Retinal Cone Photoreceptors Research Articles

A parafoveal retinal cones analysis using adaptive-optics retinal camera in patients with primary open angle glaucoma

ObjectivesTo study the density, spacing, and regularity of retinal cone photoreceptors using an Adaptive Optics (AO) retinal camera (Rtx1TM, Imagine Eyes, Orsay, France) in patients with Primary Open Angle Glaucoma (POAG) and to compare the outcomes with those of healthy age-matched control subjects.MethodsThe study included 43 eyes with POAG and 31 eyes of normal subjects. POAG patients were divided into three groups according to the severity of the visual field defect. The AO Rtx1TM was used to obtain images of the parafoveal cone mosaic to calculate cone values. Analysis was performed at two and four degrees of eccentricity from the fovea along the four meridians (nasal, temporal, superior, inferior).ResultsIn POAG eyes, the mean ± standard deviation (SD) cone density at 2° considering all meridians was significantly lower than in normal controls (23,058.6 ± 3532.0 cones/mm2, and 25,511.7 ± 3157.5 cones/mm2, respectively; p = 0.003). Cone spacing was 7.3 ± 0.5 µm in POAG and 7.0 ± 0.4 µm in normal controls (p = 0.005), and cone regularity was 90.5 ± 4.9% and 93.5 ± 1.9% in POAG and normal controls, respectively (p < 0.001). At 4° similar trends were observed. However, no significant differences were found among patients with different severity of POAG (p > 0.05).ConclusionsUsing AO Rtx1TM, significant differences in retinal photoreceptors mosaic pattern were found between POAG eyes and age-matched controls, indicating a reduction in photoreceptors in POAG. No significant differences in retinal photoreceptor values were found among the three POAG groups.

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.

Notch pathway mutants do not equivalently perturb mouse embryonic retinal development.

In the vertebrate eye, Notch ligands, receptors, and ternary complex components determine the destiny of retinal progenitor cells in part by regulating Hes effector gene activity. There are multiple paralogues for nearly every node in this pathway, which results in numerous instances of redundancy and compensation during development. To dissect such complexity at the earliest stages of eye development, we used seven germline or conditional mutant mice and two spatiotemporally distinct Cre drivers. We perturbed the Notch ternary complex and multiple Hes genes to understand if Notch regulates optic stalk/nerve head development; and to test intracellular pathway components for their Notch-dependent versus -independent roles during retinal ganglion cell and cone photoreceptor competence and fate acquisition. We confirmed that disrupting Notch signaling universally blocks progenitor cell growth, but delineated specific pathway components that can act independently, such as sustained Hes1 expression in the optic stalk/nerve head. In retinal progenitor cells, we found that among the genes tested, they do not uniformly suppress retinal ganglion cell or cone differentiation; which is not due differences in developmental timing. We discovered that shifts in the earliest cell fates correlate with expression changes for the early photoreceptor factor Otx2, but not with Atoh7, a factor required for retinal ganglion cell formation. During photoreceptor genesis we also better defined multiple and simultaneous activities for Rbpj and Hes1 and identify redundant activities that occur downstream of Notch. Given its unique roles at the retina-optic stalk boundary and cone photoreceptor genesis, our data suggest Hes1 as a hub where Notch-dependent and -independent inputs converge.

Deep phenotyping of PROM1-associated retinal degeneration

Background/aimsThe purpose of this study was to investigate retinal structure in detail of subjects with autosomal-dominant (AD) and autosomal-recessive (AR) PROM1-associated retinal degeneration (PROM1-RD), study design: institutional, cross-sectional study.MethodsFour eyes...

Knockout of DLIC1 leads to retinal cone degeneration via disturbing Rab8 transport in zebrafish

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1−/− zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1−/− zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1−/− retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1−/− zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1−/− retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.

Ageing changes in retinal outer nuclear layer thickness and cone photoreceptor density using adaptive optics-free imaging.

To investigate age-related changes of the outer nuclear layer (ONL) thickness and cone density, and their associations in healthy participants using a modified, narrow scan-angle Heidelberg Retina Angiograph (HRA2). Retinal cones were imaged outside the fovea at 8.8° eccentricity and cone density was compared to ONL thickness measurements obtained by Spectral-Domain Optical Coherence Tomography (SD-OCT) at the same locations. Fifty-six eyes of 56 healthy participants with a median age (interquartile range, IQR) of 37 years (29-55) were included. Median (IQR) cone count was 7,472 (7,188, 7,746) cones/mm2 and median (IQR) ONL thickness was 56 (52, 60) µm for healthy participants. Both cone density and ONL thickness were negatively associated with age: cone density, R2 = 0.16 (F(1,54) = 10.41, P = 0.002); ONL thickness, R2 = 0.12 (F(1,54) = 7.41, P = 0.009). No significant association was seen between cone density and ONL thickness (R2 = 0.03; F(1,54) = 1.66, P = 0.20). Cone density was lower, and ONL thinner, in older compared to younger participants, therefore, image-based structural measures should be compared to age-related data. However, cone density and ONL thickness were not strongly associated, indicating that determinants of ONL thickness measurements other than cone density measurements, and including measurement error, have a major influence.

Associative colour learning and discrimination in the South African Cape rock sengi Elephantulus edwardii (Macroscelidea, Afrotheria, Mammalia)

Abstract Beside insects, sengis also consume plant material such as leaves, fruits, seeds and floral nectar. It is known that they use olfaction for foraging, but little is known about their vision and visual learning capabilities. Colour vision has been tested in two species, showing that they are likely dichromats (green- and blue-sensitive retinal cone-photoreceptors, meaning red-green colour blind). Our aim was to examine the learning and colour discrimination abilities of another species, Elephantulus edwardii. Using training procedures and choice experiments, we tested the hypotheses that the animals can associate a reward with trained colours and that they can discriminate between different colour hues. The sengis preferred the trained colours over the others, indicating associative learning. They could discriminate between all tested colours (blue, red, green, yellow). The sengis’ colour choice behaviour indicates that the animals can use also colour features to find food plant material. Additionally, learning abilities most likely are essential for the sengis’ foraging activities, for instance by associating floral or fruit shape, colour or scent with nectar or ripe fruit, to increase the efficiency to locate food sources.

Effect of Direct Current Electric Fields on Cone Like Retinal Photoreceptor Cells.

Studies show that electric fields are used as therapy during nerve and tissue injuries along with trans-retinal stimulation. However, cellular and molecular changes induced by such treatments remain largely unknown especially in retinal photoreceptor cells. In vitro studies show that direct current electric fields (dcEF) were known to influence cell division, polarity, shape, and motility. Here we could characterize for the first time the reactions of 661W, a retinal cone photoreceptor especially regarding organelle polarization, membrane polarization of mitochondria, O2 consumption, ATP/ADP ratio and gene expression. The 661W cells were stimulated with a constant dcEF of field strength 5 V/cm during 30 min or 5 h depending on the parameters studied. In response to dcEF, the cells aligned perpendicular to the field by forming a leading edge with extended membrane protrusions towards the cathode. Using immunofluorescence and live cell imaging, we show that the cell membrane depolarized at the cathodal side. The microtubules spread into the direction of migration. Also, the microtubule organization center re-oriented into this direction. Concomitantly with the microtubules, actin filaments reorganized in an asymmetrical fashion mainly at the cathodal side. The Golgi apparatus, which is involved in many steps of actin synthesis, moved to the cathodal side. In the last 2 h of the 5 h experiment, microtubules positioned themselves at the rear (anodal side), like the nucleus. The averaged displacement of the whole cells under dcEF was 155% of control for 3 V/cm and 235% for 5 V/cm. The average speed increased by 142% and 243% respectively. Inside the cells mitochondria moved to the cathodal side, where the energy consuming producing processes take place. In this line, we measured an increase in ATP production and O2 consumption. Mitochondrial calcium was found more on the anodal side, at the site of the nucleus with its calcium delivering endoplasmic reticulum. In addition, oxymetry studies reveal an increased ATP synthesis by 115.2% and oxygen consumption by 113.3% 3 h after dcEF stimulation. An analysis of differentially expressed genes by RNA sequencing revealed an upregulation of genes involved in cellular movement, cell to cell and intracellular signaling, molecular transport, assembly and organization. The mechanisms found can enhance our understanding regarding the beneficial effects of EF treatment in retinal diseases.

Regulation of rod photoreceptor function by farnesylated G-protein γ-subunits.

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.

Mettl14-mediated m6A modification is essential for visual function and retinal photoreceptor survival

BackgroundAs the most abundant epigenetic modification of eukaryotic mRNA, N6-methyladenosine (m6A) modification has been shown to play a role in mammalian nervous system development and function by regulating mRNA synthesis and degeneration. However, the role of m6A modification in retinal photoreceptors remains unknown.ResultsWe generated the first retina-specific Mettl14-knockout mouse models using the Rho-Cre and HRGP-Cre lines and investigated the functions of Mettl14 in retinal rod and cone photoreceptors. Our data showed that loss of Mettl14 in rod cells causes a weakened scotopic photoresponse and rod degeneration. Further study revealed the ectopic accumulation of multiple outer segment (OS) proteins in the inner segment (IS). Deficiency of Mettl14 in cone cells led to the mislocalization of cone opsin proteins and the progressive death of cone cells. Moreover, Mettl14 depletion resulted in drastic decreases in METTL3/WTAP levels and reduced m6A methylation levels. Mechanistically, transcriptomic analyses in combination with MeRIP-seq illustrated that m6A depletion via inactivation of Mettl14 resulted in reduced expression levels of multiple phototransduction- and cilium-associated genes, which subsequently led to compromised ciliogenesis and impaired synthesis and transport of OS-residing proteins in rod cells.ConclusionsOur data demonstrate that Mettl14 plays an important role in regulating phototransduction and ciliogenesis events and is essential for photoreceptor function and survival, highlighting the importance of m6A modification in visual function.

Expression of rhodopsin and opsin in late‐stage epigean and hypogean salamander embryos

Evolutionarily, sensory systems have undergone extreme diversifications that have allowed vertebrates to survive diverse environments. The salamander clade Eurycea of Central Texas has branched out of its epigean ancestral roots and has successfully invaded subterranean environments. The San Marcos salamander (E. nana) and the Barton Springs salamander (E. sosorum) live above ground in springs and streams whereas the Texas blind salamander (E. rathbuni) occupies caves and recesses in the Edwards Aquifer. From an evolutionary perspective, it is thought that the hypogeal state is derived, but what remains a mystery is the developmental adaption of its sensory system to life under ground. The habitational broadening of the Eurycea salamander has been associated with the phenotype of reduced eyes exhibited in the Texas blind salamander. It is not clear whether this phenotype represents incomplete development, regression, or a combination of both. The clade of Eurycea is a prime model for comparing the molecular mechanisms that are involved in diverging neural systems, since species within the same clade present two extremes of phenotypes that are associated with their environment. Retinal development in embryonic E. rathbuni has been observed; however, the extent to which the retina develops is unknown. We identify retinal rod and cone photoreceptor cells along with their associated visual proteins (rhodopsin and opsin, respectively) in blind species and in epigean salamanders with fully developed eyes. Immunohistochemistry followed by confocal microscopy was applied for the investigation of sections of late embryonic stages of these salamander species. The results suggest opsin is expressed in the retina of E. nana and E. sosorum, and rhodopsin is expressed in all three species. Based on previous work indicating absence of visual pigments in adult Texas blind salamanders, we conclude expression of rhodopsin is transient or variable in this species. In future studies we will examine the expression of visual pigments at other stages to get a clearer picture of the time course and species‐level variability in expression.

Structure of the human cone photoreceptor cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels transduce light-induced chemical signals into electrical signals in retinal cone and rod photoreceptors. Structures of native CNG channels, which are heterotetramers formed by CNGA and CNGB subunits, have not been obtained. In the present study, we report a high-resolution cryo-electron microscopy structure of the human cone CNG channel in the apo closed state. The channel contains three CNGA3 and one CNGB3 subunits. Arg403 in the pore helix of CNGB3 projects into an asymmetric selectivity filter and forms hydrogen bonds with two pore-lining backbone carbonyl oxygens. Arg442 in S6 of CNGB3 protrudes into and occludes the pore below the hydrophobic cavity gate previously observed in homotetrameric CNGA channels. It is interesting that Arg403Gln is a disease mutation, and Arg442 is replaced by glutamine in some animal species with dichromatic or monochromatic vision. These and other unique structural features and the disease link conferred by CNGB3 indicate a critical role of CNGB3 in shaping cone photoresponses.

Toxic effects of triclocarban on larval zebrafish: A focus on visual dysfunction.

Triclocarban (TCC) is considered an endocrine disruptor and shows antagonist activity on thyroid receptors. In view of the report that thyroid hormone signaling mediates retinal cone photoreceptor specification, we hypothesize that TCC could impair visual function, which is vital to wildlife. In order to verify our hypothesis, we assessed alteration in the retinal structure (retinal layer thickness and cell density), visually-mediated behavior, cone and rod opsin gene expression, and photoreceptor immunostaining in zebrafish larvae exposed to TCC at environmentally realistic concentrations (0.16±0.005µg/L, L-group) and one-fifth of the median lethal concentrations (25.4±1.02µg/L, H-group). Significant decrease in eye size, ganglion cell density, optokinetic response, and phototactic response can be observed in the L-group, while the thickness of outer nuclear layer, where the cell bodies of cone and rod cells are located, was significantly reduced with the down-regulation of critical opsin gene (opn1sw2, opn1mw1, opn1mw3, opn1lw1, opn1lw2, and rho) expression and rhodopsin immunofluorescence in the H-group. It should be noted that TCC could affect the sensitivity of zebrafish larvae to red and green light according to the results of behavioral and opsin gene expression analysis. These findings provide the first evidence to support our hypothesis that the visual system, a novel toxicological target, is affected by TCC. Consequently, we urgently call for a more in-depth exploration of TCC-induced ocular toxicity to aquatic organisms and even to humans.

Single Cell Transcriptomic Analyses Reveal the Impact of bHLH Factors on Human Retinal Organoid Development.

The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in human ES cell-derived retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 each assert positive autoregulation, and both suppress key bHLH factors associated with the pre-neurogenic and states and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.

Advanced Retinal Imaging and Ocular Parameters of the Rhesus Macaque Eye.

PurposeTo determine the range of normal ocular biometry and perform advanced retinal imaging and functional assessment of the rhesus macaque eye.MethodsWe performed ocular phenotyping on rhesus macaques at the California National Primate Research Center. This process consisted of anterior and posterior segment eye examination by ophthalmologists, advanced retinal imaging, and functional retinal electrophysiology.ResultsFull eye examinations were performed on 142 animals, consisting of pupillary light reflex, tonometry, external examination and photography, anterior slit lamp examination, and posterior segment examination by indirect ophthalmoscopy. Ages of the rhesus macaques ranged from 0.7 to 29 years (mean, 16.4 ± 7.5 years). Anterior segment measurements such as intraocular pressure (n = 142), corneal thickness (n = 84), lens thickness (n = 114), and axial length (n = 114) were acquired. Advanced retinal imaging in the form of fundus photography (n = 78), optical coherence tomography (n = 60), and quantitative autofluorescence (n = 44) was obtained. Electroretinography (n = 75) was used to assay retinal function. Quantitative analyses of the macular structure, retinal layer segmentation, and rod and cone photoreceptor electrical responses are reported. Quantitative assessments were made and variations between sexes were analyzed to compare with established sex changes in human eyes.ConclusionsThe rhesus macaque has an ocular structure and function very similar to that of the human eye. In particular macular structure and retinal function is very similar to humans, making this species particularly useful for the study of macular biology and development of therapies for cone photoreceptor disorders.Translational RelevanceRhesus macaques are an ideal model for future vision science studies of human eye diseases.

Maternal Hypothyroidism Delayed Retinal Opsin-Development in the Neonatal Period: Analysis of TRH-Deficient Mice

Abstract Introduction: Retinal cone photoreceptor cells contain short (S) and medium (M) wavelength opsins, which are light-sensitive substances involved in color vision and visual acuity by sensing lights of different wavelengths. Thyroid hormones promote M-opsin expression and suppress S-opsin expression during the differentiation of cone photoreceptors. It was previously reported that M-opsin expression was delayed and S-opsin expression increased in TSH receptor-deficient mice and methimazole-induced hypothyroid mice. In addition, no M-opsin expression and increased S-opsin expression were observed in thyroid hormone receptor (TR) β2-deficient mice (Ng L et al, Nature Genetics. 2001; 27(1): 94-98.). This suggested that impaired thyroid function affects opsin development. We therefore examined retinal development in TRH-deficient mice, which are a model of central hypothyroidism established in our laboratory. Methods: We performed HE staining of the retina at postnatal 30 days and electroretinography at postnatal 10 weeks using TRH-/- and wild-type (WT) mice. We also examined expression levels of S/M opsin mRNA in WT, TRH-/- and TRH-/- pups born from TRH-/- dams at postnatal 12,17 and 30 days, and TRβΔ337T knock-in mice (TRβmut/mut) at postnatal 30 days. Furthermore, we performed immunohistochemistry to examine S/M opsin protein expression in these mice. Results: The retinal structures by HE staining and retinal functions by electroretinography in TRH-/- mice were unchanged compared with those in WT mice. Although M-opsin expression was not detected and S-opsin expression was higher in TRβmut/mut mice than in WT mice, the mRNA and protein expression levels of S/M-opsin did not significantly differ between TRH-/- pups born from TRH+/- dams and WT pups at all postnatal days. TRH-/- pups born from TRH-/- dams exposed to maternal hypothyroidism had similar serum total T4 levels to TRH-/- pups born from TRH+/- with normal maternal thyroid function. In contrast, the mRNA expression level of M-opsin was significantly lower (1.00±0.06 vs 0.64±0.05: mean ± SE, p&amp;lt;0.01) and the protein expression level was lower in TRH-/- pups born from TRH-/- dams than in WT pups at postnatal 12 days. However, these differences disappeared after postnatal 17 days, and there was no difference in M-opsin expression in TRH-/- pups born from TRH-/- dams compared with WT pups. Conclusions: Although no delay in opsin development was observed in TRH-/- pups born from TRH+/- dams, TRH-/- pups born from central hypothyroid dams exhibited delayed opsin development, suggesting that maternal hypothyroidism affects the development of retinal opsin in the neonatal period.

Apo-Opsin and Its Dark Constitutive Activity across Retinal Cone Subtypes

Retinal rod and cone photoreceptors mediate vision in dim and bright light, respectively, by transducing absorbed photons into neural electrical signals. Their phototransduction mechanisms are essentially identical. However, one difference is that, whereas a rod visual pigment remains stable in darkness, a cone pigment has some tendency to dissociate spontaneously into apo-opsin and retinal (the chromophore) without isomerization. This cone-pigment property is long known but has mostly been overlooked. Importantly, because apo-opsin has weak constitutive activity, it triggers transduction to produce electrical noise even in darkness. Currently, the precise dark apo-opsin contents across cone subtypes are mostly unknown, as are their dark activities. We report here a study of goldfish red (L), green (M), and blue (S) cones, finding with microspectrophotometry widely different apo-opsin percentages in darkness, being ∼30% in L cones, ∼3% in M cones, and negligible in S cones. L and M cones also had higher dark apo-opsin noise than holo-pigment thermal isomerization activity. As such, given the most likely low signal amplification at the pigment-to-transducin/phosphodiesterase phototransduction step, especially in L cones, apo-opsin noise may not be easily distinguishable from light responses and thus may affect cone vision near threshold.

Zebrafish Crb1, Localizing Uniquely to the Cell Membranes around Cone Photoreceptor Axonemes, Alleviates Light Damage to Photoreceptors and Modulates Cones' Light Responsiveness.

The crumbs (crb) apical polarity genes are essential for the development and functions of epithelia. Adult zebrafish retinal neuroepithelium expresses three crb genes (crb1, crb2a, and crb2b); however, it is unknown whether and how Crb1 differs from other Crb proteins in expression, localization, and functions. Here, we show that, unlike zebrafish Crb2a and Crb2b as well as mammalian Crb1 and Crb2, zebrafish Crb1 does not localize to the subapical regions of photoreceptors and Müller glial cells; rather, it localizes to a small region of cone outer segments: the cell membranes surrounding the axonemes. Moreover, zebrafish Crb1 is not required for retinal morphogenesis and photoreceptor patterning. Interestingly, Crb1 promotes rod survival under strong white light irradiation in a previously unreported non--cell-autonomous fashion; in addition, Crb1 delays UV and blue cones' chromatin condensation caused by UV light irradiation. Finally, Crb1 plays a role in cones' responsiveness to light through an arrestin-translocation-independent mechanism. The localization of Crb1 and its functions do not differ between male and female fish. We conclude that zebrafish Crb1 has diverged from other vertebrate Crb proteins, representing a neofunctionalization in Crb biology during evolution.SIGNIFICANCE STATEMENT Apicobasal polarity of epithelia is an important property that underlies the morphogenesis and functions of epithelial tissues. Epithelial apicobasal polarity is controlled by many polarity genes, including the crb genes. In vertebrates, multiple crb genes have been identified, but the differences in their expression patterns and functions are not fully understood. Here, we report a novel subcellular localization of zebrafish Crb1 in retinal cone photoreceptors and evidence for its new functions in photoreceptor maintenance and light responsiveness. This study expands our understanding of the biology of the crb genes in epithelia, including retinal neuroepithelium.

Dose-dependent regulation of horizontal cell fate by Onecut family of transcription factors.

Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of either Oc1 or Oc2 gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion of Oc1 and Oc2 leads to a complete loss of HCs. Here we study the genetic redundancy between Oc1 and Oc2 paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication.

Retinal cone photoreceptor distribution in the American black bear (Ursus americanus).

The distribution of cone photoreceptor subtypes (important for color vision and vision quality) varies widely in different carnivore species, but there have been limited studies on bear (ursid) cone distribution. A previous behavioral study suggests that American black bears (Ursus americanus) are dichromatic, indicating that they possess two cone subtypes, although the retinal distribution of cones is unknown. The purpose of this study was to examine the subtype and topography of cones in American black bear retinas to further predict the nature of their color vision and image resolution. We studied 10 eyes from seven individual legally hunted black bears in northeastern North Carolina. Cryosections and retinal wholemounts were labeled using antibodies targeting two cone opsin subtypes: long/medium (L/M) wavelength sensitive and short (S) wavelength sensitive. Cones in fluorescent microscopy images were counted and density maps were created for retinal wholemounts. The black bear retina contains both cone subtypes and L/M cones outnumber S cones by at least 3:1, a finding confirmed in retinal frozen sections. There are higher concentrations of S cones present than typically seen in other carnivores with some evidence for co-expression of L/M and S cones. A cone-dense area centralis is present dorsotemporal to the optic nerve, similar to other carnivores. These results confirm that American black bears are predicted to have a dichromatic vision with high acuity indicated by the presence of a dorsotemporally located area centralis.