Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (d N/d S) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur.

Monotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

To understand how the photoreceptor protein rhodopsin performs in its role as a receptor, its structure needs to be determined at the atomic level. Upon receiving a photon of light, rhodopsin undergoes a change in conformation that allows it to bind and activate the C-protein, transducin. An important future goal should be to determine the structure of both the inactive and the photoactivated state of rhodopsin, R*. This should provide the groundwork necessary for experiments on how rhodopsin achieves its signaling state R*, and how R* functions to activate transducin. To do this, the crystal structure of both rhodopsin and R* must be determined. Few membrane proteins have been successfully crystallized, so this is not a trivial undertaking. Two- or three dimensional crystals of rhodopsin must be prepared that are well ordered, to produce a high-resolution structure. Rhodopsin must be purified to homogeneity and the appropriate detergent(s) selected for crystallization experiments. Long-term thermal stability of the rhodopsin-detergent complex must be achieved in the presence of a precipitant. Two-dimensional crystals may offer advantages in investigating the structure of R*, but the structure obtained may be limited in resolution. It is necessary to work with rhodopsin in the dark, unless suitable light-stable retinal derivatives are developed. Protein engineering of rhodopsin offers attractive opportunities to improve its ability to crystallize, but is presently hindered by the absence of a high-yielding expression system. Knowledge of the structure of rhodopsin will have general importance. Because rhodopsin is a member of the family of C-protein-coupled receptors, knowledge of the structure and the mechanism of action of rhodopsin suggests by analogy how other members of the receptor family may function.

Diseases causing inherited retinal degeneration in humans, such as retinitis pigmentosa and macular dystrophy, are genetically heterogeneous and clinically diverse. More than 40 genes causing retinal degeneration have been mapped to specific chromosomal sites; of these, at least 10 have been cloned and characterized. Mutations in two proteins, rhodopsin and peripherin/RDS, account for approximately 35% of all cases of autosomal dominant retinitis pigmentosa and a lesser fraction of other retinal conditions. This target article reviews the genes and mutations causing retinal degeneration and proposes mechanisms whereby specific mutations lead to particular clinical consequences, that is, the relationship between genotype and phenotype. Retinitis pigmentosa and macular dystrophy are genetically heterogeneous diseases that cause retinal degeneration in humans and often result in severe visual impairment or blindness. Although many of the genes causing these diseases have not been identified, three photoreceptor-specific proteins have been implicated: rhodopsin, peripherin/RDS, and the P-subunit of rod phosphodiesterase. Mutations in the genes for these three proteins can cause either dominant retinitis pigmentosa, recessive retinitis pigmentosa, dominant congenital stationary night blindness, or dominant macular degeneration. Why this multiplicity of clinical phenotypes? Our target article summarizes the genetic and biochemical background to this question and proposes a number of possible explanations. Discussion focuses mainly on 73 distinct disease-causing mutations of rhodopsin. We feel that rhodopsin and other photoreceptor proteins can serve as model systems for unraveling the connection between genotype and phenotype, not only for inherited retinal diseases but for other degenerative disorders as well.

This article evaluates each of the reactions known to be involved in visual transduction as a potential site for the regulation of light adaptation. Extensive evidence suggests that calcium acts as a feedback messenger at several different points and recent work suggests a role for cGMP in regulating the primary excitatory pathway. A conclusion is that adaptation is likely to be regulated by multiple and redundant mechanisms. The goal of future experimentation will be to determine the relative importance of each of these.

Following bright flashes, rod photoreceptors exhibit a period of photocurrent saturation that increases linearly with the logarithm of flash intensity. In a recent report, Pepperberg et al. (1992) presented evidence that the slope of the function relating the saturation period (T) to the natural logarithm of flash intensity (In If) represents the exponential lifetime (τ) of photoactivated visual pigment: τ = ΔT/Δ[ln If]. In salamander rods, 11 -cis 9-desmethylretinal combines with opsin to form 9-desmethyl rhodopsin. Dim flash responses mediated by this analogue visual pigment exhibited slow recovery kinetics relative to those of native pigment (Corson et al., 1991). This observation raises the hypothesis that the physiological lifetime of photoactivated 9-desmethyl rhodopsin is substantially longer than that of native visual pigment. To test this hypothesis, we have examined the relation between the period of photocurrent saturation and flash intensity in salamander rods containing a mixture of the two pigments. Brief stimuli at two widely separated wavelengths (440 and 640 nm) elicited saturating photocurrent responses that were preferentially mediated by 9-desmethyl rhodopsin or residual native pigment, respectively. Plots of T vs. In If revealed a linear increase in the period of response saturation over a large range of saturating intensities at both wavelengths. However, the slope of the relation between T and In If with 440-nm flashes was more than twice as large (4.1 ± 0.5 s, n = 5) as that measured with 640-nm flashes (1.7 ± 0.4 s). For rods subjected only to bleaching of the native pigment, or to bleaching and resensitization with 11-cis retinal, the slope of the relation between T and In If remained independent of wavelength and indistinguishable from that of native pigment in unbleached cells. The data provide support for the hypothesis that the slope parameter τ represents the lifetime of photoactivated pigment, and specifically suggest that the lifetime of photoactivated 9-desmethyl rhodopsin is abnormally long.

In vitro mutagenesis and germline transformation were used to create a Drosophila mutant, ΔAsn20 lacking the N-linked glycosylation site near the amino terminus of the major rhodopsin (Asn20-Gly-Ser changed to Ile-Gly-Ser). Low opsin protein levels are detected in ΔAsn20 photoreceptors. Electroretinogram responses of mutant flies show that the residual rhodopsin found in this mutant is capable of initiating phototransduction. The organization of rhabdomeres, the photoreceptor organelle containing nearly all of the rhodopsin, is aberrant in the ΔAsn20 mutant and undergoes age-dependent deterioration. These results establish that an N-linked glycosylation site, and likely glycosylation itself, plays a critical role in the maturation of Drosophila rhodopsin.

In the δAsn20 Drosophila stock, the N-linked glycosylation site of opsin in Rl-6 receptors (Rhl) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rhl. MSP revealed minimal photopigment in δAsn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for δAsn20 and w9. δAsn20's Rl-6 function is decreased 100–fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller Rl-6 rhabdomeres in δAsn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE0117, a deletion of the Rhl gene, also has small rhabdomeres. δAsn20 and ninaE0117 lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rhl's coding sequence. δAsn20 and ora have surplus membrane while ninaE0117 does not. Freeze fracture reveals that δAsn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, δAsn20 interferes with either synthesis, deployment, or maintenance of opsin.

We examined a white-eyed strain of the norpA mutant (norpA;cn bw) and white (w)norpA+ controls using microspectrophotometry (MSP), electron microscopy (EM), and electroretinography (ERG). These studies revealed that light mediates receptor demise in norpA even though norpA lacks phototransduction. Rhodopsin and the rhabdomere which houses it decrease with increasing age in norpA but not in w with rearing on a 12 h light/12-h dark cycle or in constant light. At higher temperature in norpA;cn bw and w reared in constant light, visual pigment decreases, rhabdomeres diminish, and cells die. Importantly, dark rearing blocked visual pigment loss in norpA;cn bw; the M-potential, an ERG reflection of visual pigment level, corroborated this finding. MSP showed that norpA's visual pigment loss was not due to acute loss of metarhodopsin, rhodopsin's photoproduct. NorpA blocks certain processes expected to be light elicited. The alteration of visual pigment as a function of time of day, present in w controls, is absent in white-eyed norpA, suggesting that light-induced depolarization may be necessary to entrain the rhythm. Microspectrofluorometry using the fluorescent dye, Lucifer yellow, suggested that norpA lacks a light-induced uptake mechanism; using control flies, we determined the stimulus parameters required for uptake in vivo. An attempt to “cure” norpA;cn bw by replacement “therapy” using phospholipase C, missing in norpA's phototransduction cascade, was largely unsuccessful.

Visual pigment, sensitivity, and rhabdomere size were measured throughout a 12-h light/12-h dark cycle in Drosophila. Visual pigment and sensitivity were measured during subsequent constant darkness [dark/dark (D/D)]. MSP (microspectrophotometry) and the ERG (electroretinogram) revealed a cycling of visual pigment and sensitivity, respectively. A visual pigment decrease of 40% was noted at 4 h after light onset that recovered 2–4 h later in white-eyed (otherwise wild-type, w per+) flies. The ERG sensitivity [in w per+ flies in light/dark (L/D)] decreased by 75% at 4 h after light onset, more than expected if mediated by visual pigment (MSP) changes alone. ERG sensitivity begins decreasing 8 h before light onset while decreases in visual pigment begin 2 h after light onset. These cycles continue in constant darkness (D/D), suggesting a circadian rhythm. White-eyed period (per) mutants show similar cycles of visual pigment level and sensitivity in L/D; per's alterations, if any on the D/D cycles were subtle. The cross-sectional areas of rhabdomeres in w per+ were measured using electron micrographic (EM) morphometry. Area changed little through the L/D cycle.

The role of 48-kDa protein in Visual transduction remains unresolved. Two hypotheses for its role in quenching the light activation of cyclic GMP cascade suggest that the protein binds to either phosphodiesterase or phosphorylated rhodopsin. Since the protein is also reported to bind ATP, we anticipated that the protein may have ATP hydrolyzing activity, and in analogy with the GTP-binding protein of the rod outer segments, such activity may be greatly enhanced by the elements of transduction cyclic GMP cascade, permitting the protein to function cyclically as GTP-binding protein does. We found that purified 48-kDa protein hydrolyzes ATP but at a slow rate of 0.04–0.05 per min. The Km for ATP is about 45–65 μM. The activity is inhibited noncompetitively by ADP with a Ki of about 50 μM. The ATPase activity of 48-kDa protein is not affected by rhodopsin, bleached rhodopsin, phosphorylated rhodopsin, unactivated cyclic GMP phosphodiesterase, or phosphodiesterase (PDE) activated by GMP PNP-bound G-protein. These data show that although 48-kDa protein has ATPase activity, lack of regulation of this activity by the elements of visual transduction makes it unlikely for this activity to have a role in quenching the light activation of cyclic GMP cascade.

Vision begins with photoisomerization of 11-cis retinal to the all-trans conformation within the chromophore-binding pocket of opsin, leading to activation of a biochemical cascade. Release of all-trans retinal from the binding pocket curtails but does not fully quench the ability of opsin to activate transducin. All-trans retinal and some other analogs, such as β-ionone, enhance opsin’s activity, presumably on binding the empty chromophore-binding pocket. By recording from isolated salamander photoreceptors and from patches of rod outer segment membrane, we now show that high concentrations of β-ionone suppressed circulating current in dark-adapted green-sensitive rods by inhibiting the cyclic nucleotide-gated channels. There were also decreases in circulating current and flash sensitivity, and accelerated flash response kinetics in dark-adapted blue-sensitive (BS) rods and cones, and in ultraviolet-sensitive cones, at concentrations too low to inhibit the channels. These effects persisted in BS rods even after incubation with 9-cis retinal to ensure complete regeneration of their visual pigment. After long exposures to high concentrations of β-ionone, recovery was incomplete unless 9-cis retinal was given, indicating that visual pigment had been bleached. Therefore, we propose that β-ionone activates and bleaches some types of visual pigments, mimicking the effects of light.

The retinas of birds receive a substantial efferent, or centrifugal, input from a midbrain nucleus. The function of this input is presently unclear, but previous work in the pigeon has shown that efferent input is excluded from the area centralis, suggesting that the functions of the area centralis and the efferent system are incompatible. Using an antibody specific to rods, we have identified the area centralis in another species, the chicken, and mapped the distribution of the unique amacrine cells that are the postsynaptic partners of efferent fibers. Efferent target amacrine cells are found within the chicken area centralis and their density is continuous across the border of the area centralis. In contrast to the pigeon retina then, we conclude that the chicken area centralis receives efferent input. We suggest that the difference between the two species is attributable to the presence of a fovea within the area centralis of the pigeon and its absence from that of the chicken.

Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of “pigment adaptation” with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, β-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of β-ionone. To test the two hypotheses, we applied β-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of transducin, whereas all cones contain another. β-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod transducin upon the addition of β-ionone. Thus opsin structure determines the identity of β-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12–14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A nonrecombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

A fast-scanning dichroic microspectrophotometer was used to trace products of rhodopsin photolysis (metarhodopsins I/II/III and later) in structurally intact amphibian rod outer segments (ROSs) and metabolically active rods. The instrument allows the recording of absorbance spectra with a time resolution better than 1 s, and to discriminate between products with similar absorbance spectra that differ with respect to the orientation of their chromophore in the photoreceptor membrane. We demonstrate that metarhodopsin III is in a pH-reversible equilibrium with metarhodopsin II and that the metarhodopsin III chromophore is orientated with respect to the membrane plane even more strictly than the 11-cis retinal in “dark” rhodopsin. This indicates that all-trans retinal in metarhodopsin III is still attached to its native binding site on opsin. The kinetic scheme of the decay of metarhodopsins is presented in which metarhodopsin III lies in a shunt pathway from metarhodopsin II to retinal. Formation of metarhodopsin III was detected at bleaches as low as ≈ 3%, contrary to previous reports that it is not formed at below 10% bleaches. Another product that is spectrally similar to metarhodopsin III, termed P440, appears at later stages of photolysis as the result of the decay of metarhodopsin II and metarhodopsin III. The chromophoric group in P440 is orientated preferentially across the disk membrane. The final product(s) in isolated ROS, where the reduction of retinal to retinol is blocked, consists of a mixture of a free retinal and retinal possibly attached to different binding sites in the membrane. In metabolically active rods the later products are quickly converted to retinol. We conclude that metarhodopsin III represents a specific conformational state of metarhodopsin where the chromophoric binding site is still occupied by all-trans retinal. Hence, the formation and decay of metarhodopsin III may be limiting for the rate of rhodopsin regeneration and photoreceptor dark adaptation.

To determine whether the photoreceptors change structurally and functionally during aging, and to analyze whether pigmentation in the retinal pigment epithelium might be a contributing factor. Young, adult, and aged C57BL/6 and Balb/c mice (1, 4, and 17 months of age) were housed under a 12-h light/12-h dark cycle, with an ambient light intensity at the eye level of the mice of 85 ± 18 lux. Scotopic single-flash and photopic-flicker electroretinograms (ERGs) after complete dark adaptation were used to assess rod and cone function, respectively. Numbers of rod photoreceptors were counted in plastic sections, and rhodopsin levels were measured using absorption difference spectrophotometry. Numbers and types of cones were determined using lectin staining in retinal flatmounts and cone-specific antibodies in radial frozen sections. Young pigmented C57BL/6 and nonpigmented Balb/c mice had similar numbers of rods. In both mouse strains, there was an overall decline in rod photoreceptor number during aging, which was more pronounced in albino mice. Rod cell numbers correlated with a drop in the overall amount of rhodopsin and a reduction in the maximum a-wave of the rod ERG. The number of short-wavelength cones was unaffected by age and pigmentation, whereas an age-related decline was observed in mid-wavelength (MWL) cones in albino, but not in pigmented mice. In contrast, MWL cone function was reduced during aging in both strains.

Light adaptation in rod photoreceptors is thought to involve down-regulation of the signaling activity of photoactivated rhodopsin (R*). However, electrophysiological evidence in support of this notion has come largely from studies of truncated, perfused rod outer segments and of rods genetically engineered to perturb known steps in R* deactivation. To test this hypothesis within intact native rods, we examined the effect of a fixed conditioning flash on rods prepared to contain 9-demethyl rhodopsin (9dR) in addition to residual rhodopsin. 9dR, an opsin-based photopigment containing 11-cis 9-demethylretinal as its chromophore, exhibits a blue-shifted excitation spectrum and sluggish deactivation kinetics, properties that distinguish the signaling activities of photoactivated 9dR (9dR*) from those of R*. Saturating photocurrent responses mediated preferentially by R* and 9dR* were obtained with test flash stimulation at 640 and 440 nm, respectively, under dark-adapted conditions (unconditioned response) and at a fixed time after a 640-nm conditioning flash of fixed high intensity. At each test wavelength, the decrease in photocurrent saturation period induced by the conditioning flash was analyzed to determine ψ, the sensitivity of the conditioned response relative that of the unconditioned response; ψ640 /ψ440, the ratio of relative sensitivities, was then obtained. Data obtained from 12 rods yielded ψ640 /ψ440 = 0.60 ± 0.13 (mean ± SD). As common pools of transducin and other downstream components mediate transduction initiated by both R* and 9dR*, the finding that ψ640 < ψ440 provides direct evidence for the down-regulation specifically of R*'s signaling activity during rod light adaptation.

Temperature effects on spectral properties of the two types of rod photoreceptors in toad retina, “red” and “green” rods, were studied in the range 0–38°C. Absorbance spectra of the visual pigments were recorded by single-cell microspectrophotometry (MSP) and spectral sensitivities of red rods were measured by electroretinogram (ERG) recording across the isolated retina. The red-rod visual pigment is a usual rhodopsin (λmax = 503.6 nm and 502.3 nm at room temperature (21°C) in, respectively, Bufo marinus and Bufo bufo), that of green rods (λmax = 432.6 nm in Bufo marinus) belongs to the “blue” cone pigment family. In red rods, λmax depended inversely and monotonically on temperature, shifting by −2.3 nm when temperature was raised from 0°C to 38°C. Green-rod λmax showed no measurable dependence on temperature. In red rods, warming caused a relative increase of sensitivity in the long-wavelength range. This effect can be used for estimating the energy needed for photoexcitation, giving Ea = 44.3 ± 0.6 kcal/mol for Bufo marinus rhodopsin and 48.8 ± 0.5 kcal/mol for Bufo bufo rhodopsin. The values are significantly different (P < 0.001), although the two rhodopsins have very similar absorption spectra and thermal isomerization rates. Our recording techniques did not allow measurement of the corresponding effect at long wavelengths in green rods. Although spectral effects of temperature changes in the physiological range are small and of little significance for visual function, they reveal information about the energy states and different spectral tuning mechanisms of the visual pigments.

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45–50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.