Red Opsin Research Articles

Exploring RPE as a source of photoreceptors: differentiation and integration of transdifferentiating cells grafted into embryonic chick eyes.

To study the possibility of generating photoreceptors through programming RPE transdifferentiation by examining cell differentiation after transplantation into the developing chick eye. RPE was isolated, and the cells were dissociated, cultured, and guided to transdifferentiate by infection with retrovirus expressing neuroD (RCAS-neuroD), using RCAS-green fluorescence protein (GFP) as a control. The cells were then harvested and microinjected into the developing eyes of day 5 to day 7 chick embryos, and their development and integration were analyzed. Cells from the control culture integrated into the host RPE. When grafted cells were present in large number, multilayered RPE-like tissues were formed, and the extra tissues consisted of grafted cells and host cells. None of the cells from the control culture expressed photoreceptor-specific genes. In contrast, most cells from RCAS-neuroD-infected culture remained depigmented. A large number of them expressed photoreceptor-specific genes, such as visinin and opsins. Antibodies against red opsin decorated the apical tips and the cell bodies of the grafted, transdifferentiating cells. In the subretinal space, visinin(+) cells aligned along the RPE or an RPE-like structure. When integrated into the host outer nuclear layer, grafted cells emanated elaborate, axonal arborization into the outer plexiform layer of the host retina. Cultured RPE cells retained their remarkable regenerative capabilities. Cells guided to transdifferentiate along the photoreceptor pathway by neuroD developed a highly ordered cellular structure and could integrate into the outer nuclear layer. These data suggest that, through genetic programming, RPE cells could be a potential source of photoreceptor cells.

Rod and Cone Opsin Mislocalization in an Autopsy Eye From a Carrier of X-linked Retinitis Pigmentosa With a Gly436Asp Mutation in the RPGR Gene

We investigated whether opsin mislocalization occurs in photoreceptors in a female carrier of X-linked retinitis pigmentosa with a Gly436Asp mutation in the retinitis pigmentosa GTPase regulator gene (RPGR). Histologic findings in autopsy eyes from a carrier were compared with those from a normal female. Frozen retinal sections from the periphery of one eye of the carrier and the normal were stained with antibodies against either human red or green opsins, blue cone opsin, or rhodopsin and labeled with fluorochrome conjugated secondary antibodies. Cell nuclei were counterstained with Hoechst dye. Fellow eyes were evaluated with light microscopy. Fluorescent labeling showed mislocalized cone and rod opsins in photoreceptor cells only in the carrier. The carrier also showed some loss of photoreceptor nuclei. A defect in trafficking of opsins to outer segments exists in a carrier with the RPGR Gly436Asp mutation.

Exploring the Molecular Mechanism for Color Distinction in Humans

We examine here the role of the red, green, and blue human opsin structures in modulating the absorption properties of 11-cis-retinal bonded to the protein via a protonated Schiff base (PSB). We built the three-dimensional structures of the human red, green, and blue opsins using homology modeling techniques with the crystal structure of bovine rhodopsin as the template. We then used quantum mechanics (QM) combined with molecular mechanics (MM) (denoted as QM/MM) techniques in conjunction with molecular dynamics to determine how the room temperature molecular structures of the three human color opsin proteins modulate the absorption frequency of the same bound 11-cis-retinal chromophore to account for the differences in the observed absorption spectra. We find that the conformational twisting of the 11-cis-retinal PSB plays an important role in the green to blue opsin shift, whereas the dipolar side chains in the binding pocket play a surprising role of red-shifting the blue opsin with respect to the green opsin, as a fine adjustment to the opsin shift. The dipolar side chains play a large role in the opsin shift from red to green.

Conserved cis-elements in the Xenopus red opsin promoter necessary for cone-specific expression

The long-wavelength sensitive (red) opsin genes encode proteins which play a central role in daytime and color vision in vertebrates. We used transgenic Xenopus to identify 5′ cis-elements in the red cone opsin promoter necessary for cone-specific expression. We found a highly conserved extended region (−725 to −173) that was required for restricting GFP transgene expression to cones. We further identified a short element (5′-CCAATTAAGAGAT-3′) highly conserved amongst tetrapods, including humans, necessary to restrict expression to cones in the retina. These results identify novel conserved elements that regulate spatial expression of tetrapod red cone opsin genes.

Rhodopsin, Violet and Blue Opsin Expressions in the Chick Are Highly Dependent on Tissue and Serum Conditions

The molecular, cellular or tissue environment can influence the expression of genes and thereby regulate processes of tissue formation. Here we determined the tissue and serum dependence of the expression of all photopigments in the chick by a series of distinct retinal cell cultures, analyzed by RT-PCR using specific primers for all four opsins and rhodopsin followed by quantitative scanning of the respective gel bands. For comparison, we first determined expression of all opsins during normal chick retinogenesis, which began with red and violet opsins at E12, shortly followed by blue and green opsins and finally rhodopsin at E14. This period corresponds to the time of synaptogenesis in the inner retina. All cultures were started with 6-day-old dissociated retinal cells. Cells were kept at low or high cell density (called LoDens or HiDens), or they were reaggregated as retinal spheres, whereby all of them were raised at low (2%) or high serum (12%) levels (called LoSer or HiSer). In LoDens/HiSer cultures, expression of all opsins was weak. At HiDens/LoSer red and green opsin expression was strong, while rhodopsin and violet/blue remained low. In HiDens/HiSer cultures the expression of red and green was strong; rhodopsin was almost normal, while violet and green were low. In reaggregates at high serum the expression came closest to a normal retina, but violet and blue opsins were still below normal. At low serum, however, violet and blue were negligible and rhodopsin was low. This in vitro study shows that rhodopsin, followed by violet and blue opsin expressions is highly dependent on serum, cell density and tissue conditions, while red and green opsins are more autonomous.

The signaling pathway in photoresponses that may be mediated by visual pigments in erythrophores of Nile tilapia

The ability to increase the synthesis or vary the distribution of pigment in response to light is an important feature of many pigment cells. Unlike other light-sensitive pigment cells, erythrophores of Nile tilapia change the direction of pigment migration depending on the peak wavelength of incident light: light near 365, 400 or 600 nm induces pigment aggregation, while dispersion occurs in response to light at 500 nm. How these phenomena are achieved is currently unknown. In the present study, the phototransduction involved in the pigment dispersion caused by light at 500 nm or the aggregation by light at 600 nm was examined, using pertussis toxin, cholera toxin, blockers of ion channels, various chemicals affecting serial steps of signaling pathways and membrane-permeable cAMP analog. The results show that light-induced bidirectional movements in tilapia erythrophores may be controlled by cytosolic cAMP levels via Gi- or Gs-type G proteins. In addition, RT-PCR demonstrated for the first time the expression of mRNAs encoding red and green opsins in tilapia fins, only where erythrophores exist. Here, we suggest that multiple cone-type visual pigments may be present in the erythrophores, and that unique cascades in which such opsins couple to Gi or Gs-type G proteins are involved in the photoresponses in these pigment cells. Thus, tilapia erythrophore system seems to be a nice model for understanding the photoresponses of cells other than visual cells.

Coupling and decoupling of evolutionary mode between X- and Y-chromosomal red-green opsin genes in owl monkeys

We previously discovered Y-chromosomal red-green opsin genes in two types of owl monkeys with different chromosomal characteristics. In one type, the Y-linked opsin gene is a single-copy intact gene and in the other, the genes exist as multiple pseudogenes on a Y/autosome fusion chromosome. In the present study, we first distinguished the two types of monkeys as distinct allopatric species on the basis of karyotypic characteristics: Aotus lemurinus griseimembra (Karyotype III, diploid chromosome number [2 n] = 53) and Aotus azarae boliviensis (Karyotype VI; male 2 n = 49; female 2 n = 50), belonging to the northern and southern species groups, respectively, separated by the Amazon River system. Our sequence analysis revealed a common L1-Alu-Alu insertion between the two species in the 3′-flanking region of the X-linked opsin genes. The insertion was absent in the Y-linked opsin genes and in the human red and green opsin genes, indicating that it occurred in the X copy before the split into northern and southern species and after the X to Y duplication, i.e. duplication preceded speciation. We also show that in the northern species, the Y-linked opsin gene has evolved concomitantly with the X-linked copy whereas in the southern species, the Y-autosome fusion possibly led to decoupling evolutionary processes between X- and Y-linked copies and subsequent degeneration and duplications of the Y-linked opsin gene.

Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development

Zebrafish have two red, LWS-1 and LWS-2, and four green, RH2-1, RH2-2, RH2-3 and RH2-4, opsin genes encoding photopigments with distinct absorption spectra. Occurrence of opsin subtypes by gene duplication is characteristic of fish but little is known whether the subtypes are expressed differently in the retina, either spatially or temporally. Here we show by in situ hybridization the dynamic expression patterns of the opsin subtypes in the zebrafish retina. Expression of red type opsins is initiated with the shorter-wavelength subtype LWS-2, followed by the longer-wavelength subtype LWS-1. In the adult retina, LWS-2 was expressed in the central to dorsal area and LWS-1 in the ventral and peripheral areas. Expression patterns of green type opsins were similar to those of the red type opsins. The expression started with the shortest wavelength subtype RH2-1 followed by the longer wavelength ones, and in the adult retina, the shorter wavelength subtypes (RH2-1 and RH2-2) were expressed in the central to dorsal area and longer wavelength subtypes (RH2-3 and RH2-4) in the ventral and peripheral areas. These results provide the framework for subsequent studies of opsin gene regulation and for probing functional rationale of the developmental changes by using the power of zebrafish genetics.

Molecular cloning of cone opsin genes and their expression in the retina of a smelt, Ayu ( Plecoglossus altivelis, Teleostei)

Five cone opsin genes of landlocked ayu fish ( Plecoglossus altivelis) were cloned, and the expression patterns of these genes were investigated. AYU-LWS, -RH2-1, -RH2-2, -SWS1-1, and -SWS1-2 were isolated and had high (more than 75%) identity with red, green, green, UV, and UV-sensitive opsin, respectively, genes of other fish reported previously. The results of Southern blotting experiments showed that each gene is present as a single copy. Gene expression was measured by RT–PCR using four populations collected from rivers and a lake in spring and summer. The results of the RT–PCR experiment showed that AYU-SWS1-2 was highly expressed, whereas AYU-SWS1-1 was scarce. Two RH2 opsins were expressed simultaneously in the same individual, and the expression ratio between these opsins changed among populations. In situ hybridization revealed that AYU-LWS and -RH2-1 were expressed in the double cones and that AYU-RH2-2 and -SWS1-2 were expressed in the long and short single cones (LSC and SSC), respectively. It was shown that an individual ayu expresses two RH2 opsins simultaneously in different types of cone cells.

Signatures of Selection and Gene Conversion Associated with Human Color Vision Variation

Trichromatic color vision in humans results from the combination of red, green, and blue photopigment opsins. Although color vision genes have been the targets of active molecular and psychophysical research on color vision abnormalities, little is known about patterns of normal genetic variation in these genes among global human populations. The current study presents nucleotide sequence analyses and tests of neutrality for a 5.5-kb region of the X-linked long-wave "red" opsin gene (OPN1LW) in 236 individuals from ethnically diverse human populations. Our analysis of the recombination landscape across OPN1LW reveals an unusual haplotype structure associated with amino acid replacement variation in exon 3 that is consistent with gene conversion. Compared with the absence of OPN1LW amino acid replacement fixation since divergence from chimpanzee, the human population exhibits a significant excess of high-frequency OPN1LW replacements. Our results suggest that subtle changes in L-cone opsin wavelength absorption may have been adaptive during human evolution.

Gene duplication and spectral diversification of cone visual pigments of zebrafish.

Zebrafish is becoming a powerful animal model for the study of vision but the genomic organization and variation of its visual opsins have not been fully characterized. We show here that zebrafish has two red (LWS-1 and LWS-2), four green (RH2-1, RH2-2, RH2-3, and RH2-4), and single blue (SWS2) and ultraviolet (SWS1) opsin genes in the genome, among which LWS-2, RH2-2, and RH2-3 are novel. SWS2, LWS-1, and LWS-2 are located in tandem and RH2-1, RH2-2, RH2-3, and RH2-4 form another tandem gene cluster. The peak absorption spectra (lambdamax) of the reconstituted photopigments from the opsin cDNAs differed markedly among them: 558 nm (LWS-1), 548 nm (LWS-2), 467 nm (RH2-1), 476 nm (RH2-2), 488 nm (RH2-3), 505 nm (RH2-4), 355 nm (SWS1), 416 nm (SWS2), and 501 nm (RH1, rod opsin). The quantitative RT-PCR revealed a considerable difference among the opsin genes in the expression level in the retina. The expression of the two red opsin genes and of three green opsin genes, RH2-1, RH2-3, and RH2-4, is significantly lower than that of RH2-2, SWS1, and SWS2. These findings must contribute to our comprehensive understanding of visual capabilities of zebrafish and the evolution of the fish visual system and should become a basis of further studies on expression and developmental regulation of the opsin genes.

Novel missense mutations in red/green opsin genes in congenital color-vision deficiencies

The DNAs from 217 Japanese males with congenital red/green color-vision deficiencies were analyzed. Twenty-three subjects had the normal genotype of a single red gene, followed by a green gene. Four of the 23 were from the 69 protan subject group and 19 of the 23 were from the 148 deutan subject group. Three of the 23 subjects had missense mutations. The mutation Asn94Lys (AAC→AAA) occurred in the single green gene of a deutan subject (A155). The Arg330Gln (CGA→CAA) mutation was detected in both green genes of another deutan subject (A164). The Gly338Glu (GGG→GAG) mutation occurred in the single red gene of a protan subject (A89). Both normal and mutant opsins were expressed in cultured COS-7 cells and visual pigments were regenerated with 11- cis-retinal. The normal red and green opsins showed absorbance spectra with λ max of 560 and 530 nm, respectively, but the three mutant opsins had altered spectra. The mutations in Asn94Lys and Gly338Glu resulted in no absorbance and the Arg330Gln mutation gave a low absorbance spectrum with a λ max of 530 nm. Therefore these three mutant opsins are likely to be affected in the folding process, resulting in a loss of function as a visual pigment.

Microenvironmental Regulation of Visual Pigment Expression in the Chick Retina

Visual pigment (VP) expression in the chick embryo retina was investigated in ovo, in dissociated and explant cultures, and in cDNAs from individual cells. While VP mRNA is not detectable by in situ hybridization until embryonic day (ED) 14–16 in ovo, analysis of VP expression by RT-PCR showed that VP messages are present in the retina as many as 7–10 days before they become detectable by in situ hybridization, and are also detected in other regions of the embryonic CNS. On the other hand, red opsin expression is markedly accelerated when cells are isolated from their intraocular microenvironment at ED 6, and placed in pigment epithelium-free dissociated or explant cultures. This acceleration occurs regardless of cell density, birth date, or serum presence in the medium, suggesting that many photoreceptors are already programmed to express red opsin on or before ED 6, and that microenvironmental inhibitory factors prevent implementation of this program until ED 14 in ovo. The selectivity of this phenomenon is suggested by the finding that other VPs are not observed by in situ hybridization in ED 6 cultures, although they are detectable in cultures of older retinas. Taken together, these findings suggest that red opsin expression may be constitutive for many developing photoreceptor cells in the chick.

Expression of Sonic Hedgehog and Retinal Opsin Genes in Experimentally-induced Myopic Chick Eyes

The purpose of this study was to evaluate changes in the expression of different genes in chick retinal tissues after induction of experimental myopia and to evaluate the roles of these genes in the regulation of postnatal eye growth and myopia. Form-deprivation using occlusive goggles and hyperopic defocus by negative spectacle lenses were used to induce myopia in hatched chicks.Expression levels of Sonic hedgehog, its receptor complex, and other retinal cell genes were evaluated by semi-quantitative reverse transcription-polymerase chain reaction. Levels of Sonic hedgehog protein were further evaluated by Western blot analysis. The induction of myopia caused significant increase in expression of Sonic hedgehog mRNA and protein and increased expression of blue and red opsin mRNA. In contrast, the expression of mRNA for Sonic hedgehog receptor complex (Patched–Smoothened), rhodopsin,vimentin , green opsin, violet opsin, and HPC-1 were unaffected by the induction of myopia. The increase in expression of Sonic hedgehog in chick retinas in experimentally-induced myopia suggests involvement in the retina control of postnatal eye growth. Furthermore, Sonic hedgehog may influence the expression of blue andred opsins under myopic conditions.

The molecular genetics of red and green color vision in mammals.

To elucidate the molecular mechanisms of red-green color vision in mammals, we have cloned and sequenced the red and green opsin cDNAs of cat (Felis catus), horse (Equus caballus), gray squirrel (Sciurus carolinensis), white-tailed deer (Odocoileus virginianus), and guinea pig (Cavia porcellus). These opsins were expressed in COS1 cells and reconstituted with 11-cis-retinal. The purified visual pigments of the cat, horse, squirrel, deer, and guinea pig have lambdamax values at 553, 545, 532, 531, and 516 nm, respectively, which are precise to within +/-1 nm. We also regenerated the "true" red pigment of goldfish (Carassius auratus), which has a lambdamax value at 559 +/- 4 nm. Multiple linear regression analyses show that S180A, H197Y, Y277F, T285A, and A308S shift the lambdamax values of the red and green pigments in mammals toward blue by 7, 28, 7, 15, and 16 nm, respectively, and the reverse amino acid changes toward red by the same extents. The additive effects of these amino acid changes fully explain the red-green color vision in a wide range of mammalian species, goldfish, American chameleon (Anolis carolinensis), and pigeon (Columba livia).

Cloning and characterization of six zebrafish photoreceptor opsin cDNAs and immunolocalization of their corresponding proteins

Zebrafish (Danio rerio) represents an excellent genetic model for vertebrate visual system studies. Because the opsin proteins are ideal markers of specific photoreceptor cell types, we cloned six different zebrafish opsin cDNAs. Based on pairwise alignments and phylogenetic comparisons between the predicted zebrafish opsin amino acid sequences and other vertebrate opsins, the cDNAs encode rhodopsin, two different green opsins (zfgr1 and zfgr2), a red, a blue, and an ultraviolet opsin. Phylogenetic analysis indicates the zfgr1 protein occupies a well-resolved dendrogram branch separate from the other green opsins examined, while zebrafish ultraviolet opsin is closely related to the human blue- and chicken violet-sensitive proteins. Polyclonal antisera were generated against individual bacterial fusion proteins containing either the red, blue, or ultraviolet amino termini or the rod or green opsin carboxyl termini. Immunolocalization on adult zebrafish frozen sections demonstrates the green and red opsins are each expressed in different members of the double cone cell pair, the blue opsin is detected in long single cones, and the ultraviolet opsin protein is expressed in the short single cones. In 120-h postfertilization wholemounts, green, red, blue, and ultraviolet opsin-positive cells are detected in an orderly arrangement throughout the entire retina. The antibodies' photoreceptor-type specificity indicates they will be useful for characterizing both wild-type and mutant zebrafish retinas.

Mutagenesis studies of human red opsin: trp-281 is essential for proper folding and protein-retinal interactions.

Human red and green opsins contain a strikingly large number of tryptophan residues. These tryptophans are highly conserved among all red and green opsins. To investigate possible roles of these tryptophans in folding and structure, we have systematically replaced each tryptophan of human red opsin. When expressed in COS cells, wild-type red opsin undergoes N-linked glycosylation, forms a light-sensitive pigment with absorption maximum at 560 nm upon reconstitution with 11-cis-retinal, and is transported to the plasma membrane. We used the extent of glycosylation, pigment generation, and intracellular localization of mutant red opsins as our criteria for assessing the effect of substitution. Replacement of eight tryptophans, Trp-59, Trp-90, Trp-149, Trp-152, Trp-183, Trp-191, Trp-195, and Trp-243, with Phe or Ala did not affect the wild-type phenotype significantly. However, replacement of Trp-5 and Trp-51 in the putative N-terminal domain and Trp-142, Trp-177, Trp-179, and Trp-281 in the transmembrane domain with Phe had profound effects, indicating that these substitutions affected red opsin folding. Judged by the severity of the effects, we propose that Trp-5, Trp-51, Trp-177, and Trp-281 are important for red opsin folding. Although substitution of Trp-281 with Phe and Cys did not permit normal glycosylation and transport, substitution with Tyr and His permitted these processes but resulted in blue-shifted pigment. Thus, polar aromatics appear to substitute for Trp-281 to allow red opsin folding. The large spectral shift indicates that Trp-281 is essential for the proper interaction of the protein with 11-cis-retinal.

Butyrylcholinesterase Antisense Transfection Increases Apoptosis in Differentiating Retinal Reaggregates of the Chick Embryo

To investigate the roles of the enzymes butyryl- and acetylcholinesterase (BChE and AChE) in retinal proliferation and differentiation, we use reaggregated spheres from retinal cells of the 6-day-old chick embryo, forming cellular and fibrous areas homologous to all layers of a normal retina. Recently, we could suppress BChE expression by transfecting these so-called retinospheroids during their proliferation period with a pSVK3 expression vector containing a 5' fragment of the rabbit BChE gene in antisense orientation. Along with morphological changes, proliferation was significantly decreased. Here, we have studied the effect of antisense BChE suppression during the differentiation period of retinospheroids. As BChE is suppressed, the differentiation of AChE-positive cells is increased, whereas the immunoreactivities for red and green cone-specific opsins are strongly reduced. Concomitantly, the rate of apoptosis as determined by propidium iodide uptake, by increased CPP 32-like caspase expression, and by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA fragmentation assays is roughly doubled, predominantly at the expense of degenerating photoreceptor precursors. This is further strong evidence that the proliferation marker BChE regulates an intricate balance between cell proliferation, cell differentiation, and programmed cell death in this in vitro retinal system.

Cloning and expression of the red visual pigment gene of goat ( Capra hircus)

We have cloned and sequenced the red opsin gene, r Ch , of goat ( Capra hircus). When r Ch is expressed in cultured cells and reconstituted with 11- cis retinal, the resulting visual pigment has a wavelength of maximal absorption ( λmax) of 553 nm. This result and the deduced aa sequence of the goat red opsin suggest that the λmax values of the red pigments of goat and human are based mainly on Y277 and T285. The slightly lower λmax value of the red pigment in goat than in human (~560 nm) can be explained by a single aa difference between the goat (A180) and human (S180) red pigments.

A New Form of Inherited Red-Blindness Identified in Zebrafish

A red-blind zebrafish mutant, partial optokinetic response b (pob), has been isolated by measuring eye movements of larvae in a three-generation screen for recessive mutations affecting the visual system. pob larvae exhibit eye movements in response to rotating black and white stripes illuminated with white light, but they do not move their eyes when the stripes are illuminated with red light. Physiological, immunohistochemical, and in situ hybridization analyses of pob retinas showed a selective loss of red-sensitive cones at 5 days postfertilization (dpf). At 3 dpf, cells expressing red opsin are present, suggesting that red-sensitive cones form initially but then disappear rapidly, whereas other photoreceptors remain. Linkage analysis indicated that the mutation identified in the pob mutant is not at the red opsin locus. Because red opsin is the only known molecule unique to red cones, these data suggest that a novel gene is required for the maintenance or function of red cones.