Search

Description, FGF and Wnt signaling controls the phase transition of the ocular fate., The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE), and the ciliary margin (CM). By single-cell analysis, we showed that fibroblast growth factor (FGF) signaling regulates the CM in its stem cell–like property of self-renewal, differentiation, and survival, which is balanced by an evolutionarily conserved Wnt signaling gradient. FGF promotes Wnt signaling in the CM by stabilizing β-catenin in a GSK3β-independent manner. While Wnt signaling converts the NR to either the CM or the RPE depending on FGF signaling, FGF transforms the RPE to the NR or CM dependent on Wnt activity. The default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in human retinal organoid. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE) and the ciliary margin (CM). By single cell analysis, we showed that a gradient of FGF signaling regulates demarcation and subdivision of the CM and controls its stem cell-like property of self-renewal, differentiation and survival. This regulation by FGF is balanced by an evolutionarily conserved Wnt signaling gradient induced by the lens ectoderm and the periocular mesenchyme, which specifies the CM and the distal RPE. These two morphogen gradients converge in the CM where FGF signaling promotes Wnt signaling by stabilizing β-catenin in a GSK3β-independent manner. We further showed that activation of Wnt signaling converts the NR to either the CM or the RPE depending on the level of FGF signaling. Conversely, activation of FGF transforms the RPE to the NR or CM dependent on Wnt activity. We demonstrated that the default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in retinal organoid culture of human iPS cells. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.

Macular degeneration (MD) is characterized by the progressive deterioration of the macula and represents one of the most prevalent causes of blindness worldwide. Abnormal intracellular accumulation of lipid droplets and pericellular deposits of lipid-rich material in the retinal pigment epithelium (RPE) called drusen are clinical hallmarks of different forms of MD including Doyne honeycomb retinal dystrophy (DHRD) and age-related MD (AMD). However, the appropriate molecular therapeutic target underlying these disorder phenotypes remains elusive. Here, we address this knowledge gap by comparing the proteomic profiles of induced pluripotent stem cell (iPSC)-derived RPEs (iRPE) from individuals with DHRD and their isogenic controls. Our analysis and follow-up studies elucidated the mechanism of lipid accumulation in DHRD iRPE cells. Specifically, we detected significant downregulation of carboxylesterase 1 (CES1), an enzyme that converts cholesteryl ester to free cholesterol, an indispensable process in cholesterol export. CES1 knockdown or overexpression of EFEMP1(R345W), a variant of EGF-containing fibulin extracellular matrix protein 1 that is associated with DHRD and attenuated cholesterol efflux and led to lipid droplet accumulation. In iRPE cells, we also found that EFEMP1(R345W) has a hyper-inhibitory effect on epidermal growth factor receptor (EGFR) signaling when compared to EFEMP1(WT) and may suppress CES1 expression via the downregulation of transcription factor SP1. Taken together, these results highlight the homeostatic role of cholesterol efflux in iRPE cells and identify CES1 as a mediator of cholesterol efflux in MD.

Recent advances have driven the development of stem cell-derived, self-organizing, three-dimensional miniature organs, termed organoids, which mimic different eye tissues including the retina, cornea, and lens. Organoids and engineered microfluidic organ-on-chips (organ chips) are transformative technologies that show promise in simulating the architectural and functional complexity of native organs. Accordingly, they enable exploration of facets of human disease and development not accurately recapitulated by animal models. Together, these technologies will increase our understanding of the basic physiology of different eye structures, enable us to interrogate unknown aspects of ophthalmic disease pathogenesis, and serve as clinically-relevant surrogates for the evaluation of ocular therapeutics. Both the burden and prevalence of monogenic and multifactorial ophthalmic diseases, which can cause visual impairment or blindness, in the human population warrants a paradigm shift towards organoids and organ chips that can provide sensitive, quantitative, and scalable phenotypic assays. In this article, we review the current situation of organoids and organ chips in ophthalmology and discuss how they can be leveraged for translational applications.

Retinitis pigmentosa (RP) is a genetically heterogenous group of eye diseases in which initial degeneration of rods triggers secondary degeneration of cones, leading to significant loss of daylight, color, and high-acuity vision. Gene complementation with adeno-associated viral (AAV) vectors is one strategy to treat RP. Its implementation faces substantial challenges, however; for example, the tremendous number of loci with causal mutations. Gene therapy targeting secondary cone degeneration is an alternative approach that could provide a much-needed generic treatment for many patients with RP. Here, we show that microglia are required for the upregulation of potentially neurotoxic inflammatory factors during cone degeneration in RP, creating conditions that might contribute to cone dysfunction and death. To ameliorate the effects of such factors, we used AAV vectors to express isoforms of the antiinflammatory cytokine transforming growth factor beta (TGF-β). AAV-mediated delivery of TGF-β1 rescued degenerating cones in 3 mouse models of RP carrying different pathogenic mutations. Treatment with TGF-β1 protected vision, as measured by 2 behavioral assays, and could be pharmacologically disrupted by either depleting microglia or blocking the TGF-β receptors. Our results suggest that TGF-β1 may be broadly beneficial for patients with cone degeneration, and potentially other forms of neurodegeneration, through a pathway dependent upon microglia.

Mutations in the PTEN-induced kinase 1 (PINK1) and Parkin RBR E3 ubiquitin-protein ligase (PARKIN) genes are associated with familial forms of Parkinson’s disease (PD). PINK1, a protein kinase, and PARKIN, an E3 ubiquitin ligase, control the specific elimination of dysfunctional or superfluous mitochondria, thus fine-tuning mitochondrial network and preserving energy metabolism. PINK1 regulates PARKIN translocation in impaired mitochondria and drives their removal via selective autophagy, a process known as mitophagy. As knowledge obtained using different PINK1 and PARKIN transgenic animal models is being gathered, growing evidence supports the contribution of mitophagy impairment to several human pathologies, including PD and Alzheimer’s diseases (AD). Therefore, therapeutic interventions aiming to modulate PINK1/PARKIN signalling might have the potential to treat these diseases. In this review, we will start by discussing how the interplay of PINK1 and PARKIN signalling helps mediate mitochondrial physiology. We will continue by debating the role of mitochondrial dysfunction in disorders such as amyotrophic lateral sclerosis, Alzheimer’s, Huntington’s and Parkinson’s diseases, as well as eye diseases such as age-related macular degeneration and glaucoma, and the causative factors leading to PINK1/PARKIN-mediated neurodegeneration and neuroinflammation. Finally, we will discuss PINK1/PARKIN gene augmentation possibilities with a particular focus on AD, PD and glaucoma.

Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form “functional” CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.

The Crumbs complex has prominent roles in the control of apical cell polarity, in the coupling of cell density sensing to downstream cell signaling pathways, and in regulating junctional structures and cell adhesion. The Crumbs complex acts as a conductor orchestrating multiple downstream signaling pathways in epithelial and neuronal tissue development. These pathways lead to the regulation of cell size, cell fate, cell self-renewal, proliferation, differentiation, migration, mitosis, and apoptosis. In retinogenesis, these are all pivotal processes with important roles for the Crumbs complex to maintain proper spatiotemporal cell processes. Loss of Crumbs function in the retina results in loss of the stratified appearance resulting in retinal degeneration and loss of visual function. In this review, we begin by discussing the physiology of vision. We continue by outlining the processes of retinogenesis and how well this is recapitulated between the human fetal retina and human embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-derived retinal organoids. Additionally, we discuss the functionality of in utero and preterm human fetal retina and the current level of functionality as detected in human stem cell-derived organoids. We discuss the roles of apical-basal cell polarity in retinogenesis with a focus on Leber congenital amaurosis which leads to blindness shortly after birth. Finally, we discuss Crumbs homolog (CRB)-based gene augmentation.

Summary: Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments. : Wijnholds and colleagues show that the key Crumbs complex members, CRB1 and CRB2, are recapitulated between human fetal retina and iPSC-derived retinal organoids. CRB2 is expressed earlier than CRB1 in fetal and iPSC-derived human retina. CRB1-RP patient iPSC-derived retinas show a morphological phenotype. In addition, they show that AAV5 infects photoreceptors and Müller glial cells in adult and iPSC-derived human retina. Keywords: CRB1, CRB2, organoids, cell polarity, Müller glial cells, photoreceptors, adeno-associated virus, human induced pluripotent stem cells, retina, CRB1 patients

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells, while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.

Tissue-specific progenitor cells form a potential population for cell-based transplantation therapy. Müller glial cells are suggested to contain, or represent the progenitor pool in the retina and can dedifferentiate and regenerate neuronal cells in fish and amphibians. However, the Müller glial cells of mammals have a more limited regenerative capacity in response to injury and disease. Transplantation experiments with Müller glial cells have shown limited success. In this study we transplant NTPDase2- positive Müller glial cells labelled with CellTracker Green or NTPDase2-positive Müller glial cells sorted from transgenic tdTomato mouse retina. We found that NTPDase2-sorted Müller glial cells do not have the intrinsic capacity to integrate into the mouse retina upon subretinal injection under the conditions used. Interestingly, we also found that CellTracker Green labelled NTPDase2-sorted Müller glial cells release CellTracker Green conjugates. NTPDase2-sorted negative cells do not. Injection of CellTracker Green alone did not lead to labelling of any retinal cell types. However, when NTPDase2-sorted cells are injected into the mouse retina these conjugates are preferentially taken up by Starburst amacrine cells. This highlights a potentially novel import transporter on the plasma membrane of Starburst amacrine cells.

Müller glial cells are large neuroglial cells that extend throughout the entire retina, they function to maintain homeostasis and retinal integrity. In teleost fish, in response to retinal damage, Müller cells can re-enter the cell cycle, dedifferentiate and regenerate neuronal cells. Therefore, increasing our knowledge about these cells might open new avenues to regenerative medicine. Here, we present a reliable method to isolate Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) positive Müller glial cells by FluorescenceActivated Cell Sorting (FACS). For this, cell suspensions were made from immature and mature mouse neural retinae by incubation in collagenase I and DNase I and repeated trituration. We confirmed by flow cytometry that glutamine synthetase positive Müller glial cells were also NTPDase2 positive. Thereafter, the surface marker NTPDase2 was used to sort by FACS a population enriched for Müller glial cells. NTPDase2 successfully marks an enriched population of glutamine synthetase positive cells. This is a reproducible method to sort Müller glial cells from mouse retina. The FACS isolated Müller glial cells can be used to study their properties in retinal degeneration, regeneration and cell function.

The mammalian apical-basal determinant Crumbs homolog-1 (CRB1) plays a crucial role in retinal structure and function by the maintenance of adherens junctions between photoreceptors and Muller glial cells. Patients with mutations in the CRB1 gene develop retinal dystrophies, including early-onset retinitis pigmentosa and Leber congenital amaurosis. Previously, we showed that Crb1 knockout mice developed a slow-progressing retinal phenotype at foci in the inferior retina, although specific ablation of Crb2 in immature photoreceptors leads to an early-onset phenotype throughout the retina. Here, we conditionally disrupted one or both alleles of Crb2 in immature photoreceptors, on a genetic background lacking Crb1, and studied the retinal dystrophies thereof. Our data showed that disruption of one allele of Crb2 in immature photoreceptors caused a substantial aggravation of the Crb1 phenotype in the entire inferior retina. The photoreceptor layer showed early-onset progressive thinning limited to the inferior retina, although the superior retina maintained intact. Surprisingly, disruption of both alleles of Crb2 in immature photoreceptors further aggravated the phenotype. Throughout the retina, photoreceptor synapses were disrupted and photoreceptor nuclei intermingled with nuclei of the inner nuclear layer. In the superior retina, the ganglion cell layer appeared thicker because of ectopic nuclei of photoreceptors. In conclusion, the data suggest that CRB2 is required to maintain retinal progenitor and photoreceptor cell adhesion and prevent photoreceptor ingression into the immature inner retina. We hypothesize, from these animal models, that decreased levels of CRB2 in immature photoreceptors adjust retinitis pigmentosa because of the loss of CRB1 into Leber congenital amaurosis phenotype.

In vitro retinal organoid modeling from human pluripotent stem cells is becoming more common place in many ophthalmic laboratories worldwide. These organoids mimic human retinogenesis through formation of organized layered retinal structures that display markers for typical retinal cell types. Pivotally these humanized retinal models provide a stepping stone to the clinic as therapeutic tools and are expected to provide a promising alternative to current animal models. Thus pluripotent stem cell based healthy as well as diseased human retinal organoids are attractive for use in drug potency assays and gene augmentation therapeutics. Here we outline an established protocol for generation of these retinal organoids and how they can be used in conjunction with adeno-associated virus vectors for transgene expression assays.

Once considered science fiction, gene therapy is rapidly becoming scientific reality, targeting a growing number of the approximately 250 genes linked to hereditary retinal disorders such as retinitis pigmentosa and Leber's congenital amaurosis. Powerful new technologies have emerged, leading to the development of humanized models for testing and screening these therapies, bringing us closer to the goal of personalized medicine. These tools include the ability to differentiate human induced pluripotent stem cells (iPSCs) to create a "retina-in-a-dish" model and the self-formed ectodermal autonomous multi-zone, which can mimic whole eye development. In addition, highly specific gene-editing tools are now available, including the CRISPR/Cas9 system and the recently developed homology-independent targeted integration approach, which allows gene editing in non-dividing cells. Variants in the CRB1 gene have long been associated with retinopathies, and more recently the CRB2 gene has also been shown to have possible clinical relevance with respect to retinopathies. In this review, we discuss the role of the CRB protein complex in patients with retinopathy. In addition, we discuss new opportunities provided by stem cells and gene-editing tools, and we provide insight into how the retinal therapeutic pipeline can be improved. Finally, we discuss the current state of adeno-associated virus-mediated gene therapy and how it can be applied to treat retinopathies associated with mutations in CRB1.

International audience; Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in Crb1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in Crb1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.

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., Author Summary Mutations in the human CRB1 gene lead to one of the most severe forms of retinal dystrophies, called Leber congenital amaurosis. Here, we report that ablation of CRB1 and the second family member CRB2 are crucial for proper retinal development. These mice display severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. The thickening of the retina is due to increased cell proliferation during late retinal development leading to an increased number of late-born retinal cells. We describe in these CRB1 Leber congenital amaurosis mouse models the molecular and cellular events involving CRB proteins during the development of the retina.

The writer argues for the removal of Rule 47(21) from Prison Rules. It is contrary to the principle o/nulla poena sine lege and also to that of legal certainty. It is unnecessary in that acts constituting offences against good order and discipline may already be punished under other paragraphs of the Rule. An opportunity to review the question will arise with the drafting of new primary and subordinate legislation.

The writer considers the Home Office responses to Lord Justice Woolf's report on the 1990 prison disturbances insofar as they affect one aspect of prison lift, nameb, the adjudication. He fears that, with the removal from boards of visitors of their adjudicatosy function, elements of natural justice have been placed in jeopardy. It may not be possible for a governor to come to a hearing without bias or without hints that the governor is a judge in his or her own cause not being sufficiently independent. Lord Bridge's dictum in ex parte Leech (1988) to the effect that the governor adjudicates as something other than a servant of the Secretary of State is diyficulty to sustain in practice. The adjudicating governor may be in breach of Article 6 of the European Convention on Human Rights. The writer offers a way forward whereby discipline may be maintained in prisons through the application of a redrafted Prison Rule 47 and a revision of the present procedure.