-Activators/metabolismABSTRACT
The zinc finger transcription factor Blimp1 plays fundamentally important roles in many cell lineages and in the early development of several cell types, including B and T lymphocytes and germ cells. Although Blimp1 expression in developing retinal photoreceptor cells has been reported, its function remains unclear. We identified Blimp1 as a downstream factor of Otx2, which plays an essential role in photoreceptor cell fate determination. To investigate Blimp1 function in the mouse retina, we ablated Blimp1 in the developing retina by conditional gene targeting. In the Blimp1 conditional knockout (CKO) retina, the number of photoreceptor cells was markedly reduced in the differentiated retina. We found that the numbers of both bipolar-like cells and proliferating retinal cells increased noticeably, with ectopic localizations in the postnatal developing retina. In contrast, a reduction of the number of photoreceptor precursors was observed during development. Forced expression of Blimp1 by in vivo electroporation suppressed bipolar cell genesis in the developing retina. Multiple genes involved in bipolar development, including Chx10, were upregulated in the Blimp1 CKO retina. Furthermore, we showed that Blimp1 can bind to the Chx10 enhancer and repress Chx10 enhancer activity. These results suggest that Blimp1 plays a crucial role in photoreceptor development by repressing genes involved in bipolar cell fate specification and retinal cell proliferation in differentiating photoreceptor precursors.
Subject(s)
Cell Differentiation/physiology , Homeodomain Proteins/metabolism , Photoreceptor Cells, Vertebrate/physiology , Retina/growth & development , Stem Cells/physiology , Transcription Factors/metabolism , Animals , Cell Count , Cell Lineage , Electroporation , Gene Expression Regulation, Developmental , Gene Targeting , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Models, Neurological , Oligonucleotide Array Sequence Analysis , Positive Regulatory Domain I-Binding Factor 1 , RNA, Messenger/metabolism , Retina/embryology , Retina/physiology , Retinal Bipolar Cells/physiology , Retinal Cone Photoreceptor Cells/physiology , Transcription Factors/deficiency , Transcription Factors/geneticsABSTRACT
PURPOSE: To identify human transient receptor potential cation channel, subfamily M, member 1 (TRPM1) gene mutations in patients with congenital stationary night blindness (CSNB). METHODS: We analyzed four different Japanese patients with complete CSNB in whom previous molecular examination revealed no mutation in either nyctalopin (NYX) or glutamate receptor, metabotropic 6 (GRM6). The ophthalmologic examination included best-corrected visual acuity, refraction, biomicroscopy, ophthalmoscopy, fundus photography, Goldmann kinetic perimetry, color vision tests, and electroretinography (ERG). Exons 2 through 27 and the exon-intron junction regions of human TRPM1 were sequenced. RESULTS: Five different mutations in human TRPM1 were identified. Mutations were present in three unrelated patients with complete CSNB. All three patients were compound heterozygotes. Fundus examination revealed no abnormalities other than myopic changes, and the single bright-flash, mixed rod-cone ERG showed a "negative-type" configuration with a reduced normal a-wave and a significantly reduced b-wave amplitude. Our biochemical and cell biologic analyses suggest that the two identified IVS mutations lead to abnormal TRPM1 protein production, and imply that the two identified missense mutations lead to the mislocalization of the TRPM1 protein in bipolar cells (BCs). CONCLUSIONS: Human TRPM1 mutations are associated with the complete form of CSNB in Japanese patients, suggesting that TRPM1 plays an essential role in mediating the photoresponse in ON BCs in humans as well as in mice.