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Key Points Vision begins in the retina at the rod and cone photoreceptors, which are sensory neurons with specialized visual pigments for capturing light quanta. Most mammals have one type of rod and two types of cone (M and S) photoreceptors that confer dichromatic vision. Humans have one type of rod and three cone subtypes that confer trichromacy. All retinal neurons, including photoreceptors, are generated from multipotent progenitor cells through a step-wise process that increasingly restricts lineage choices and commits cells to a particular fate. The balanced actions of six key transcription factors (the paired-type homeodomain transcription factor OTX2, cone–rod homeobox protein CRX, neural retina leucine zipper protein (NRL), photoreceptor-specific nuclear receptor (NR2E3), nuclear receptor RORβ and thyroid hormone receptor β2 (TRβ2)) are crucial as retinal progenitors commit to a rod or cone lineage. We propose a 'transcriptional dominance' model of photoreceptor fate determination that includes three fundamental attributes: that all photoreceptor types originate from a common postmitotic photoreceptor precursor that has the potential to form rods or any cone type; that such precursors differentiate by 'default' as S cones unless additional signals promote acquisition of a rod or M cone identity; and that the particular fate acquired by a precursor results from a contest among specific transcription factors. We predict that transcriptional signals control two key points during fate specification: first, the decision to form a rod or a cone — dictated by NRL and its downstream target NR2E3; second, the decision for a cone to acquire an S cone or M cone identity, largely determined by thyroid hormone receptor TRβ2. OTX2 and RORβ act upstream of NRL, whereas CRX induces both rod and cone genes during photoreceptor maturation. Abnormalities, dysfunction and/or death of photoreceptors constitute the primary cause of visual impairment or blindness in most retinal diseases. Many retinal disease genes are targets of the differentiation factors NRL, CRX and NR2E3, which also maintain rod homeostasis. Studies of transcriptional regulation underlying photoreceptor development should further advance gene- and small-molecule-based interventions and cell-based transplantation therapies for retinal degenerative diseases. All retinal neurons are generated from multipotent progenitor cells through a step-wise process that increasingly restricts lineage choices. Swaroop and colleagues discuss our current understanding of the transcription factors and gene-regulatory networks involved in photoreceptor subtype specification and photoreceptor development. In the developing vertebrate retina, diverse neuronal subtypes originate from multipotent progenitors in a conserved order and are integrated into an intricate laminated architecture. Recent progress in mammalian photoreceptor development has identified a complex relationship between six key transcription-regulatory factors (RORβ, OTX2, NRL, CRX, NR2E3 and TRβ2) that determine rod versus M cone or S cone cell fate. We propose a step-wise 'transcriptional dominance' model of photoreceptor cell fate determination, with the S cone representing the default state of a generic photoreceptor precursor. Elucidation of gene-regulatory networks that dictate photoreceptor genesis and homeostasis will have wider implications for understanding the development of nervous system function and for the treatment of neurodegenerative diseases.

In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa. Retinitis pigmentosa is mainly caused by mutations that initially affect survival of rod photoreceptors, leading to secondary loss of cones. Here the authors use gene editing to prevent rod degeneration, leading to survival of cones and improved vision in mice.

Chromatin organization and enhancer-promoter contacts establish unique spatiotemporal gene expression patterns in distinct cell types. Non-coding genetic variants can influence cellular phenotypes by modifying higher-order transcriptional hubs and consequently gene expression. To elucidate genomic regulation in human retina, we mapped chromatin contacts at high resolution and integrated with super-enhancers (SEs), histone marks, binding of CTCF and select transcription factors. We show that topologically associated domains (TADs) with central SEs exhibit stronger insulation and augmented contact with retinal genes relative to TADs with edge SEs. Merging genome-wide expression quantitative trait loci (eQTLs) with topology map reveals physical links between 100 eQTLs and corresponding eGenes associated with retinal neurodegeneration. Additionally, we uncover candidate genes for susceptibility variants linked to age-related macular degeneration and glaucoma. Our study of high-resolution genomic architecture of human retina provides insights into genetic control of tissue-specific functions, suggests paradigms for missing heritability, and enables the dissection of common blinding disease phenotypes. The genome-wide high-resolution chromatin contact of the human retina identifies genetic control of cell-type specific gene expression pattern, missing heritability in retinopathies, and candidate genes/variants for diseases including AMD and glaucoma.

Both genetic and environmental factors influence the etiology of age-related macular degeneration (AMD), a leading cause of blindness. AMD severity is primarily measured by images of the fundus of the retina and recently developed machine learning methods can successfully predict AMD progression using image data. However, none of these methods have used both genetic and image data for predicting AMD progression. Here we used both genotypes and fundus images to predict whether an eye had progressed to late AMD with a modified deep convolutional neural network. In total, we used 31,262 fundus images and 52 AMD-associated genetic variants from 1,351 subjects from the Age-Related Eye Disease Study, which provided disease severity phenotypes and fundus images available at baseline and follow-up visits over a period of 12 years. Our results showed that fundus images coupled with genotypes could predict late AMD progression with an averaged area-under-the-curve value of 0.85 (95% confidence interval 0.83–0.86). The results using fundus images alone showed an averaged area under the receiver operating characteristic curve value of 0.81 (95% confidence interval 0.80–0.83). We implemented our model in a cloud-based application for individual risk assessment. Age-related macular degeneration is a serious eye disease which should be detected as early as possible. Using both fundus images and genetic information, a deep neural network is able to detect the severity of the disease and predict its progression seven years into the future.

Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases.

Inherited retinal degenerations (IRDs) constitute a group of clinically and genetically diverse vision-impairing disorders. Retinitis pigmentosa (RP), the most common form of IRD, is characterized by gradual dysfunction and degeneration of rod photoreceptors, followed by the loss of cone photoreceptors. Recently, we identified reserpine as a lead molecule for maintaining rod survival in mouse and human retinal organoids as well as in the rd16 mouse, which phenocopy Leber congenital amaurosis caused by mutations in the cilia-centrosomal gene CEP290 (Chen et al., 2023). Here, we show the therapeutic potential of reserpine in a rhodopsin P23H rat model of autosomal dominant RP. At postnatal day (P) 68, when males and females are analyzed together, the reserpine-treated rats exhibit higher rod-derived scotopic b-wave amplitudes compared to the controls with little or no change in scotopic a-wave or cone-derived photopic b-wave. Interestingly, the reserpine-treated female rats display enhanced scotopic a- and b-waves and photopic b-wave responses at P68, along with a better contrast threshold and increased outer nuclear layer thickness. The female rats demonstrate better preservation of both rod and cone photoreceptors following reserpine treatment. Retinal transcriptome analysis reveals sex-specific responses to reserpine, with significant upregulation of phototransduction genes and proteostasis-related pathways, and notably, genes associated with stress response. This study builds upon our previously reported results reaffirming the potential of reserpine for gene-agnostic treatment of IRDs and emphasizes the importance of biological sex in retinal disease research and therapy development.

RNA-binding proteins (RBPs) perform diverse functions including the regulation of chromatin dynamics and the coupling of transcription with RNA processing. However, our understanding of their actions in mammalian neurons remains limited. Using affinity purification, yeast-two-hybrid and proximity ligation assays, we identified interactions of multiple RBPs with neural retina leucine (NRL) zipper, a Maf-family transcription factor critical for retinal rod photoreceptor development and function. In addition to splicing, many NRL-interacting RBPs are associated with R-loops, which form during transcription and increase during photoreceptor maturation. Focusing on DHX9 RNA helicase, we demonstrate that its expression is modulated by NRL and that the NRL–DHX9 interaction is positively influenced by R-loops. ssDRIP-Seq analysis reveals both stranded and unstranded R-loops at distinct genomic elements, characterized by active and inactive epigenetic signatures and enriched at neuronal genes. NRL binds to both types of R-loops, suggesting an epigenetically independent function. Our findings suggest additional functions of NRL during transcription and highlight complex interactions among transcription factors, RBPs, and R-loops in regulating photoreceptor gene expression in the mammalian retina.

Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290 . We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration. Leber congenital amaurosis (LCA) is an inherited disease that affects the eyes and causes sight loss in early childhood, which generally gets worse over time. Individuals with this condition have genetic mutations that result in the death of light-sensitive cells, known as photoreceptors, in a region called the retina at the back of the eye. Patients carrying a genetic change in the gene CEP290 account for 20-25% of all LCA. At present, treatment options are only available for a limited number of patients with LCA. One option is to use small molecules as drugs that may target or bypass the faulty processes within the eye to help the photoreceptors survive in many different forms of LCA and other retinal diseases. However, over 90% of new drug candidates fail the first phase of clinical trials for human diseases. This in part due to the candidates having been developed using cell cultures or animal models that do not faithfully reflect how the human body works. Recent advances in cell and developmental biology are now enabling researchers to use stem cells derived from humans to grow retina tissues in a dish in the laboratory. These tissues, known as retinal organoids, behave in a more similar way to retinas in human eyes than those of traditional animal models. However, the methods for making and maintaining human retinal organoids are time-consuming and labor-intensive, which has so far limited their use in the search for new therapies. To address this challenge, Chen et al. developed a large-scale approach to grow retinal organoids from rd16 mutant mice stem cells (which are a good model for LCA caused by mutations to CEP290) and used the photoreceptors from these organoids to screen over 6,000 existing drugs for their ability to promote the survival of photoreceptors. The experiments found that the drug reserpine, which was previously approved to treat high blood pressure, also helped photoreceptors to survive in the diseased organoids. Reserpine also had a similar effect in retinal organoids derived from human patients with LCA and in the rd16 mice themselves. Further experiments suggest that reserpine may help patients with LCA by partially restoring a process by which the body destroys and recycles old and damaged proteins in the cells. The next steps following on from this work will be to perform further tests to demonstrate that this use of reserpine is safe to enter clinical trials as a treatment for LCA and other similar eye diseases.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor-specific chaperone that stabilizes the effector enzyme of phototransduction, cGMP phosphodiesterase 6 (PDE6). Mutations in the AIPL1 gene cause a severe inherited retinal dystrophy, Leber congenital amaurosis type 4 (LCA4), that manifests as the loss of vision during the first year of life. In this study, we generated three-dimensional (3D) retinal organoids (ROs) from human induced pluripotent stem cells (hiPSCs) derived from an LCA4 patient carrying a Cys89Arg mutation in AIPL1 . This study aimed to (i) explore whether the patient hiPSC-derived ROs recapitulate LCA4 disease phenotype, and (ii) generate a clinically relevant resource to investigate the molecular mechanism of disease and safely test novel therapies for LCA4 in vitro . We demonstrate reduced levels of the mutant AIPL1 and PDE6 proteins in patient organoids, corroborating the findings in animal models; however, patient-derived organoids maintained retinal cell cytoarchitecture despite significantly reduced levels of AIPL1.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype–environment interaction in retina. Here, the authors perform genome-wide mapping of DNA methylation and expression quantitative trait loci, revealing associations among genotype, epigenome and transcriptome, uncovering genes and gene-environment interactions contributing to age-related macular degeneration (AMD).