Cone-dominant Retinas Research Articles

Deep-sea fish reveal alternative pathway for vertebrate visual development.

Vertebrate vision is accomplished by two phenotypically distinct types of photoreceptors in the retina: the saturation-resistant cones for the detection of bright light and the highly sensitive rods for dim light conditions [1]. The current dogma is that, during development, all vertebrates initially feature a cone-dominated retina, and rods are added later [2, 3]. By studying the ontogeny of vision in three species of deep-sea fishes, we show that their larvae express cone-specific genes in photoreceptors with rod-like morphologies. Through development, these fishes either retain this rod-like cone retina (Maurolicus mucronatus) or switch to a retina with true rod photoreceptors with expression of rod-specific genes and transcription factors (Vinciguerria mabahiss and Benthosema pterotum). In contrast to the larvae of most marine fishes, which inhabit the bright upper layer of the open ocean, the larvae of deep-sea fishes occur deeper, exposing them to a dimmer light environment [4-7]. Spectral maxima predictions from molecular dynamics simulations and environmental light estimations suggest that using transmuted photoreceptors that combine the characteristics of both cones and rods maximises visual performance in these dimmer light conditions. Our findings provide molecular, morphological, and functional evidence for the evolution of an alternative developmental pathway for vertebrate vision.

A Novel Tree Shrew Model of Chronic Experimental Autoimmune Uveitis and Its Disruptive Application.

BackgroundPrevious studies have established several animal models for experimental autoimmune uveitis (EAU) in rodents without the fovea centralis in the human retina. This study aimed to develop and explore the application of a novel EAU model in tree shrews with a cone-dominated retina resembling the human fovea.MethodsTree shrews were clinically and pathologically evaluated for the development and characteristics of EAU immunized with six inter-photoreceptor retinoid-binding proteins (IRBPs). IRBP-specific T-cell proliferation and serum cytokine of tree shrews were evaluated to determine the immune responses. Differentially expressed genes (DEGs) were identified in the eyes of tree shrews with EAU by RNA-sequencing. The disruptive effects of the DEG RGS4 inhibitor CCG 203769 and dihydroartemisinin on the EAU were investigated to evaluate the potential application of tree shrew EAU.ResultsIRBP1197–1211 and R14 successfully induced chronic EAU with subretinal deposits and retinal damage in the tree shrews. The immunological characteristics presented the predominant infiltration of microglia/macrophages, dendritic cells, and CD4-T-cells into the uvea and retina and pathogenic T helper (Th) 1 and Th17 responses. The subretinal deposits positively expressed amyloid β-protein (Aβ), CD8, and P2Y purinoceptor 12 (P2RY12). The crucial DEGs in R14-induced EAU, such as P2RY2 and adenylate cyclase 4 (ADCY4), were enriched for several pathways, including inflammatory mediator regulation of transient receptor potential (TRP) channels. The upregulated RGS4 in IRBP-induced EAU was associated with mitogen-activated protein kinase (MAPK) activity. RGS4 inhibition and dihydroartemisinin could significantly alleviate the retinal pathological injuries of IRBP1197-1211-induced EAU by decreasing the expression of CD4 T-cells.ConclusionOur study provides a novel chronic EAU in tree shrews elicited by bovine R14 and tree shrew IRBP1197-1211 characterized by retinal degeneration, retinal damage with subretinal Aβ deposits and microglia/macrophage infiltration, and T-cell response, probably by altering important pathways and genes related to bacterial invasion, inflammatory pain, microglial phagocytosis, and lipid and glucose metabolism. The findings advance the knowledge of the pathogenesis and therapeutics of the fovea-involved visual disturbance in human uveitis.

A Novel Tree Shrew Model of Diabetic Retinopathy.

Existing animal models with rod-dominant retinas have shown that hyperglycemia injures neurons, but it is not yet clearly understood how blue cone photoreceptors and retinal ganglion cells (RGCs) deteriorate in patients because of compromised insulin tolerance. In contrast, northern tree shrews (Tupaia Belangeri), one of the closest living relatives of primates, have a cone-dominant retina with short wave sensitivity (SWS) and long wave sensitivity (LWS) cones. Therefore, we injected animals with a single streptozotocin dose (175 mg/kg i.p.) to investigate whether sustained hyperglycemia models the features of human diabetic retinopathy (DR). We used the photopic electroretinogram (ERG) to measure the amplitudes of A and B waves and the photopic negative responses (PhNR) to evaluate cone and RGC function. Retinal flat mounts were prepared for immunohistochemical analysis to count the numbers of neurons with antibodies against cone opsins and RGC specific BRN3a proteins. The levels of the proteins TRIB3, ISR-1, and p-AKT/p-mTOR were measured with western blot. The results demonstrated that tree shrews manifested sustained hyperglycemia leading to a slight but significant loss of SWS cones (12%) and RGCs (20%) 16 weeks after streptozotocin injection. The loss of BRN3a-positive RGCs was also reflected by a 30% decline in BRN3a protein expression. These were accompanied by reduced ERG amplitudes and PhNRs. Importantly, the diabetic retinas demonstrated increased expression of TRIB3 and level of p-AKT/p-mTOR axis but reduced level of IRS-1 protein. Therefore, a new non-primate model of DR with SWS cone and RGC dysfunction lays the foundation to better understand retinal pathophysiology at the molecular level and opens an avenue for improving the research on the treatment of human eye diseases.

Cone photoreceptor reflectance variation in the northern tree shrew and thirteen-lined ground squirrel.

In vivo images of human cone photoreceptors have been shown to vary in their reflectance both spatially and temporally. While it is generally accepted that the unique anatomy and physiology of the photoreceptors themselves drives this behavior, the exact mechanisms have not been fully elucidated as most studies on these phenomena have been limited to the human retina. Unlike humans, animal models offer the ability to experimentally manipulate the retina and perform direct in vivo and ex vivo comparisons. The thirteen-lined ground squirrel and northern tree shrew are two emerging animal models being used in vision research. Both models feature cone-dominant retinas, overcoming a key limitation of traditional rodent models. Additionally, each possesses unique but well-documented anatomical differences in cone structure compared to human cones, which can be leveraged to further constrain theoretical models of light propagation within photoreceptors. Here we sought to characterize the spatial and temporal reflectance behavior of cones in these species. Adaptive optics scanning light ophthalmoscopy (AOSLO) was used to non-invasively image the photoreceptors of both species at 5 to 10 min intervals over the span of 18 to 25 min. The reflectance of individual cone photoreceptors was measured over time, and images at individual time points were used to assess the variability of cone reflectance across the cone mosaic. Variability in spatial and temporal photoreceptor reflectance was observed in both species, with similar behavior to that seen in human AOSLO images. Despite the unique cone structure in these animals, these data suggest a common origin of photoreceptor reflectance behavior across species. Such data may help constrain models of the cellular origins of photoreceptor reflectance signals. These animal models provide an experimental platform to further explore the morphological origins of light capture and propagation.

Expression patterns of iron regulatory proteins after intense light exposure in a cone-dominated retina.

Iron is implicated in ocular diseases such as in age-related macular degeneration. Light is also considered as a pathological factor in this disease. Earlier, two studies reported the influence of constant light environment on the pattern of expressions of iron-handling proteins. Here, we aimed to see the influence of light in 12-h light-12-h dark (12L:12D) cycles on the expression of iron-handling proteins in chick retina. Chicks were exposed to 400 lx (control) and 5000 lx (experimental) light at 12L:12D cycles and sacrificed at variable timepoints. Retinal ferrous ion (Fe2+) level, ultrastructural changes, lipid peroxidation level, immunolocalization and expression patterns of iron-handling proteins were analysed after light exposure. Both total Fe2+ level (p = 0.0004) and lipid peroxidation (p = 0.002) significantly increased at 12-, 48- and 168-h timepoint (for Fe2+) and 48- and 168-h timepoint (for lipid peroxidation), and there were degenerative retinal changes after 168 h of light exposure. Intense light exposure led to an increase in the levels of transferrin and transferrin receptor-1 (at 168-h) and ferroportin-1, whereas the levels of ferritins, hephaestin, (at 24-, 48- and 168-h timepoint) and ceruloplasmin (at 168-h timepoint) were decreased. These changes in iron-handling proteins after light exposure are likely due to a disturbance in the iron storage pool evident from decreased ferritin levels, which would result in increased intracellular Fe2+ levels. To counteract this, Fe2+ is released into the extracellular space, an observation supported by increased expression of ferroportin-1. Ceruloplasmin was able to convert Fe2+ into Fe3+ until 48 h of light exposure, but its decreased expression with time (at 168-h timepoint) resulted in increased extracellular Fe2+ that might have caused oxidative stress and retinal cell damage.

Loss of Class III Phosphoinositide 3-Kinase Vps34 Results in Cone Degeneration.

Simple SummaryCone photoreceptors are the class of neurons in the retina that support daylight and color vision. In humans and rodents, the cone photoreceptors constitute a small percentage of total retinal photoreceptors; in some retinal diseases, these cells malfunction over time and cease to work, and eventually die. Class III phosphoinositide 3-kinase, also known as vacuole protein sorting 34 (Vps34), generates phosphoinositide 3-phosphate (PI(3)P), a lipid molecule that transmits information inside of the cell. PI(3)P plays an essential role in removing injured cells, a process called autophagy, which maintains a healthy environment, as well as in protein trafficking inside of the cell. Furthermore, PI(3)P can act as a bridging molecule for proteins to bind to each other. We eliminated the class III phosphoinositide 3-kinase in mouse cones, which resulted in the loss of visual function and death of cone cells. Our studies suggest that PI(3)P generated by class III phosphoinositide 3-kinase is essential for cone photoreceptor function and survival.The major pathway for the production of the low-abundance membrane lipid phosphatidylinositol 3-phosphate (PI(3)P) synthesis is catalyzed by class III phosphoinositide 3-kinase (PI3K) Vps34. The absence of Vps34 was previously found to disrupt autophagy and other membrane-trafficking pathways in some sensory neurons, but the roles of phosphatidylinositol 3-phosphate and Vps34 in cone photoreceptor cells have not previously been explored. We found that the deletion of Vps34 in neighboring rods in mouse retina did not disrupt cone function up to 8 weeks after birth, despite diminished rod function. Immunoblotting and lipid analysis of cones isolated from the cone-dominant retinas of the neural retina leucine zipper gene knockout mice revealed that both PI(3)P and Vps34 protein are present in mouse cones. To determine whether Vps34 and PI(3)P are important for cone function, we conditionally deleted Vps34 in cone photoreceptor cells of the mouse retina. Overall retinal morphology and rod function appeared to be unaffected. However, the loss of Vps34 in cones resulted in the loss of structure and function. There was a substantial reduction throughout the retina in the number of cones staining for M-opsin, S-opsin, cone arrestin, and peanut agglutinin, revealing degeneration of cones. These studies indicate that class III PI3K, and presumably PI(3)P, play essential roles in cone photoreceptor cell function and survival.

Noninvasive imaging of the tree shrew eye: Wavefront analysis and retinal imaging with correlative histology

Tree shrews are small mammals with excellent vision and are closely related to primates. They have been used extensively as a model for studying refractive development, myopia, and central visual processing and are becoming an important model for vision research. Their cone dominant retina (∼95% cones) provides a potential avenue to create new damage/disease models of human macular pathology and to monitor progression or treatment response. To continue the development of the tree shrew as an animal model, we provide here the first measurements of higher order aberrations along with adaptive optics scanning light ophthalmoscopy (AOSLO) images of the photoreceptor mosaic in the tree shrew retina. To compare intra-animal in vivo and ex vivo cone density measurements, the AOSLO images were matched to whole-mount immunofluorescence microscopy. Analysis of the tree shrew wavefront indicated that the optics are well-matched to the sampling of the cone mosaic and is consistent with the suggestion that juvenile tree shrews are nearly emmetropic (slightly hyperopic). Compared with in vivo measurements, consistently higher cone density was measured ex vivo, likely due to tissue shrinkage during histological processing. Tree shrews also possess massive mitochondria (“megamitochondria”) in their cone inner segments, providing a natural model to assess how mitochondrial size affects in vivo retinal imagery. Intra-animal in vivo and ex vivo axial distance measurements were made in the outer retina with optical coherence tomography (OCT) and transmission electron microscopy (TEM), respectively, to determine the origin of sub-cellular cone reflectivity seen on OCT. These results demonstrate that these megamitochondria create an additional hyper-reflective outer retinal reflective band in OCT images. The ability to use noninvasive retinal imaging in tree shrews supports development of this species as a model of cone disorders.

Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors

Long-chain PUFAs (LC-PUFAs; C20-C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C 28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

Distribution and diversity of intrinsically photosensitive retinal ganglion cells in tree shrew.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate the pupillary light reflex, circadian entrainment, and may contribute to luminance and color perception. The diversity of ipRGCs varies from rodents to primates, suggesting differences in their contributions to retinal output. To further understand the variability in their organization and diversity across species, we used immunohistochemical methods to examine ipRGCs in tree shrew (Tupaia belangeri). Tree shrews share membership in the same clade, or evolutionary branch, as rodents and primates. They are highly visual, diurnal animals with a cone-dominated retina and a geniculo-cortical organization resembling that of primates. We identified cells with morphological similarities to M1 and M2 cells described previously in rodents and primates. M1-like cells typically had somas in the ganglion cell layer, with 23% displaced to the inner nuclear layer (INL). However, unlike M1 cells, they had bistratified dendritic fields ramifying in S1 and S5 that collectively tiled space. M2-like cells had dendritic fields restricted to S5 that were smaller and more densely branching. A novel third type of melanopsin immunopositive cell was identified. These cells had somata exclusively in the INL and monostratified dendritic fields restricted to S1 that tiled space. Surprisingly, these cells immunolabeled for tyrosine hydroxylase, a key component in dopamine synthesis. These cells immunolabeled for an RGC marker, not amacrine cell markers, suggesting that they are dopaminergic ipRGCs. We found no evidence for M4 or M5 ipRGCs, described previously in rodents. These results identify some organizational features of the ipRGC system that are canonical versus species-specific.

The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina.

The tumor form of pyruvate kinase M2 (PKM2) undergoes tyrosine phosphorylation and gives rise to the Warburg effect. The Warburg effect defines a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose, even in the presence of oxygen. Retinal photoreceptors are highly metabolic and their energy consumption is equivalent to that of a multiplying tumor cell. In the present study, we found that PKM2 is the predominant isoform in both rod- and cone-dominant retina, and that it undergoes a light-dependent tyrosine phosphorylation. We also discovered that PKM2 phosphorylation is signaled through photobleaching of rhodopsin. Our findings suggest that phosphoinositide 3-kinase activation promotes PKM2 phosphorylation. Light and tyrosine phosphorylation appear to regulate PKM2 to provide a metabolic advantage to photoreceptor cells, thereby promoting cell survival.

Immunohistochemical Localization of GFAP and Glutamate Regulatory Proteins in Chick Retina and Their Levels of Expressions in Altered Photoperiods.

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate-aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.

Expressions of visual pigments and synaptic proteins in neonatal chick retina exposed to light of variable photoperiods.

Light causes damage to the retina, which is one of the supposed factors for age-related macular degeneration in human. Some animal species show drastic retinal changes when exposed to intense light (e.g. albino rats). Although birds have a pigmented retina, few reports indicated its susceptibility to light damage. To know how light influences a cone-dominated retina (as is the case with human), we examined the effects of moderate light intensity on the retina of white Leghorn chicks (Gallus g. domesticus). The newly hatched chicks were initially acclimatized at 500 lux for 7 days in 12 h light: 12 h dark cycles (12L:12D). From posthatch day (PH) 8 until PH 30, they were exposed to 2000 lux at 12L:12D, 18L:6D (prolonged light) and 24L:0D (constant light) conditions. The retinas were processed for transmission electron microscopy and the level of expressions of rhodopsin, S- and L/M cone opsins, and synaptic proteins (Synaptophysin and PSD-95) were determined by immunohistochemistry and Western blotting. Rearing in 24L:0D condition caused disorganization of photoreceptor outer segments. Consequently, there were significantly decreased expressions of opsins and synaptic proteins, compared to those seen in 12L:12D and 18L:6D conditions. Also, there were ultrastructural changes in outer and inner plexiform layer (OPL, IPL) of the retinas exposed to 24L:0D condition. Our data indicate that the cone-dominated chick retina is affected in constant light condition, with changes (decreased) in opsin levels. Also, photoreceptor alterations lead to an overall decrease in synaptic protein expressions in OPL and IPL and death of degenerated axonal processes in IPL.

α-Melanocyte-stimulating hormone prevents glutamate excitotoxicity in developing chicken retina via MC4R-mediated down-regulation of microRNA-194.

Glutamate excitotoxicity is a common pathology to blinding ischemic retinopathies, such as diabetic retinopathy, glaucoma, and central retinal vein or artery occlusion. The development of an effective interventional modality to glutamate excitotoxicity is hence important to preventing blindness. Herein we showed that α-melanocyte-stimulating hormone (α-MSH) time-dependently protected against glutamate-induced cell death and tissue damage in an improved embryonic chicken retinal explant culture system. α-MSH down-regulated microRNA-194 (miR-194) expression during the glutamate excitotoxicity in the retinal explants. Furthermore, pharmacological antagonists to melanocortin 4 receptor (MC4R) and lentivirus-mediated overexpression of pre-miR-194 abrogated the suppressing effects of α-MSH on glutamate-induced activities of caspase 3 or 7, the ultimate enzymes for glutamate-induced cell death. These results suggest that the protective effects of α-MSH may be due to the MC4R mediated-down-regulation of miR-194 during the glutamate-induced excitotoxicity. Finally, α-MSH attenuated cell death and recovered visual functions in glutamate-stimulated post-hatch chick retinas. These results demonstrate the previously undescribed protective effects of α-MSH against glutamate-induced excitotoxic cell death in the cone-dominated retina both in vitro and in vivo, and indicate a novel molecular mechanism linking MC4R-mediated signaling to miR-194.

Identification of the 11- cis- specific retinyl-ester synthase in retinal Müller cells as multifunctional O- acyltransferase (MFAT)

Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.

Die Fledermaus: regarding optokinetic contrast sensitivity and light-adaptation, chicks are mice with wings.

BackgroundThrough adaptation, animals can function visually under an extremely broad range of light intensities. Light adaptation starts in the retina, through shifts in photoreceptor sensitivity and kinetics plus modulation of visual processing in retinal circuits. Although considerable research has been conducted on retinal adaptation in nocturnal species with rod-dominated retinas, such as the mouse, little is known about how cone-dominated avian retinas adapt to changes in mean light intensity.Methodology/Principal FindingsWe used the optokinetic response to characterize contrast sensitivity (CS) in the chick retina as a function of spatial frequency and temporal frequency at different mean light intensities. We found that: 1) daytime, cone-driven CS was tuned to spatial frequency; 2) nighttime, presumably rod-driven CS was tuned to temporal frequency and spatial frequency; 3) daytime, presumably cone-driven CS at threshold intensity was invariant with temporal and spatial frequency; and 4) daytime photopic CS was invariant with clock time.Conclusion/SignificanceLight- and dark-adaptational changes in CS were investigated comprehensively for the first time in the cone-dominated retina of an avian, diurnal species. The chick retina, like the mouse retina, adapts by using a “day/night” or “cone/rod” switch in tuning preference during changes in lighting conditions. The chick optokinetic response is an attractive model for noninvasive, behavioral studies of adaptation in retinal circuitry in health and disease.

The cone-dominant retina and the inner ear of zebrafish express the ortholog of CLRN1, the causative gene of human Usher syndrome type 3A

Clarin-1 (CLRN1) is the causative gene in Usher syndrome type 3A, an autosomal recessive disorder characterized by progressive vision and hearing loss. CLRN1 encodes Clarin-1, a glycoprotein with homology to the tetraspanin family of proteins. Previous cell culture studies suggest that Clarin-1 localizes to the plasma membrane and interacts with the cytoskeleton. Mouse models demonstrate a role for the protein in mechanosensory hair bundle integrity, but the function of Clarin-1 in hearing remains unclear. Even less is known of its role in vision, because the Clrn1 knockout mouse does not exhibit a retinal phenotype and expression studies in murine retinas have provided conflicting results. Here, we describe cloning and expression analysis of the zebrafish clrn1 gene, and report protein localization of Clarin-1 in auditory and visual cells from embryonic through adult stages. We detect clrn1 transcripts as early as 24h post-fertilization, and expression is maintained through adulthood. In situ hybridization experiments show clrn1 transcripts enriched in mechanosensory hair cells and supporting cells of the inner ear and lateral line organ, photoreceptors, and cells of the inner retina. In mechanosensory hair cells, Clarin-1 is polarized to the apical cell body and the synapses. In the retina, Clarin-1 localizes to lateral cell contacts between photoreceptors and is associated with the outer limiting membrane and subapical processes emanating from Müller glial cells. We also find Clarin-1 protein in the outer plexiform, inner nuclear and ganglion cell layers of the retina. Given the importance of Clarin-1 function in the human retina, it is imperative to find an animal model with a comparable requirement. Our data provide a foundation for exploring the role of Clarin-1 in retinal cell function and survival in a diurnal, cone-dominant species.

Rod Photoreceptors Protect from Cone Degeneration-Induced Retinal Remodeling and Restore Visual Responses in Zebrafish

Humans are largely dependent upon cone-mediated vision. However, death or dysfunction of rods, the predominant photoreceptor subtype, results in secondary loss of cones, remodeling of retinal circuitry, and blindness. The changes in circuitry may contribute to the vision deficit and undermine attempts at restoring sight. We exploit zebrafish larvae as a genetic model to specifically characterize changes associated with photoreceptor degenerations in a cone-dominated retina. Photoreceptors form synapses with two types of second-order neurons, bipolar cells, and horizontal cells. Using cell-specific reporter gene expression and immunolabeling for postsynaptic glutamate receptors, significant remodeling is observed following cone degeneration in the pde6c(w59) larval retina but not rod degeneration in the Xops:mCFP(q13) line. In adults, rods and cones are present in approximately equal numbers, and in pde6c(w59) mutants glutamate receptor expression and synaptic structures in the outer plexiform layer are preserved, and visual responses are gained in these once blind fish. We propose that the abundance of rods in the adult protects the retina from cone degeneration-induced remodeling. We test this hypothesis by genetically manipulating the number of rods in larvae. We show that an increased number and uniform distribution of rods in lor/tbx2b(p25bbtl) or six7 morpholino-injected larvae protect from pde6c(w59)-induced secondary changes. The observations that remodeling is a common consequence of photoreceptor death across species, and that in zebrafish a small number of surviving photoreceptors afford protection from degeneration-induced changes, provides a model for systematic analysis of factors that slow or even prevent the secondary deteriorations associated with neural degenerative disease.

Cone outer segment extracellular matrix as binding domain for interphotoreceptor retinoid‐binding protein

Cones are critically dependent on interphotoreceptor retinoid-binding protein (IRBP) for retinoid delivery in the visual cycle. Cone-dominant vertebrates offer an opportunity to uncover the molecular basis of IRBP's role in this process. Here, we explore the association of IRBP with the interphotoreceptor matrix (IPM) of cones vs. rods in cone dominant retinas from chicken (Gallus domesticus), turkey (Meleagris gallopavo), and pig (Sus scrofa). Retinas were detached and fixed directly or washed in saline prior to fixation. Disassociated photoreceptors with adherent matrix were also prepared. Under 2 mM CaCl(2) , insoluble matrix was delaminated from saline washed retinas. The distribution of IRBP, as well as glycans binding peanut agglutinin (cone matrix) and wheat germ agglutinin (rod/cone matrix), was defined by confocal microscopy. Retina flat mounts showed IRBP diffusely distributed in an interconnecting, lattice-like pattern throughout the entire matrix. Saline wash replaced this pattern with fluorescent annuli surrounding individual cone outer segments. In isolated cones and matrix sheets, IRBP colocalized with the peanut agglutinin binding matrix glycans. Our results reveal a wash-resistant association of IRBP with a matrix domain immediately surrounding cone outer segments. The cone matrix sheath may be responsible for IRBP-mediated cone targeting of 11-cis retinoids.

A rod-dominated visual system in leptocephalus larvae of elopomorph fishes (Elopomorpha: Teleostei)

The nature and distributions of photoreceptor cell types were investigated in the retinas of 12 species (5 families) of elopomorph anguilliform leptocephalus larvae. Anti-opsin immunofluorescence, light microscopy and transmission electron microscopy (TEM) were used to assess opsin distribution across the retinas and to associate photoreceptor morphology and opsin content. Retinas of all species were immunoreactive with anti-rhodopsin throughout, while anti-cone opsin immunoreactivity was restricted only to the ventral region of the retina in all specimens. Rod and cone photoreceptors were morphologically indistinguishable at low magnifications; TEM revealed that nearly all photoreceptors had rod-like ultrastructure, with only rare examples of cone-like cells identified in the ventral retina. These results indicate a rhodopsin/rod-dominated retina in leptocephalus larvae of anguilliform eels in the teleost subdivision Elopomorpha, contrasting with the cone-dominated retinas of nearly all other species of teleost larvae. This distinctive developmental pattern shared among elopomorph larvae has important evolutionary and ecological implications, indicating a shared ancestor and/or ecological characteristics that are very different from most other teleost larvae.

MicroRNA Profile of the Developing Mouse Retina

Purpose. MicroRNAs (miRNAs) are short, noncoding transcripts that negatively regulate gene expression. They are implicated in diverse cellular processes. The purpose of this study was to obtain a global expression profile of miRNAs in the developing retina and identify differences in miRNA expression between adult rod and cone photoreceptors. Methods. Locked nucleic acid (LNA) microarrays were used to investigate the miRNA transcriptome of the developing mouse retina and brain. Real-time PCR was used to validate the array findings. Laser capture microdissection was used to determine the miRNA spatial pattern of expression. Results. One hundred thirty-eight miRNAs were expressed at at least one of the investigated time points. Several miRNAs showed significant changes in expression between embryonic day 15 and adult age in both retina and brain. Cluster analysis identified subgroups of miRNAs showing defined expression profiles. Globally, correlation of expression was higher, with increasing sequence similarity of the mature miRNAs. The miRNAs with identical seed sequences exhibited highly correlated expression profiles. The co-expression of selected host gene and intronic miRNA pairs was confirmed in adult retina. In some cases, expression profiles of miRNAs showed weak correlation with those of their host transcripts, suggesting posttranscriptional regulation of miRNAs during development. In addition, the miRNA transcriptome of rod- and cone-dominant retinas showed only minor differences, and no miRNAs specific for either cell-type were identified. Conclusions. Global expression profiling revealed dozens of miRNAs with significant expression changes in the developing retina. Precise patterns of expression of miRNAs suggest their specific roles in development.