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PubMed1996

Development of the pattern of photoreceptors in the chick retina.

The various classes of photoreceptor cells found in vertebrate retinae are organized in specific patterns, which are important for visual function. It is not known how these patterns are achieved during development. The chick retina provides an excellent model system in which to investigate this issue, containing cone opsins red, green, blue, and violet, as well as the rod-specific opsin rhodopsin. In this study, whole-mount in situ hybridization has revealed striking differences among opsins in both spatial and temporal aspects of expression. The long-wavelength cone opsins, red and green, were first detected in a small spot within the area centralis at embryonic day 14 (E14). In contrast, the short-wavelength cone opsins, blue and violet, were not detected until 2 d later and showed domains of expression both within the area centralis and in temporal retina. The first rhodopsin transcripts were seen at E15 in inferior retina. When opsin expression was first detected, there were differences in the localization of RNA within the inner segment of cone photoreceptors, suggesting that morphological differentiation preceded the expression of photopigment molecules. Marked differences in the distribution of rods and cones were also found. Within the area centralis, a circular rod-free zone bisected by a narrow rod-sparse region along the nasal-temporal axis was evident as soon as rhodopsin RNA could be detected. Such specialized regions appear to be set aside soon after photoreceptor cells become postmitotic, as evidenced by a spatially restricted pattern of visinin RNA observed at E7. The onset of particular opsins in restricted regions of the retina suggest an underlying pattern related to visual function in the chick.

Animals↗
PubMed2004

Chromatin immunoprecipitation assay on the rainbow trout opsin proximal promoters illustrates binding of NF-kappaB and c-jun to the SWS1 promoter in the retina.

Misexpression of opsins has been linked to apoptosis of photoreceptor cells in the vertebrate retina. Salmonid fish lose their ultraviolet-sensitive (UVS) cones through post-natal developmental apoptosis mediated by thyroid hormone (TH). In order to identify genetic mechanisms that may play a role in the loss of UVS cones, the transcriptional regulation of the SWS1 opsin in the rainbow trout (Oncorhynchus mykiss) was investigated. The Transfac database was interrogated with promoter sequence acquired by genome-walking PCR using MatInspector V2.2 to identify putative transcription factor (TF) binding sites. Putative binding sites for AP-1 (c-jun) and NF-kappaB were found in the SWS1 opsin promoter and were chosen for further investigation due to their high MatInspector scores, their established role in photoreceptor apoptosis, and their relative exclusion from other opsin promoters. NF-kappaB and c-jun proteins were visualized in rainbow trout retinal tissue with immunohistochemistry and c-jun was identified in rainbow trout retinal protein homogenate by immunoblot. A chromatin immunoprecipitation-polymerase chain reaction technique was employed to examine the in vivo interaction of c-jun and NF-kappaB proteins with their proposed binding sites in the opsin promoters. This analysis demonstrated that NF-kappaB and c-jun bind to the SWS1 opsin promoter, but not to the other rod and cone opsin promoters tested. Given the role of NF-kappaB and c-jun during photoreceptor apoptosis, the influence of their activity through TH and their selective binding to the SWS1 opsin promoter in rainbow trout, these TFs represent good candidates of mechanisms underlying UVS cone degeneration in salmonids.

Animals↗
PubMed1984

Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors.

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin-ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.

Age Factors↗
PubMed1996

Ret 1, a cis-acting element of the rat opsin promoter, can direct gene expression in rod photoreceptors.

The Ret 1 element, located at -136 to -110 in the rat opsin promoter, binds developmentally regulated retinal nuclear proteins. A similar sequence is found up-stream of opsin genes, from humans to Drosophila, as well as many other photoreceptor-specific genes. The function of the Ret 1 element was tested both in vitro and in two sets of transgenic mice. A mutated Ret 1 element did not bind retinal nuclear proteins in vitro. The same mutations in an otherwise normal 1.9-kb rat opsin promoter failed to drive expression of a lacZ reporter gene in nine of 12 lines. In the three other lines, expression in photoreceptors was very faint. Four tandem copies of the Ret 1 element maintained the Ret 1 binding specificity in vitro and were able to direct expression of a lacZ transgene in photoreceptors of all nine mouse lines obtained. In two lines, expression was also detected in the ganglion cell layer and the ciliary epithelium. In three lines, a characteristic pattern of expression was found in the nervous system in addition to the normal retinal expression. These results indicate that Ret 1 can and is necessary to drive gene expression in rod photoreceptors. Furthermore, our results suggest that Ret 1-like elements may also be important in the developing nervous system.

Animals↗
PubMed1998

Evolution of colour vision in vertebrates.

The expression of five major families of visual pigments occurred early in vertebrae evolution, probably about 350-400 million years ago, before the separation of the major vertebrate classes. Phylogenetic analysis of opsin gene sequences suggests that the ancestral pigments were cone pigments, with rod pigments evolving last. Modern teleosts, reptiles and birds have genera that possess rods and four spectral classes of cone each representing one of the five visual pigment families. The complement of four spectrally distinct cone classes endows these species with the potential for tetrachromatic colour vision. In contrast, probably because of their nocturnal ancestry, mammals have rod-dominated retinas with colour vision reduced to a basic dichromatic system subserved by only two spectral classes of cone. It is only within primates, about 35 millions years ago, that mammals 're-evolved' a higher level of colour vision: trichromacy. This was achieved by a gene duplication within the longer-wave cone class to produce two spectrally distinct members of the same visual pigment family which, in conjunction with a short-wavelength pigment, provide the three spectral classes of cone necessary to subserve trichromacy.

Animals↗
PubMedDate not supplied

Diurnal rhythm of cone opsin expression in the teleost fish Haplochromis burtoni.

The biochemical and morphological specializations of rod and cone photoreceptors reflect their roles in sight. The apoprotein opsin, which converts photons into chemical signals, functions at one end of these highly polarized cells, in the outer segment. Previous work has shown that the mRNA of rod opsin, the opsin specific to rods, is renewed in the outer segment with a diurnal rhythm in the retina of the teleost fish Haplochromis burtoni. Here we show that in the same species, all three cone opsin mRNAs (blue, green, and red) also have a diurnal rhythm of expression. Quantitative real-time polymerase chain reaction (PCR) with primer pairs specific for the cone photoreceptor opsin subtypes was used to detect opsin mRNA abundance in animals sacrificed at 3-h intervals around the clock. All three cone opsins were expressed with diurnal rhythms similar to each other but out of phase with the rod opsin rhythm. Specifically, cone opsin expression occurs at a higher level near the onset of the dark period, when cones are not used for vision. Finally, we found that the rhythm of cone opsin expression in fish appears to be light dependent, as prolonged darkness changes normal diurnal expression patterns.

Animals↗
PubMed2002

Visualization of rod photoreceptor development using GFP-transgenic zebrafish.

Zebrafish retina contains five morphologically distinct classes of photoreceptors, each expressing a distinct type of opsin gene. Molecular mechanisms underlying specification of opsin expression and differentiation among the cell types are largely unknown. This is partly because mutants affected with expression of a particular class of opsin gene are difficult to find. In this study we established the transgenic lines of zebrafish carrying green fluorescent protein (GFP) gene under the 1.1-kb and 3.7-kb upstream regions of the rod-opsin gene. In transgenic fish, GFP expression initiated and proceeded in the same spatiotemporal pattern with rod-opsin gene. The retinal section from adult transgenic fish showed GFP expression throughout the rod cell layer. These results indicate that the proximal 1.1-kb region is sufficient to drive gene expression in all rod photoreceptor cells. These transgenic fish should facilitate screening of mutants affected specifically with rod-opsin expression or rod cell development by visualization of rod cells by GFP.

5' Untranslated Regions↗
PubMed1998

Functional characterization of visual and nonvisual pigments of American chameleon (Anolis carolinensis).

Using only 11-cis 3, 4-dehydroretinal as a chromophore in the pure-cone retina, American chameleon (Anolis carolinensis) detects a wide range of color from ultraviolet (UV) to infrared. We previously characterized its visual opsin genes sws1Ac, sws2Ac, rh1Ac, rh2Ac, and LwsAc that encode SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, and LWSAc opsins, respectively, and the pineal gland-specific opsin (PAc) gene. Here we present the light absorption profiles of the visual pigments obtained by expressing these opsins and reconstituting them with 11-cis retinal using the COS1 cell cDNA expression system. The purified SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, LWSAc, and PAc pigments have the wavelengths of maximal absorption at 358, 437, 491, 495, 560, and 482 nm, respectively. SWS1Ac is the first vertebrate UV opsin whose spectral sensitivity has been directly evaluated. RH1 pigments, orthologous to the rod pigments of other vertebrates, are sensitive to hydroxylamine in the dark, exhibiting a cone pigment-like characteristic, probably reflecting their adaptation to the pure cone retina. Interestingly, the blue-sensitive SWS2Ac pigment shows an exceptionally low level of sensitivity to hydroxylamine, possessing a rod pigment-like characteristic.

Animals↗
PubMed1992

Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.

With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes.

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.

Amino Acid Sequence↗
PubMed1994

Photoreceptor outer segment development in Xenopus laevis: influence of the pigment epithelium.

Opsin gene expression, synthesis, and photoreceptor outer segment morphology were evaluated during retinal development in Xenopus laevis. Retinal rudiments were harvested during in vivo development from embryonic stages 31 through 46 or were allowed to develop in vitro after removal from stage 33/34 embryos for 1, 2, or 3 days either with or without an investing pigment epithelium. Opsin mRNA was detected at stage 33/34 and the transcript level increased until stage 40 and remained at this level through stage 46. Opsin was first detected at stage 37/38 and progressively increased through stage 46. Rudimentary photoreceptor outer segment membranes occasionally appeared as early as stage 33/34 and they gradually increased in length, forming well-defined stacks of collapsed membranous saccules (discs) during in vivo development. The maturation of eye rudiments in culture was followed to determine how closely in vivo and in vitro development compare and to examine the ability of photoreceptors to differentiate when maintained in the absence of an overlying pigment epithelium (PE) layer. With the PE present, opsin mRNA as well as opsin content steadily increased over the entire culture period. After 1 day of culture, short cilia with minimal amounts of outer segment membranous material were present. By Day 3, the degree of outer segment differentiation corresponded morphologically to approximately stage 43 of in vivo development. When cultured in the absence of an investing PE, the opsin mRNA level increased minimally during the 3 days in culture. Opsin content increased, yet the relative amount was approximately 50% less than that present in retinas developing in the presence of the PE. Membranous material was elaborated; however, the outer segments appeared to be highly disorganized and formed whorl-like structures rather than the normal stacked disc morphology. These results suggest that the PE may be involved in regulating opsin at the transcriptional and/or translational levels and also participates in the organization of rod outer segment membranes.

Animals↗
PubMed1991

Purification of retinol dehydrogenase from bovine retinal rod outer segments.

We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds. In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37-kDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140.

Alcohol Oxidoreductases↗
PubMed2006

Parietal-eye phototransduction components and their potential evolutionary implications.

The parietal-eye photoreceptor is unique because it has two antagonistic light signaling pathways in the same cell-a hyperpolarizing pathway maximally sensitive to blue light and a depolarizing pathway maximally sensitive to green light. Here, we report the molecular components of these two pathways. We found two opsins in the same cell: the blue-sensitive pinopsin and a previously unidentified green-sensitive opsin, which we name parietopsin. Signaling components included gustducin-alpha and Galphao, but not rod or cone transducin-alpha. Single-cell recordings demonstrated that Go mediates the depolarizing response. Gustducin-alpha resembles transducin-alpha functionally and likely mediates the hyperpolarizing response. The parietopsin-Go signaling pair provides clues about how rod and cone phototransduction might have evolved.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1986

Tunicamycin-induced dysgenesis of retinal rod outer segment membranes. I. A scanning electron microscopy study.

Incubation of Xenopus retinas with tunicamycin has been shown to block the glycosylation of opsin, the rod visual pigment apoglycoprotein, with concomitant accumulation of vesicular membrane material in the compartment between the rod inner and outer segments (i.e., the intersegmental space) (Fliesler et al, J Cell Biol 100:574-587, 1985). To further assess the morphology, topology, and cellular origin of this membranous material, Xenopus retinas were incubated in the presence or absence of tunicamycin and the photoreceptor cells were examined by scanning electron microscopy. The material which accumulated in the intersegmental space appeared to be a complex of membranous structures consisting of cisternae with numerous tubular projections, as well as closely associated individual vesicles of various sizes. This tubulo-vesicular material was exclusively associated with the basal surface of the rod outer segment. The connecting cilium, periciliary ridge complex, and the apical surface of the rod inner segment were devoid of such membrane material. Nascent (open) discs (i.e., evaginations of the plasma membrane at the base of the outer segment) often observed in control retinas were not present in tunicamycin-treated tissue. These results support the hypothesis that the membranous material which accumulates in the intersegmental space of rods in tunicamycin-treated retinas represents incompletely and aberrantly formed nascent disc membranes. The formation of this material is apparently a consequence of a deficiency in newly synthesized, asparagine-linked membrane glycoconjugates (e.g., the oligosaccharide chains of opsin) at the site of disc assembly.

Animals↗
PubMed1994

Decreased opsin mRNA and immunoreactivity in progressive rod-cone degeneration (prcd): cytochemical studies of early disease and degeneration.

Opsin mRNA level and immunoreactivity were examined by in situ hybridization and immunocytochemistry in normal and progressive rod cone degeneration (prcd)-affected dogs. In situ hybridization used 35S- and/or 3H-labeled bovine opsin cRNA probes; immunocytochemistry used six monoclonal mouse anti-bovine opsin antibodies (MAb1) that are specific to different regions of the N-terminal, loop v-vi and the C-terminal domains. Optimal labeling and histological resolution at the single cell level were achieved with semi-thin sections of DGD wax-embedded tissues; it was possible to correlate the cytochemical observations with the disease staging in topographically defined regions that exhibited different disease severity. In early disease (stages 0-1), opsin mRNA levels and immunoreactivity were normal. During the transition from disease to degeneration (stage 2), however, opsin mRNA was reduced sharply; it then rapidly became undetectable in late stages of degeneration (stages 3, 4). Reduction of immunoreactivity was seen with all the MAbs in stages 2 and 3, but the degree of reduction varied remarkably in different regions of the protein molecule; immunoreactivity was reduced more in the cytoplasmic regions, particularly in the phosphorylation sites and the far end of the C-terminal domain. In contrast, the epitopes of the N-terminal domain that are located in the intradiscal compartment were better preserved. It is noteworthy that, in stages 2 and 3, many rod cells still survived despite the decrease in the mRNA level and immunoreactivity. The results indicate that early disease in prcd-affected rods is not initiated by a reduction of opsin mRNA or the protein quantity. However, opsin expression disappears in early degeneration and before cell death. The differences in immunoreactivity with disease may result either from alterations in the protein structure or configuration, or from selective loss of epitopes located in the cytoplasmic domains of the molecule.

Animals↗
PubMed2005

Lens opacity and photoreceptor degeneration in the zebrafish lens opaque mutant.

The zebrafish lens opaque (lop) mutant was identified in a chemical mutagenesis screen. The lop mutant, which develops normally through 4 days postfertilization (dpf), exhibits several signs of lens and retinal degeneration at 7 dpf. Histology revealed disrupted lens fibers and increased numbers of nucleated cells within the mutant lens and anterior chamber. The mutant lens also exhibited aberrant epithelial cell morphologies and lacked a definitive transition zone, which suggests that secondary fiber differentiation was interrupted. In addition, the mutant exhibits severely reduced photoreceptors and a reduction in the number of horizontal cells at 7 dpf. Other retinal cell classes appeared unaffected in the mutant. Transmission electron microscopy and opsin immunohistochemistry showed that the different photoreceptor types were generated at the retinal margin, but the rods and cones failed to mature and disappeared. The mutant lens and retina also displayed increased cell proliferation based on proliferating cell nuclear antigen immunolabeling, suggesting that the lens opacity was due to unregulated cell proliferation and undifferentiated cell accumulation within the mutant lens. The lop mutant phenotype supports recent studies showing the lens has a role in regulating teleost retinal development.

Animals↗
PubMed1999

Influence of UVA light stress on photoreceptor cell metabolism: decreased rates of rhodopsin regeneration and opsin synthesis.

There is considerable evidence indicating that rhodopsin is the chromophore mediating light damage to the rat retina caused by exposure to mid-visible wavelengths. Retinal damage is, however, more effectively produced by short-wavelength light, and little is known about the initiating events for this damage class. The present study sought to determine the involvement of rhodopsin bleaching in short-wavelength damage by examining rhodopsin levels and opsin synthesis at early time points following acute ultraviolet-A (UVA) exposures of the pigmented rat eye. A gradual decline in rhodopsin to 8% of the level in non-exposed control eyes occurred over a 1 hr exposure to 1500 microW cm-2of UVA light. When animals were placed in darkness following this exposure, rhodopsin had recovered to only 27% of control levels by 2 hr post-exposure indicating a very slow rate of regeneration. For later time points, animals were returned to dim cyclic light and by 2 days following exposure, rhodopsin levels had risen to 57% of control. In contrast, opsin levels at this same time point were unaffected by UVA exposure. Other observations indicating the UVA exposure affected photoreceptor cell metabolism included a 27% decrease in the rate of opsin synthesis between 1 and 2 days following exposure, and a 69% reduction in the rate of rod outer segment disk renewal during the initial 3 days following exposure. These data show that UVA light stress in the retina causes a gradual bleaching of rhodopsin followed by a slow rate of recovery and altered photoreceptor cell metabolism. These results are consistent with the concept that rhodopsin mediates UVA-induced retinal damage and the possible mechanisms by which this might occur are discussed in relation to alternative hypotheses currently in the literature.

Animals↗