Genetic manipulation of rod-cone differences in mouse retina.

Though rod and cone photoreceptors use similar phototransduction mechanisms, previous model calculations have indicated that the most important differences in their light responses are likely to be differences in amplification of the G-protein cascade, different decay rates of phosphodiesterase (PDE) and pigment phosphorylation, and different rates of turnover of cGMP in darkness. To test this hypothesis, we constructed TrUx;GapOx rods by crossing mice with decreased transduction gain from decreased transducin expression, with mice displaying an increased rate of PDE decay from increased expression of GTPase-activating proteins (GAPs). These two manipulations brought the sensitivity of TrUx;GapOx rods to within a factor of 2 of WT cone sensitivity, after correcting for outer-segment dimensions. These alterations did not, however, change photoreceptor adaptation: rods continued to show increment saturation though at a higher background intensity. These experiments confirm model calculations that rod responses can mimic some (though not all) of the features of cone responses after only a few changes in the properties of transduction proteins.

Retinal metabolism displays evidence for uncoupling of glycolysis and oxidative phosphorylation via Cori-, Cahill-, and mini-Krebs-cycle.

The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilised 1H-NMR spectroscopy-based metabolomics, in situ enzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: (1) the mini-Krebs-cycle, fuelled by glutamine and branched chain amino acids, generating N-acetylaspartate; (2) the alanine-generating Cahill-cycle; (3) the lactate-releasing Cori-cycle. Moreover, the metabolomics data indicated a shuttling of taurine and hypotaurine between the retinal pigment epithelium and photoreceptors, likely resulting in an additional net transfer of reducing power to photoreceptors. These findings expand our understanding of retinal physiology and pathology and shed new light on neuronal energy homeostasis and the pathogenesis of neurodegenerative diseases.

Chemically induced cone degeneration in the 13-lined ground squirrel.

Animal models of retinal degeneration are critical for understanding disease and testing potential therapies. Inducing degeneration commonly involves the administration of chemicals that kill photoreceptors by disrupting metabolic pathways, signaling pathways, or protein synthesis. While chemically induced degeneration has been demonstrated in a variety of animals (mice, rats, rabbits, felines, 13-lined ground squirrels (13-LGS), pigs, chicks), few studies have used noninvasive high-resolution retinal imaging to monitor the in vivo cellular effects. Here, we used longitudinal scanning light ophthalmoscopy (SLO), optical coherence tomography, and adaptive optics SLO imaging in the euthermic, cone-dominant 13-LGS (46 animals, 52 eyes) to examine retinal structure following intravitreal injections of chemicals, which were previously shown to induce photoreceptor degeneration, throughout the active season of 2019 and 2020. We found that iodoacetic acid induced severe pan-retinal damage in all but one eye, which received the lowest concentration. While sodium nitroprusside successfully induced degeneration of the outer retinal layers, the results were variable, and damage was also observed in 50% of contralateral control eyes. Adenosine triphosphate and tunicamycin induced outer retinal specific damage with varying results, while eyes injected with thapsigargin did not show signs of degeneration. Given the variability of damage we observed, follow-up studies examining the possible physiological origins of this variability are critical. These additional studies should further advance the utility of chemically induced photoreceptor degeneration models in the cone-dominant 13-LGS.

Txnip deletions and missense alleles prolong the survival of cones in a retinitis pigmentosa mouse model.

Retinitis pigmentosa (RP) is an inherited retinal disease in which there is a loss of cone-mediated daylight vision. As there are >100 disease genes, our goal is to preserve cone vision in a disease gene-agnostic manner. Previously we showed that overexpressing TXNIP, an α-arrestin protein, prolonged cone vision in RP mouse models, using an AAV to express it only in cones. Here, we expressed different alleles of Txnip in the retinal pigmented epithelium (RPE), a support layer for cones. Our goal was to learn more of TXNIP's structure-function relationships for cone survival, as well as determine the optimal cell type expression pattern for cone survival. The C-terminal half of TXNIP was found to be sufficient to remove GLUT1 from the cell surface, and improved RP cone survival, when expressed in the RPE, but not in cones. Knock-down of HSP90AB1, a TXNIP-interactor which regulates metabolism, improved the survival of cones alone and was additive for cone survival when combined with TXNIP. From these and other results, it is likely that TXNIP interacts with several proteins in the RPE to indirectly support cone survival, with some of these interactions different from those that lead to cone survival when expressed only in cones.

Longitudinal Assessment of OCT-Based Measures of Foveal Cone Structure in Achromatopsia.

Purpose: Achromatopsia (ACHM) is an autosomal recessive retinal disease associated with reduced or absent cone function. There is debate regarding the extent to which cone structure shows progressive degeneration in patients with ACHM. Here, we used optical coherence tomography (OCT) images to evaluate outer nuclear layer (ONL) thickness and ellipsoid zone (EZ) integrity over time in individuals with ACHM. Methods: Sixty-three individuals with genetically confirmed ACHM with follow-up ranging from about 6 months to 10 years were imaged using either Bioptigen or Cirrus OCT. Foveal cone structure was evaluated by assessing EZ integrity and ONL thickness. Results: A total of 470 OCT images were graded, 243 OD and 227 OS. The baseline distribution of EZ grades was highly symmetrical between eyes (P = 0.99) and there was no significant interocular difference in baseline ONL thickness (P = 0.12). The EZ grade remained unchanged over the follow-up period for 60 of 63 individuals. Foveal ONL thickness showed a clinically significant change in only 1 of the 61 individuals analyzed, although detailed adaptive optics imaging revealed no changes in cone density in this individual. Conclusions: ACHM appears to be a generally stable condition, at least over the follow-up period assessed here. As cones are the cellular targets for emerging gene therapies, stable EZ and ONL thickness demonstrate therapeutic potential for ACHM, although other aspects of the visual system need to be considered when determining the best timing for therapeutic intervention.

Statistical Evaluation of ERG Responses: A New Method to Validate Cycle-by-Cycle Recordings in Advanced Retinal Degenerations.

Purpose: To describe and evaluate a novel method to determine the validity of measurements made using cycle-by-cycle (CxC) recording techniques in patients with advanced retinal degenerations (RD) having low-amplitude flicker electroretinogram (ERG) responses. Methods: The method extends the original CxC recording algorithm introduced by Sieving et al., retaining the original recording setup and the preliminary analysis of raw data. Novel features include extended use of spectrum analysis, reduction of errors due to known sources, and a comprehensive statistical assessment using three different tests. The method was applied to ERG recordings from seven patients with RD and two patients with CNGB3 achromatopsia. Results: The method was implemented as a Windows application to processes raw data obtained from a commercial ERG system, and it features a computational toolkit for statistical assessment of ERG recordings with amplitudes as low as 1 µV, commonly found in advanced RD patients. When recorded using conditions specific for eliciting cone responses, none of the CNGB3 patients had a CxC validated response, indicating that no signal artifacts were present with our recording conditions. A comparison of the presented method with conventional 30 Hz ERG was performed. Bland-Altman plots indicated good agreement (mean difference, -0.045 µV; limits of agreement, 0.193 to -0.282 µV) between the resulting amplitudes. Within-session test-retest variability was 15%, comparing favorably to the variability of standard ERG amplitudes. Conclusions: This novel method extracts highly reliable clinical recordings of low-amplitude flicker ERGs and effectively detects artifactual responses. It has potential value both as a cone outcome variable and planning tool in clinical trials on natural history and treatment of advanced RDs.

Parafoveal cone function in choroideremia assessed with adaptive optics optoretinography.

Choroideremia (CHM) is an X-linked retinal degeneration leading to loss of the photoreceptors, retinal pigment epithelium (RPE), and choroid. Adaptive optics optoretinography is an emerging technique for noninvasive, objective assessment of photoreceptor function. Here, we investigate parafoveal cone function in CHM using adaptive optics optoretinography and compare with cone structure and clinical assessments of vision. Parafoveal cone mosaics of 10 CHM and four normal-sighted participants were imaged with an adaptive optics scanning light ophthalmoscope. While acquiring video sequences, a 2 s 550Δ10 nm, 450 nW/deg2 stimulus was presented. Videos were registered and the intensity of each cone in each frame was extracted, normalized, standardized, and aggregated to generate the population optoretinogram (ORG) over time. A gamma-pdf was fit to the ORG and the peak was extracted as ORG amplitude. CHM ORG amplitudes were compared to normal and were correlated with bound cone density, ellipsoid zone to RPE/Bruch's membrane (EZ-to-RPE/BrM) distance, and foveal sensitivity using Pearson correlation analysis. ORG amplitude was significantly reduced in CHM compared to normal (0.22 ± 0.15 vs. 1.34 ± 0.31). In addition, CHM ORG amplitude was positively correlated with cone density, EZ-to-RPE/BrM distance, and foveal sensitivity. Our results demonstrate promise for using ORG as a biomarker of photoreceptor function.

Melatonin in the mammalian retina: Synthesis, mechanisms of action and neuroprotection.

Melatonin is an important player in the regulation of many physiological functions within the body and in the retina. Melatonin synthesis in the retina primarily occurs during the night and its levels are low during the day. Retinal melatonin is primarily synthesized by the photoreceptors, but whether the synthesis occurs in the rods and/or cones is still unclear. Melatonin exerts its influence by binding to G protein-coupled receptors named melatonin receptor type 1 (MT1) and type 2 (MT2). MT1 and MT2 receptors activate a wide variety of signaling pathways and both receptors are present in the vertebrate photoreceptors where they may form MT1/MT2 heteromers (MT1/2h). Studies in rodents have shown that melatonin signaling plays an important role in the regulation of retinal dopamine levels, rod/cone coupling as well as the photopic and scotopic electroretinogram. In addition, melatonin may play an important role in protecting photoreceptors from oxidative stress and can protect photoreceptors from apoptosis. Critically, melatonin signaling is involved in the modulation of photoreceptor viability during aging and other studies have implicated melatonin in the pathogenesis of age-related macular degeneration. Hence melatonin may represent a useful tool in the fight to protect photoreceptors-and other retinal cells-against degeneration due to aging or diseases.

Ultraviolet vision in anemonefish improves colour discrimination.

In many animals, ultraviolet (UV) vision guides navigation, foraging, and communication, but few studies have addressed the contribution of UV signals to colour vision, or measured UV discrimination thresholds using behavioural experiments. Here, we tested UV colour vision in an anemonefish (Amphiprion ocellaris) using a five-channel (RGB-V-UV) LED display. We first determined that the maximal sensitivity of the A. ocellaris UV cone was ∼386 nm using microspectrophotometry. Three additional cone spectral sensitivities had maxima at ∼497, 515 and ∼535 nm. We then behaviourally measured colour discrimination thresholds by training anemonefish to distinguish a coloured target pixel from grey distractor pixels of varying intensity. Thresholds were calculated for nine sets of colours with and without UV signals. Using a tetrachromatic vision model, we found that anemonefish were better (i.e. discrimination thresholds were lower) at discriminating colours when target pixels had higher UV chromatic contrast. These colours caused a greater stimulation of the UV cone relative to other cone types. These findings imply that a UV component of colour signals and cues improves their detectability, which likely increases the prominence of anemonefish body patterns for communication and the silhouette of zooplankton prey.

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.

Long-Term Porcine Retina Explants as an Alternative to In Vivo Experimentation.

Purpose: The porcine retina represents an optimal model system to study treatment approaches for inherited retinal dystrophies owing to close anatomical similarities to the human retina, including a cone enriched visual streak. The aim of this work was to establish a protocol to keep explants in culture for up to 28 days with good morphological preservation. Methods: Two to four retina explants per eye were obtained from the central part of the retina and transferred onto a membrane insert with the photoreceptors facing down. Different medium compositions using Neurobasal-A medium containing 100 or 450 mg/dL glucose and combinations of fetal calf serum, B-27 with or without insulin and N-2 were tested. We developed a tissue quality score with robust markers for different retinal cell types (protein kinase C alpha, peanut agglutinin and 4',6-diamidino-2-phenylindol). Results: Retinae were kept until 28 days with only little degradation. The best results were attained using Neurobasal-A medium containing 100 mg/dL glucose supplemented with B-27 containing insulin and N-2. For an easy preparation process, it is necessary to minimize transport time and keep the eyes on ice until dissected. Heat-mediated decontamination by the butcher has to be avoided. Conclusions: Using a standardized protocol, porcine retina explants represent an easy to handle intermediate model between in vitro and in vivo experimentation. This model system is robustly reproducible and contributes to the implementation of the 3R principle to minimize animal experimentation. Translational Relevance: This model can be used to test future therapeutic approaches for inherited retinal dystrophies.

Age-Related Macular Degeneration, a Mathematically Tractable Disease.

A progression sequence for age-related macular degeneration onset may be determinable with consensus neuroanatomical nomenclature augmented by drusen biology and eye-tracked clinical imaging. This narrative review proposes to supplement the Early Treatment of Diabetic Retinopathy Study (sETDRS) grid with a ring to capture high rod densities. Published photoreceptor and retinal pigment epithelium (RPE) densities in flat mounted aged-normal donor eyes were recomputed for sETDRS rings including near-periphery rich in rods and cumulatively for circular fovea-centered regions. Literature was reviewed for tissue-level studies of aging outer retina, population-level epidemiology studies regionally assessing risk, vision studies regionally assessing rod-mediated dark adaptation (RMDA), and impact of atrophy on photopic visual acuity. The 3 mm-diameter xanthophyll-rich macula lutea is rod-dominant and loses rods in aging whereas cone and RPE numbers are relatively stable. Across layers, the largest aging effects are accumulation of lipids prominent in drusen, loss of choriocapillary coverage of Bruch's membrane, and loss of rods. Epidemiology shows maximal risk for drusen-related progression in the central subfield with only one third of this risk level in the inner ring. RMDA studies report greatest slowing at the perimeter of this high-risk area. Vision declines precipitously when the cone-rich central subfield is invaded by geographic atrophy. Lifelong sustenance of foveal cone vision within the macula lutea leads to vulnerability in late adulthood that especially impacts rods at its perimeter. Adherence to an sETDRS grid and outer retinal cell populations within it will help dissect mechanisms, prioritize research, and assist in selecting patients for emerging treatments.

Hyperglycemia and circadian disruption lead to retinal dysfunction in a stabilized colony of the fat sand rat Psammomys obesus.

PURPOSE: The Fat Sand Rat (Psammomys obesus) recapitulates several features of human pre-proliferative diabetic retinopathy, but data are restricted to wild animals, incompatible with stringent biomedical research criteria. To overcome this barrier, we characterized retinal changes in a colony of P. obsesus maintained under strictly controlled housing conditions. METHODS: Animals were maintained on low or high caloric energy diets, and raised under either standard (12 h light/12 h dark) or shortened (5 h light/5 h dark) photoperiods. Visual responses were tested by electroretinography, while structural/molecular changes were assayed by immunochemistry and molecular biology (RNAseq and qPCR). RESULTS: Whereas high calorie diet alone did not induce hyperglycemia, coupled with short photoperiod >80 % animals developed severe hyper-insulinemia by 15 weeks, and 16 % animals further developed hyperglycemia. In these groups, electroretinography showed significant declines in visual responses in both hyper-insulinemic and hyperglycemic animals, especially in photopic (cone) responses. Transcriptomics analysis of hyperglycemic compared to low caloric controls revealed major upregulation in pathways involved in glial activation, extracellular matrix remodeling, inflammation, cytokine production, partial ischemic responses and angiogenesis. Western blotting against rhodopsin and cone opsin also showed decreased levels in both groups, overall decreases being greater for cones than rods in hyperglycemic animals. CONCLUSIONS: P. obesus maintained in rigorously monitored captive conditions, albeit showing attenuated responses to dietary overload compared to wild counterparts, nevertheless do develop some retinal features of diabetic retinopathy-like degeneration. Such a colony with known sanitary status opens their broader use for biomedical research.

Are ipRGCs involved in human color vision? Hints from physiology, psychophysics, and natural image statistics.

Human photoreceptors consist of cones, rods, and melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). First studied in circadian regulation and pupillary control, ipRGCs project to a variety of brain centers suggesting a broader involvement beyond non-visual functions. IpRGC responses are stable, long-lasting, and with a particular codification of photoreceptor signals. In comparison with the transient and adaptive nature of cone and rod signals, ipRGCs' signaling might provide an ecological advantage to different attributes of color vision. Previous studies have indicated melanopsin's influence on visual responses yet its contribution to color perception in humans remains debated. We summarized evidence and hypotheses (from physiology, psychophysics, and natural image statistics) about direct and indirect involvement of ipRGCs in human color vision, by first briefly assessing the current knowledge about the role of melanopsin and ipRGCs in vision and codification of spectral signals. We then approached the question about melanopsin activation eliciting a color percept, discussing studies using the silent substitution method. Finally, we explore various avenues through which ipRGCs might impact color perception indirectly, such as through involvement in peripheral color matching, post-receptoral pathways, color constancy, long-term chromatic adaptation, and chromatic induction. While there is consensus about the role of ipRGCs in brightness perception, confirming its direct contribution to human color perception requires further investigation. We proposed potential approaches for future research, emphasizing the need for empirical validation and methodological thoroughness to elucidate the exact role of ipRGCs in human color vision.

Predicted effectiveness of EnChroma multi-notch filters for enhancing color perception in anomalous trichromats.

EnChroma filters are aids designed to improve color vision for anomalous trichromats. Their use is controversial because the results of lab-based assessments of their effectiveness have so far largely failed to agree with positive anecdotal reports. However, the effectiveness of EnChroma filters will vary depending on the conditions of viewing, including whether the stimuli are broadband reflective surfaces or colors presented on RGB displays, whether illumination spectra are broadband or narrowband, the transmission spectra of particular filters, and the cone spectral sensitivity functions of the observer. We created a model of anomalous trichromatic color vision to predict the effects of EnChroma filters on the color signals impaired in anomalous trichromacy. Using the model we varied illumination, filter type and observer cone sensitivity functions, and tested the effect of presenting colors as broadband reflective surfaces or on RGB displays. We also used hyperspectral images to assess the impact of the filters on anomalous trichromats' color vision for natural scenes. Model results predicted that the filters should be broadly effective at enhancing anomalous trichromats' equivalent to L/(L + M) chromatic contrasts under a range of viewing conditions, but are substantially more effective for deuteranomals than for protanomals. The filters are predicted to be more effective for broadband reflective surfaces presented under broadband illuminants than for surfaces presented under narrowband illuminants or for colors presented on RGB displays. Since the potential impacts of contrast adaptation and perceptual learning are not considered in the model, it needs to be empirically validated. Results of empirical tests of the effects of EnChroma filters on deuteranomalous color vision in comparison with model predictions are presented in an accompanying paper (Somers et al., in prep.).

CRISPR editing of anti-anemia drug target rescues independent preclinical models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.

Specific photoreceptor cell fate pathways are differentially altered in NR2E3-associated diseases.

Mutations in NR2E3, a gene encoding an orphan nuclear transcription factor, cause two retinal dystrophies with a distinct phenotype, but the precise role of NR2E3 in rod and cone transcriptional networks remains unclear. To dissect NR2E3 function, we performed scRNA-seq in the retinas of wildtype and two different Nr2e3 mouse models that show phenotypes similar to patients carrying NR2E3 mutations. Our results reveal that rod and cone populations are not homogeneous and can be separated into different sub-classes. We identify a previously unreported cone pathway that generates hybrid cones co-expressing both cone- and rod-related genes. In mutant retinas, this hybrid cone subpopulation is more abundant and includes a subpopulation of rods transitioning towards a cone cell fate. Hybrid photoreceptors with high misexpression of cone- and rod-related genes are prone to regulated necrosis. Overall, our results shed light on the role of NR2E3 in modulating photoreceptor differentiation towards cone and rod fates and explain how different mutations in NR2E3 lead to distinct visual disorders in humans.

Non-image-forming vision as measured through ipRGC-mediated pupil constriction is not modulated by covert visual attention.

In brightness, the pupil constricts, while in darkness, the pupil dilates; this is known as the pupillary light response (PLR). The PLR is driven by all photoreceptors: rods and cones, which contribute to image-forming vision, and intrinsically photosensitive retinal ganglion cells (ipRGCs), which mainly contribute to non-image-forming vision. Rods and cones cause immediate pupil constriction upon light exposure, whereas ipRGCs cause sustained constriction throughout light exposure. Recent studies have shown that covert attention modulated the initial PLR; however, it remains unclear whether the same holds for the sustained PLR. We tested this by leveraging ipRGCs' responsiveness to blue light, causing the most prominent sustained constriction. While replicating previous studies by showing that pupils constricted more when either directly looking at, or covertly attending to, bright as compared to dim stimuli (with the same color), we also found that the pupil constricted more when directly looking at blue as compared to red stimuli (with the same luminosity). Crucially, however, in two high-powered studies (n = 60), we did not find any pupil-size difference when covertly attending to blue as compared to red stimuli. This suggests that ipRGC-mediated pupil constriction, and possibly non-image-forming vision more generally, is not modulated by covert attention.

Germline knockout of Nr2e3 protects photoreceptors in three distinct mouse models of retinal degeneration.

Retinitis pigmentosa (RP) is a common form of retinal dystrophy that can be caused by mutations in any one of dozens of rod photoreceptor genes. The genetic heterogeneity of RP represents a significant challenge for the development of effective therapies. Here, we present evidence for a potential gene-independent therapeutic strategy based on targeting Nr2e3, a transcription factor required for the normal differentiation of rod photoreceptors. Nr2e3 knockout results in hybrid rod photoreceptors that express the full complement of rod genes, but also a subset of cone genes. We show that germline deletion of Nr2e3 potently protects rods in three mechanistically diverse mouse models of retinal degeneration caused by bright-light exposure (light damage), structural deficiency (rhodopsin-deficient Rho-/- mice), or abnormal phototransduction (phosphodiesterase-deficient rd10 mice). Nr2e3 knockout confers strong neuroprotective effects on rods without adverse effects on their gene expression, structure, or function. Furthermore, in all three degeneration models, prolongation of rod survival by Nr2e3 knockout leads to lasting preservation of cone morphology and function. These findings raise the possibility that upregulation of one or more cone genes in Nr2e3-deficient rods may be responsible for the neuroprotective effects we observe.

Photoreceptor calyceal processes accompany the developing outer segment, adopting a stable length despite a dynamic core.

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). Although CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the CPs remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, yet heat-shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Müller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development and surrounding environment of photoreceptor microvilli in the zebrafish retina.