Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single-cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCβ, secretagogin) was species-specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.
Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.
Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.
Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.
- MeSH
- buněčná diferenciace genetika MeSH
- buněčné linie MeSH
- editace genu * MeSH
- fagocytóza * MeSH
- indukované pluripotentní kmenové buňky patologie ultrastruktura MeSH
- lidé MeSH
- mutace genetika MeSH
- regulace genové exprese MeSH
- retinální pigmentový epitel patologie ultrastruktura MeSH
- retinopathia pigmentosa genetika patologie MeSH
- tyrosinkinasa c-Mer genetika metabolismus MeSH
- zevní segment fotoreceptoru sítnice metabolismus patologie ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of either Oc1 or Oc2 gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion of Oc1 and Oc2 leads to a complete loss of HCs. Here we study the genetic redundancy between Oc1 and Oc2 paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication.
- MeSH
- alely MeSH
- amakrinní buňky metabolismus patologie MeSH
- bipolární buňky sítnice metabolismus patologie MeSH
- čípky retiny metabolismus patologie MeSH
- ependymální buňky metabolismus patologie MeSH
- genetické lokusy MeSH
- genotyp MeSH
- hepatocytární jaderný faktor 6 genetika metabolismus MeSH
- homeodoménové proteiny genetika metabolismus MeSH
- myši transgenní MeSH
- myši MeSH
- oči růst a vývoj patologie MeSH
- retina cytologie patologie fyziologie MeSH
- retinální gangliové buňky cytologie metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: The aim of this work was to study the association of serum ionized calcium with retinal photoreceptor apoptosis on spectral domain optical coherence tomography (SD-OCT) in diabetic retinopathy (DR). METHODS: Sixty consecutive cases with Type 2 diabetes mellitus were categorized into three groups: no diabetic retinopathy; non-proliferative DR; proliferative DR. The eye with more severe form of the disease was considered. Twenty healthy controls were also included. Best corrected visual acuity (BCVA) was measured on logMAR scale. Retinal photoreceptor apoptosis was defined as disruption of retinal photoreceptor ellipsoid zone (EZ). Ellipsoid zone disruption was assessed using SD-OCT. Serum levels of total and ionized calcium were measured using standard protocol. RESULTS: EZ disruption was found to be positively associated with serum total calcium and ionized calcium. Also, EZ disruption was found to be positively associated with logMAR BCVA. CONCLUSION: Increased serum ionized calcium induces retinal photoreceptor apoptosis resulting in increased EZ disruption in DR.
- MeSH
- apoptóza * MeSH
- diabetická retinopatie krev patologie MeSH
- fotoreceptory obratlovců patologie MeSH
- homeostáza MeSH
- lidé středního věku MeSH
- lidé MeSH
- optická koherentní tomografie MeSH
- vápník krev MeSH
- zraková ostrost MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Three-dimensional (3D) retinal organoids, in vitro tissue structures derived from self-organizing cultures of differentiating human embryonic stem cells or induced pluripotent stem cells, could recapitulate some aspects of the cytoarchitectural structure and function of the retina in vivo. 3D retinal organoids display huge potential for the investigation of the pathogenesis of monogenic hereditary eye diseases that are related to the malfunction or degeneration of photoreceptors or retinal ganglion cells by providing an effective in vitro tool with multiple applications. In combination with recent genome editing tools, 3D retinal organoids could also represent a reliable and renewable source of transplantable cells for personalized therapies. In this review, we describe the recent advances in human pluripotent stem cells-derived retinal organoids, determination of their histoarchitecture, complexity, and maturity. We also discuss their application as a means to decipher the pathogenesis of retinal diseases, as well as the main drawbacks and challenges. Stem Cells 2019;37:1496-1504.
- MeSH
- editace genu metody MeSH
- indukované pluripotentní kmenové buňky cytologie transplantace MeSH
- nemoci retiny patologie MeSH
- organoidy cytologie ultrastruktura MeSH
- retina patologie ultrastruktura MeSH
- tyčinky retiny cytologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The Crb1 and 2 (Crumbs homolog 1 & 2) genes encode large, single-pass transmembrane proteins essential for the apicobasal polarity and adhesion of epithelial cells. Crb1 mutations cause degenerative retinal diseases in humans, including Leber congenital amaurosis type 8 (LCA8) and retinitis pigmentosa type 12 (RP12). In LCA8, impaired photoreceptor development and/or survival is thought to cause blindness during early infancy, whereas, in RP12, progressive photoreceptor degeneration damages peripheral vision later in life. There are multiple animal models of RP12 pathology, but no experimental model of LCA8 recapitulates the full spectrum of its pathological features. To generate a mouse model of LCA8 and identify the functions of Crb1/2 in developing ocular tissues, we used an mRx-Cre driver to generate allelic combinations that enabled conditional gene ablation from the optic vesicle stage. In this series only Crb1/2 double knockout (dKO) mice exhibited characteristics of human LCA8 disease: locally thickened retina with spots devoid of cells, aberrant positioning of retinal cells, severely disrupted lamination, and depigmented retinal-pigmented epithelium. Retinal defects antedated E12.5, which is far earlier than the stage at which photoreceptor cells mainly differentiate. Most remarkably, Crb1/Crb2 dKO showed a severely attenuated electroretinogram at the eye opening stage. These results suggest that human LCA8 can be modeled in the mouse by simultaneously ablating Crb1/2 from the beginning of eye development. Importantly, they also indicate that LCA8 is caused by malfunction of retinal progenitor cells during early ocular development rather than by defective photoreceptor-Muller glial interaction, a mechanism proposed for RP12.
- MeSH
- delece genu * MeSH
- dospělí MeSH
- elektroretinografie MeSH
- fotoreceptory obratlovců metabolismus MeSH
- Leberova kongenitální amauróza genetika patologie MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- modely nemocí na zvířatech MeSH
- mutace genetika MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- oči metabolismus patologie MeSH
- orgánová specificita MeSH
- pigmentace MeSH
- proteiny nervové tkáně genetika metabolismus MeSH
- retinální pigmentový epitel metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
