RDH12 allows cone photoreceptors to regenerate opsin visual pigments from a chromophore precursor to escape competition with rods.

Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.

Cone photoreceptor differentiation regulated by thyroid hormone transporter MCT8 in the retinal pigment epithelium.

The key role of a thyroid hormone receptor in determining the maturation and diversity of cone photoreceptors reflects a profound influence of endocrine signaling on the cells that mediate color vision. However, the route by which hormone reaches cones remains enigmatic as cones reside in the retinal photoreceptor layer, shielded by the blood-retina barrier. Using genetic approaches, we report that cone differentiation is regulated by a membrane transporter for thyroid hormone, MCT8 (SLC16A2), in the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier. Mct8-deficient mice display hypothyroid-like cone gene expression and compromised electroretinogram responses. Mammalian color vision is typically facilitated by cone types that detect medium-long (M) and short (S) wavelengths of light but Mct8-deficient mice have a partial shift of M to S cone identity, resembling the phenotype of thyroid hormone receptor deficiency. RPE-specific ablation of Mct8 results in similar shifts in cone identity and hypothyroid-like gene expression whereas reexpression of MCT8 in the RPE in Mct8-deficient mice partly restores M cone identity, consistent with paracrine-like control of thyroid hormone signaling by the RPE. Our findings suggest that in addition to transport of essential solutes and homeostatic support for photoreceptors, the RPE regulates the thyroid hormone signal that promotes cone-mediated vision.

Insights into the evolution of photoreceptor oil droplets in frogs and toads.

Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

Ablation of Fatty Acid Transport Protein-4 Enhances Cone Survival, M-cone Vision, and Synthesis of Cone-Tropic 9-cis-Retinal in rd12 Mouse Model of Leber Congenital Amaurosis.

The canonical visual cycle employing RPE65 as the retinoid isomerase regenerates 11-cis-retinal to support both rod- and cone-mediated vision. Mutations of RPE65 are associated with Leber congenital amaurosis that results in rod and cone photoreceptor degeneration and vision loss of affected patients at an early age. Dark-reared Rpe65-/- mouse has been known to form isorhodopsin that employs 9-cis-retinal as the photosensitive chromophore. The mechanism regulating 9-cis-retinal synthesis and the role of the endogenous 9-cis-retinal in cone survival and function remain largely unknown. In this study, we found that ablation of fatty acid transport protein-4 (FATP4), a negative regulator of 11-cis-retinol synthesis catalyzed by RPE65, increased the formation of 9-cis-retinal, but not 11-cis-retinal, in a light-independent mechanism in both sexes of RPE65-null rd12 mice. Both rd12 and rd12;Fatp4-/- mice contained a massive amount of all-trans-retinyl esters in the eyes, exhibiting comparable scotopic vision and rod degeneration. However, expression levels of M- and S-opsins as well as numbers of M- and S-cones surviving in the superior retinas of rd12;Fatp4-/ - mice were at least twofold greater than those in age-matched rd12 mice. Moreover, FATP4 deficiency significantly shortened photopic b-wave implicit time, improved M-cone visual function, and substantially deaccelerated the progression of cone degeneration in rd12 mice, whereas FATP4 deficiency in mice with wild-type Rpe65 alleles neither induced 9-cis-retinal formation nor influenced cone survival and function. These results identify FATP4 as a new regulator of synthesis of 9-cis-retinal, which is a "cone-tropic" chromophore supporting cone survival and function in the retinas with defective RPE65.

Evolutionary and developmental specialization of foveal cell types in the marmoset.

In primates, high-acuity vision is mediated by the fovea, a small specialized central region of the retina. The fovea, unique to the anthropoid lineage among mammals, undergoes notable neuronal morphological changes during postnatal maturation. However, the extent of cellular similarity across anthropoid foveas and the molecular underpinnings of foveal maturation remain unclear. Here, we used high-throughput single-cell RNA sequencing to profile retinal cells of the common marmoset (Callithrix jacchus), an early divergent in anthropoid evolution from humans, apes, and macaques. We generated atlases of the marmoset fovea and peripheral retina for both neonates and adults. Our comparative analysis revealed that marmosets share almost all their foveal types with both humans and macaques, highlighting a conserved cellular structure among primate foveas. Furthermore, by tracing the developmental trajectory of cell types in the foveal and peripheral retina, we found distinct maturation paths for each. In-depth analysis of gene expression differences demonstrated that cone photoreceptors and Müller glia (MG), among others, show the greatest molecular divergence between these two regions. Utilizing single-cell ATAC-seq and gene-regulatory network inference, we uncovered distinct transcriptional regulations differentiating foveal cones from their peripheral counterparts. Further analysis of predicted ligand-receptor interactions suggested a potential role for MG in supporting the maturation of foveal cones. Together, these results provide valuable insights into foveal development, structure, and evolution.

Structural changes in laser-induced retinopathy examined by adaptive optics optical coherence tomography.

Purpose: We report a case of laser-induced retinopathy that posed diagnostic challenges with conventional spectral domain optical coherence tomography (SD-OCT), but was successfully diagnosed using adaptive optics-optical coherence tomography (AO-OCT). Observations: A 27-year-old man with a history of occupational laser device use presented with central scotoma and visual disturbances in the right eye. Conventional SD-OCT only revealed decreased reflectivity in parts of the foveal ellipsoidal zone band. However, other multimodal observations indicated damage to the retinal pigment epithelium (RPE) and choriocapillaris. Additionally, a well-defined circular, dark lesion, approximately 80 µm in diameter, was identified in the outer retina. AO-OCT demonstrated the absence of the RPE and Bruch's membrane, accompanied by the loss of inner and outer segments of cone photoreceptors and dropout of cone cell nuclei, with Müller cells remaining unaffected. Conclusions and Importance: This case of laser-induced retinopathy advances our understanding of the pathophysiological effect of laser exposure on the retina, suggesting a higher incidence of laser-induced retinopathy than previously diagnosed. It also serves as a crucial reminder for laser users to exercise caution and highlights the necessity for ophthalmologists to carefully observe and examine such cases.

GRF2 Is Crucial for Cone Photoreceptor Viability and Ribbon Synapse Formation in the Mouse Retina.

Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuclear migration in retinal cone photoreceptors. To evaluate the functional relevance of that cellular process for two putative targets of the GEF activity of GRF2 (RAC1 and CDC42), here we compared the structural and functional retinal phenotypes resulting from conditional targeting of RAC1 or CDC42 in the cone photoreceptors of constitutive GRF2KO and GRF2WT mice. We observed that single RAC1 disruption did not cause any obvious morphological or physiological changes in the retinas of GRF2WT mice, and did not modify either the phenotypic alterations previously described in the retinal photoreceptor layer of GRF2KO mice. In contrast, the single ablation of CDC42 in the cone photoreceptors of GRF2WT mice resulted in clear alterations of nuclear movement that, unlike those of the GRF2KO retinas, were not accompanied by electrophysiological defects or slow, progressive cone cell degeneration. On the other hand, the concomitant disruption of GRF2 and CDC42 in the cone photoreceptors resulted, somewhat surprisingly, in a normalized pattern of nuclear positioning/movement, similar to that physiologically observed in GRF2WT mice, along with worsened patterns of electrophysiological responses and faster rates of cell death/disappearance than those previously recorded in single GRF2KO cone cells. Interestingly, the increased rates of cone cell apoptosis/death observed in single GRF2KO and double-knockout GRF2KO/CDC42KO retinas correlated with the electron microscopic detection of significant ultrastructural alterations (flattening) of their retinal ribbon synapses that were not otherwise observed at all in single-knockout CDC42KO retinas. Our observations identify GRF2 and CDC42 (but not RAC1) as key regulators of retinal processes controlling cone photoreceptor nuclear positioning and survival, and support the notion of GRF2 loss-of-function mutations as potential drivers of cone retinal dystrophies.

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

Fluorescent reporter pluripotent stem cell-derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of guanine nucleotide-binding protein subunit alpha transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP alleles robustly and exclusively labeled immature and mature cones. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of the morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 µm3 per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

Gpr75 knockout mice display age-dependent cone photoreceptor cell loss.

GPR75 is an orphan G protein-coupled receptor for which there is currently limited information and its function in physiology and disease is only recently beginning to emerge. This orphan receptor is expressed in the retina but its function in the eye is unknown. The earliest studies on GPR75 were conducted in the retina, where the receptor was first identified and cloned and mutations in the receptor were identified as a possible contributor to retinal degenerative disease. Despite these sporadic reports, the function of GPR75 in the retina and in retinal disease has yet to be explored. To assess whether GPR75 has a functional role in the retina, the retina of Gpr75 knockout mice was characterized. Knockout mice displayed a mild progressive retinal degeneration, which was accompanied by oxidative stress. The degeneration was because of the loss of both M-cone and S-cone photoreceptor cells. Housing mice under constant dark conditions reduced oxidative stress but did not prevent cone photoreceptor cell loss, indicating that oxidative stress is not a primary cause of the observed retinal degeneration. Studies here demonstrate an important role for GPR75 in maintaining the health of cone photoreceptor cells and that Gpr75 knockout mice can be used as a model to study cone photoreceptor cell loss.

Etiology of Idiopathic Macular Holes in the Light of Estrogen Hormone.

The aim of this review was to identify a new potential explanation for the development of macular holes in relation to the female sex and to explain the possible underlying pathways. This approach was based on the evaluation of anatomical, physiological, and morphological analyses currently available in the literature. The findings showed that estrogen exerts a protective effect on the neuroretina and may influence Müller and cone cells. Both cell types are responsible for the building of the fovea structure. However, this protection may be lost due to the sudden decrease in estrogen levels during menopause. In conclusion, the fovea cones, through its sensitivity to estrogen and high energy consumption, may be very vulnerable to damage caused by a sudden changes in the concentration of estrogen in menopausal females. Such changes may result in cone degeneration, and thus a destroyed structure of the fovea, and may lead to the development of a hole in the fovea, as in the case of macular holes. This review revealed that under the decreasing influence of estrogen may cones play a key role with regard to the etiology of the development of macular holes. This aspect may be of strategic importance in prophylactic therapy for the prevention of the development of macular holes in premenopausal females or after ocular trauma.

Molecular and Clinical Characterization of CNGA3 and CNGB3 Genes in Brazilian Patients Affected with Achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by reduced visual acuity, nystagmus, photophobia, and very poor or absent color vision. Pathogenic variants in six genes encoding proteins composing the cone phototransduction cascade (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2) and of the unfolded protein response (ATF6) have been related to ACHM cases, while CNGA3 and CNGB3 alone are responsible for most cases. Herein, we provide a clinical and molecular overview of 42 Brazilian patients from 38 families affected with ACHM related to biallelic pathogenic variants in the CNGA3 and CNGB3 genes. Patients' genotype and phenotype were retrospectively evaluated. The majority of CNGA3 variants were missense, and the most prevalent CNGB3 variant was c.1148delC (p.Thr383Ilefs*13), resulting in a frameshift and premature stop codon, which is compatible with previous publications in the literature. A novel variant c.1893T>A (p.Tyr631*) in the CNGB3 gene is reported for the first time in this study. A great variability in morphologic findings was observed in our patients, although no consistent correlation with age and disease stage in OCT foveal morphology was found. The better understanding of the genetic variants landscape in the Brazilian population will help in the diagnosis of this disease.

Modeling of dielectric resonator antenna array for retina photoreceptors.

The retina encompasses several cone and rod photoreceptors at fovea region i.e., 90 million cells of rod photoreceptors and 4.5million cells of cone photoreceptors. The overall photoreceptors determine the vision of every human. An electromagnetic dielectric resonator antenna has been presented for retina photoreceptors in order to model them at fovea and its peripheral retina with the respected angular spectrum. Three coloring primary system of human eye (R, G, B) can be realized based on the model. Three miscellaneous models i.e., simple, graphene coated, and interdigital models have been presented in this paper. The nonlinear property of interdigital structures is one of the best advantages to use for creating the capacitor. The capacitance property helps improving the upper band of visible spectrum. The absorption of light for graphene as an energy harvesting material and its conversion into electrochemical signals is making it one of the best models. The mentioned three electromagnetic models of human photoreceptors have been expressed as a receiver antenna. The proposed electromagnetic models based on dielectric resonator antenna (DRA) are being analyzed for cones and rods photoreceptors of retina in the human eye by Finite Integral Method (FIM) utilized by CST MWS. The results show that the models are so fine for vision spectrum due to its localized near field enhancement property. The results indicate fine parameters of S 11 (return loss below -10 dB) with invaluable resonants in a wide range of frequencies from 405 THz to 790 THz (vision spectrum), appropriate S 21 (insertion loss 3-dB bandwidth), very good field distribution of electric and magnetic fields for flowing the power and electrochemical signals. Finally, mfERG clinical and experimental results validate the numeric results by the normalized output to input ratio of these models and it points out that these models can stimulate the electrochemical signals in photoreceptor cells for the best suiting of realizing the new retinal implants.

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of Guanine Nucleotide-Binding Protein Subunit Alpha Transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 cubic microns per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

Mechanism of Cone Degeneration in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of genetic disorders resulting in inherited blindness due to the degeneration of rod and cone photoreceptors. The various mechanisms underlying rod degeneration primarily rely on genetic mutations, leading to night blindness initially. Cones gradually degenerate after rods are almost eliminated, resulting in varying degrees of visual disability and blindness. The mechanism of cone degeneration remains unclear. An understanding of the mechanisms underlying cone degeneration in RP, a highly heterogeneous disease, is essential to develop novel treatments of RP. Herein, we review recent advancements in the five hypotheses of cone degeneration, including oxidative stress, trophic factors, metabolic stress, light damage, and inflammation activation. We also discuss the connection among these theories to provide a better understanding of secondary cone degeneration in RP. Five current mechanisms of cone degenerations in RP Interactions among different pathways are involved in RP.

The medaka mutant deficient in eyes shut homolog exhibits opsin transport defects and enhanced autophagy in retinal photoreceptors.

Eyes shut homolog (EYS) encodes a proteoglycan and the human mutation causes retinitis pigmentosa type 25 (RP25) with progressive retinal degeneration. RP25 most frequently affects autosomal recessive RP patients with many ethnic backgrounds. Although studies using RP models have facilitated the development of therapeutic medications, Eys has been lost in rodent model animals. Here we examined the roles for Eys in the maintenance of photoreceptor structure and function by generating eys-null medaka fish using the CRISPR-Cas9 system. Medaka EYS protein was present near the connecting cilium of wild-type photoreceptors, while it was absent from the eys-/- retina. The mutant larvae exhibited a reduced visual motor response compared with wild-type. In contrast to reported eys-deficient zebrafish at the similar stage, no retinal cell death was detected in the 8-month post-hatching (8-mph) medaka eys mutant. Immunohistochemistry showed a significant reduction in the length of cone outer segments (OSs), retention of OS proteins in the inner segments of photoreceptors, and abnormal filamentous actin network at the base of cone OSs in the mutant retina by 8 mph. Electron microscopy revealed aberrant structure of calyceal processes, numerous vesiculation and lamellar interruptions, and autophagosomes in the eys-mutant cone photoreceptors. In situ hybridization showed an autophagy component gene, gabarap, was ectopically expressed in the eys-null retina. These results suggest eys is required for regeneration of OS, especially of cone photoreceptors, and transport of OS proteins by regulating actin filaments. Enhanced autophagy may delay the progression of retinal degeneration when lacking EYS in the medaka retina.

Investigations into photoreceptor energy metabolism during experimental retinal detachment.

Retinal detachment is a sight-threatening disorder, which occurs when the photoreceptors are separated from their vascular supply. The aim of the present study was to shed light on photoreceptor energy metabolism during experimental detachment in rats. Retinal detachment was induced in the eyes of rats via subretinal injection of sodium hyaluronate. Initially, we investigated whether detachment caused hypoxia within photoreceptors, as evaluated by the exogenous and endogenous biomarkers pimonidazole and HIF-1α, as well as by qPCR analysis of HIF target genes. The results showed no unequivocal staining for pimonidazole or HIF-1α within any detached retina, nor upregulation of HIF target genes, suggesting that any reduction in pO2 is of insufficient magnitude to produce hypoxia-induced covalent protein adducts or HIF-1α stabilisation. Subsequently, we analysed expression of cellular bioenergetic enzymes in photoreceptors during detachment. We documented loss of mitochondrial, and downregulation of glycolytic enzymes during detachment, indicating that photoreceptors have reduced energetic requirements and/or capacity. Given that detachment did not cause widespread hypoxia, but did result in downregulated expression of bioenergetic enzymes, we hypothesised that substrate insufficiency may be critical in terms of pathogenesis, and that boosting metabolic inputs may preserve photoreceptor bioenergetic production and, protect against their degeneration. Thus, we tested whether supplementation with the bioavailable energy substrate pyruvate mitigated rod and cone injury and degeneration. Despite protecting photoreceptors in culture from nutrient deprivation, pyruvate failed to protect against apoptotic death of rods, loss of cone opsins, and loss of inner segment mitochondria, in situ, when evaluated at 3 days after detachment. The regimen was also ineffective against cumulative photoreceptor deconstruction and degeneration when evaluated after 4 weeks. Retinal metabolism, particularly the bioenergetic profiles and pathological responses of the various cellular subtypes still presents a considerable knowledge gap that has important clinical consequences. While our data do not support the use of pyruvate supplementation as a means of protecting detached photoreceptors, they do provide a foundation and motivation for future research in this area.

Transcriptional control of cone photoreceptor diversity by a thyroid hormone receptor.

Cone photoreceptor diversity allows detection of wavelength information in light, the first step in color (chromatic) vision. In most mammals, cones express opsin photopigments for sensitivity to medium/long (M, "green") or short (S, "blue") wavelengths and are differentially arrayed over the retina. Cones appear early in retinal neurogenesis but little is understood of the subsequent control of diversity of these postmitotic neurons, because cone populations are sparse and, apart from opsins, poorly defined. It is also a challenge to distinguish potentially subtle differences between cell subtypes within a lineage. Therefore, we derived a Cre driver to isolate individual M and S opsin-enriched cones, which are distributed in counter-gradients over the mouse retina. Fine resolution transcriptome analyses identified expression gradients for groups of genes. The postnatal emergence of gradients indicated divergent differentiation of cone precursors during maturation. Using genetic tagging, we demonstrated a role for thyroid hormone receptor ß2 (TRß2) in control of gradient genes, many of which are enriched for TRß2 binding sites and TRß2-regulated open chromatin. Deletion of TRß2 resulted in poorly distinguished cones regardless of retinal location. We suggest that TRß2 controls a bipotential transcriptional state to promote cone diversity and the chromatic potential of the species.

Synaptic vesicle release during ribbon synapse formation of cone photoreceptors.

Mammalian cone photoreceptors enable through their sophisticated synapse the high-fidelity transfer of visual information to second-order neurons in the retina. The synapse contains a proteinaceous organelle, called the synaptic ribbon, which tethers synaptic vesicles (SVs) at the active zone (AZ) close to voltage-gated Ca2+ channels. However, the exact contribution of the synaptic ribbon to neurotransmission is not fully understood, yet. In mice, precursors to synaptic ribbons appear within photoreceptor terminals shortly after birth as free-floating spherical structures, which progressively elongate and then attach to the AZ during the following days. Here, we took advantage of the process of synaptic ribbon maturation to study their contribution to SV release. We performed whole-cell patch-clamp recordings from cone photoreceptors at three postnatal (P) development stages (P8-9, P12-13, >P30) and measured evoked SV release, SV replenishment rate, recovery from synaptic depression, domain organization of voltage-sensitive Ca2+ channels, and Ca2+-sensitivity of exocytosis. Additionally, we performed electron microscopy to determine the density of SVs at ribbon-free and ribbon-occupied AZs. Our results suggest that ribbon attachment does not organize the voltage-sensitive Ca2+ channels into nanodomains or control SV release probability. However, ribbon attachment increases SV density at the AZ, increases the pool size of readily releasable SVs available for evoked SV release, facilitates SV replenishment without changing the SV pool refilling time, and increases the Ca2+- sensitivity of glutamate release.

Novel OPN1LW/OPN1MW Exon 3 Haplotype-Associated Splicing Defect in Patients with X-Linked Cone Dysfunction.

Certain combinations of common variants in exon 3 of OPN1LW and OPN1MW, the genes encoding the apo-protein of the long- and middle-wavelength sensitive cone photoreceptor visual pigments in humans, induce splicing defects and have been associated with dyschromatopsia and cone dysfunction syndromes. Here we report the identification of a novel exon 3 haplotype, G-C-G-A-T-T-G-G (referring to nucleotide variants at cDNA positions c.453, c.457, c.465, c.511, c.513, c.521, c.532, and c.538) deduced to encode a pigment with the amino acid residues L-I-V-V-A at positions p.153, p.171, p.174, p.178, and p.180, in OPN1LW or OPN1MW or both in a series of seven patients from four families with cone dysfunction. Applying minigene assays for all observed exon 3 haplotypes in the patients, we demonstrated that the novel exon 3 haplotype L-I-V-V-A induces a strong but incomplete splicing defect with 3-5% of residual correctly spliced transcripts. Minigene splicing outcomes were similar in HEK293 cells and the human retinoblastoma cell line WERI-Rb1, the latter retaining a cone photoreceptor expression profile including endogenous OPN1LW and OPN1MW gene expression. Patients carrying the novel L-I-V-V-A haplotype presented with a mild form of Blue Cone Monochromacy or Bornholm Eye Disease-like phenotype with reduced visual acuity, reduced cone electroretinography responses, red-green color vision defects, and frequently with severe myopia.

An enhancer located in a Pde6c intron drives transient expression in the cone photoreceptors of developing mouse and human retinas.

How cone photoreceptors are formed during retinal development is only partially known. This is in part because we do not fully understand the gene regulatory network responsible for cone genesis. We reasoned that cis-regulatory elements (enhancers) active in nascent cones would be regulated by the same upstream network that controls cone formation. To dissect this network, we searched for enhancers active in developing cones. By electroporating enhancer-driven fluorescent reporter plasmids, we observed that a sequence within an intron of the cone-specific Pde6c gene acted as an enhancer in developing mouse cones. Similar fluorescent reporter plasmids were used to generate stable transgenic human induced pluripotent stem cells that were then grown into three-dimensional human retinal organoids. These organoids contained fluorescently labeled cones, demonstrating that the Pde6c enhancer was also active in human cones. We observed that enhancer activity was transient and labeled a minor population of developing rod photoreceptors in both mouse and human systems. This cone-enriched pattern argues that the Pde6c enhancer is activated in cells poised between rod and cone fates. Additionally, it suggests that the Pde6c enhancer is activated by the same regulatory network that selects or stabilizes cone fate choice. To further understand this regulatory network, we identified essential enhancer sequence regions through a series of mutagenesis experiments. This suggested that the Pde6c enhancer was regulated by transcription factor binding at five or more locations. Binding site predictions implicated transcription factor families known to control photoreceptor formation and families not previously associated with cone development. These results provide a framework for deciphering the gene regulatory network that controls cone genesis in both human and mouse systems. Our new transgenic human stem cell lines provide a tool for determining which cone developmental mechanisms are shared and distinct between mice and humans.