Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A 1 (the precursor of 11-cis retinal) into vitamin A 2 (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A 2 , eliminating the animal’s ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A 1 and A 2 in the eye.

Despite many decades of research on the rhodopsin-porphyropsin system, the enzyme that mediates this switch has been unknown. In this study, we employed transcriptome profiling of distantly related fish and amphibian models to identify the enzyme responsible for converting retinol into 3,4-didehydroretinol (henceforth referred to as vitamin A 1 and vitamin A 2 , respectively). We confirmed that this enzyme is required for the red-shift in photoreceptor sensitivity that occurs upon chromophore exchange in vivo and demonstrated that this change in sensitivity enhances the fish’s ability to see and respond to near-infrared light.

This switch is believed to result in a better match between the sensitivity of the visual system and the spectral distribution of light in fresh water, which is often red-shifted relative to marine and terrestrial environments []. 3,4-didehydroretinoids have been identified in the eyes of the vast majority of freshwater fish and amphibian species that have been examined []. This observation suggests that thousands of vertebrate species may use chromophore switching to tune the spectral sensitivity of their visual systems. In addition, in many species of lampreys, fish, and amphibians, the visual system is dynamically tuned by altering the balance of 11-cis retinal and 11-cis 3,4-didehydroretinal in the retina when the animal moves between different light environments []. For example, the ratio of 11-cis 3,4-didehydroretinal to 11-cis retinal increases when species such as salmon or sea lamprey migrate from the open ocean into inland freshwater environments ( Figure 1 B) []. Conversely, many amphibians use 11-cis 3,4-didehydroretinal during the freshwater tadpole stage but then switch to 11-cis retinal upon metamorphosis into a terrestrial adult ( Figure 1 B) []. Although several environmental factors, including changes in temperature or season, may affect the balance of the two chromophores [], there is clearly a strong correlation between the red-shifted light environment of freshwater habitats and the use of the red-shifted chromophore, 11-cis 3,4-didehydroretinal [].

Presence of rhodopsin and porphyropsin in the eyes of 164 fishes, representing marine, diadromous, coastal and freshwater species--a qualitative and comparative study.

Typical rod photopigments have a maximum absorbance (λ) of around 500 nm []. However, it was first noted in the 19century that certain freshwater fishes have rod photopigments whose λvalues are red-shifted by 20–30 nm relative to those of marine fishes and terrestrial vertebrates []. The spectral sensitivity of cone photoreceptors is also red-shifted in freshwater species, in some cases by as much as 60 nm []. This red-shift is caused by replacement of the chromophore, 11-cis retinal, with 11-cis 3,4-didehydroretinal, which contains an additional conjugated double bond within its β-ionone ring ( Figure 1 A) []. Rod photopigments containing 11-cis retinal are referred to as “rhodopsins” on account of the rose-colored appearance of the unbleached pigment, whereas those containing 11-cis 3,4-didehydroretinal are called “porphyropsins” on account of their purple color []. Therefore, the change in chromophore from 11-cis retinal to 11-cis 3,4-didehydroretinal is referred to as the “rhodopsin-to-porphyropsin” switch.

(H and I) The dorsal third of the RPE from two American bullfrogs was dissected, pooled, and analyzed by HPLC and found to contain a mixture of vitamin A 1 and vitamin A 2 . Only vitamin A 1 was identified in the ventral third of the RPE. All absorbance values are normalized and represented as arbitrary units (a.u.).

(G) The American bullfrog (Lithobates catesbeianus) often sits at the water’s surface and possesses exclusively A-based visual pigments in the ventral retina and a mixture of A- and A-based visual pigments in the dorsal retina, possibly facilitating downward vision into the murky, red-shifted water [].

(F) The predominant retinoid from TH-treated fish has an absorbance spectrum that matches that of the vitamin A 2 standard, with a λ max of 355 nm.

(E) The absorbance spectrum of the predominant retinoid from control fish closely matches the absorbance spectrum of a vitamin A 1 standard, with a λ max of 326 nm.

(C and D) To model this switch, zebrafish were treated with thyroid hormone (300 μg/l L-thyroxine [T4]) for 3 weeks. Retinoids were then isolated from pooled RPE and retina of three individuals, reduced using sodium borohydride, and analyzed by HPLC. Retinoids from TH-treated fish have a shorter retention time than those from vehicle-treated fish.

(B) This vitamin Ato Aswitch is widely used by a variety of vertebrates and can be deployed at key stages of the life cycle: e.g., during upstream migration in sea lamprey (Petromyzon marinus) and Coho salmon (Oncorhynchus kisutch) and upon metamorphosis in amphibians such as the northern leopard frog (Lithobates pipiens) [].

(A) The conversion of retinol (vitamin A 1 ) to 3,4-didehydroretinol (vitamin A 2 ) underlies the rhodopsin-to-porphyropsin switch and is driven by a previously unidentified dehydrogenase that catalyzes the addition of a double bond to the β-ionone ring.

The visual pigments of vertebrate rod and cone photoreceptors consist of a G-protein-coupled receptor, the opsin, and a covalently bound chromophore, most commonly 11-cis retinal []. Vision begins when a photon of light induces a cis to trans isomerization of the chromophore, thereby altering the conformation of the opsin and, in turn, activating the phototransduction cascade []. The spectral sensitivity of a visual pigment is determined by the structure of the chromophore and the electrostatic and steric interactions between the chromophore and the amino acid side chains within the binding cleft of the opsin [].

We compared the behavior of dark-adapted, vehicle- and TH-treated cyp27c1and wild-type siblings in response to 590-nm and 770-nm light ( Figure 5 ; see Figure S4 for LED characterization). Fish with vitamin A- and A-based photopigments are predicted to perceive 590-nm light similarly, based on the sensitivity curves of red cones ( Figure 5 B). As expected, in both the vehicle and TH treatments, cyp27c1mutants and wild-type fish exhibited positive phototaxis toward a 590-nm light source ( Figure 5 D). This finding indicates that normal visually mediated behaviors are intact and functional in cyp27c1mutants. In contrast, upon exposure to 770-nm light, only wild-type, TH-treated fish demonstrated a positive phototactic response, whereas cyp27c1mutants and vehicle control fish behaved as if they were in the dark ( Figure 5 E). These data indicate that the red-shift in photoreceptor spectral sensitivity caused by switching to vitamin A-based photopigments improves the animal’s ability to detect and respond to near-infrared light. This increase in long-wavelength sensitivity may provide a selective advantage under some environmental conditions and may thus have contributed to the evolution of the rhodopsin-porphyropsin system.

It has long been speculated that the switch from vitamin A- to vitamin A-based photopigments could be an adaptive response to changes in the animal’s light environment, permitting the animal to adjust its spectral sensitivity to more closely match the spectral quality of its surroundings []. The cyp27c1 mutant zebrafish present an opportunity to test the relationship between vitamin Aand behavioral responsiveness to long-wavelength light. We therefore developed an assay to measure changes in swimming behavior in response to light of various wavelengths ( Figures 5 A–5C). When exposed to a directional light source, adult zebrafish display a positive phototactic response ( Movies S1 and S2 ) []. We hypothesized that TH-treated wild-type fish would have a stronger phototactic response to near-infrared light than cyp27c1mutants due to the vitamin A-induced red-shift in spectral sensitivity.

(E) Only TH-treated wild-type fish showed a significant positive phototactic response to 770-nm light. For significantly different groups, p values are as shown. For vehicle-treated cyp27c1 Δ1/Δ3 mutant and wild-type fish at 770 nm, p values were 0.449 and 0.177, respectively. For TH-treated cyp27c1 Δ1/Δ3 mutants at 770 nm, the p value was 0.490 (paired t test; n as indicated per group; error bars = SEM). All p values remained significant after Bonferroni correction for multiple testing.

(D) TH- and vehicle-treated cyp27c1 Δ1/Δ3 mutants and wild-type siblings all showed a significant positive phototactic response to 590-nm light (paired Student’s t test; p < 0.005; n as indicated per group; error bars = SEM). All p values remained significant after Bonferroni correction for multiple testing.

(C) Summary of the experimental design. Each dark-adapted fish was acclimated to the testing tank for 30 min in the dark and then recorded during a set of three trials each at 770 and 590 nm. Each trial consisted of 3 min dark and 3 min lit conditions, with the order of light/dark presentation randomized. The light source was alternated between the left and right sides for each trial.

(A) The phototaxis assay consisted of 590- or 770-nm LED light sources placed on either the right or left side of a tank (depicted on the right side in the schematic). Light from the LEDs passed through two diffusers before illuminating one end of the tank. On the opposite end of the tank, a black occluder was inserted to prevent reflection off of the “off-side” diffuser. Fish movement was recorded using a night vision camera and a 940-nm infrared backlight located beneath the tank. The percentage of time spent within 25 mm of the lit “on” side (dashed line) was determined. The behavioral assay and subsequent data analysis were conducted in a blinded fashion with respect to the genotype of the fish.

As TH-treated cyp27c1fish fail to produce vitamin A(the precursor of 11-cis 3,4-didehydroretinal), their photoreceptors should also fail to undergo a red-shift in sensitivity. To test this hypothesis, we used single-cell suction electrode recording to measure the sensitivity of individual red cones from cyp27c1mutants and wild-type siblings, treated with either TH or vehicle control. We measured red cones because they undergo the largest red-shift upon switching to 11-cis 3,4-didehydroretinal []. The flash sensitivity of individual red cones was determined from the amplitudes of their electrical responses to a series of dim flashes of 560-, 600-, 660-, and 700-nm light and fitted with a model that incorporates contributions from both vitamin A- and A-based pigments []. No significant difference was observed between the sensitivities of vehicle-treated cyp27c1and wild-type red cones at any of the wavelengths tested, and both wild-type and mutant red cones had maximal sensitivity (λ) at 561 nm, in close agreement with a previous estimate of λ= 565 nm, determined by MSP ( Figures 4 D and 4E) []. In addition, knockout of cyp27c1 did not affect the dark current or the kinetics of the flash responses of the red cones, indicating that phototransduction remained intact ( Figure 4 E). In contrast, TH treatment induced a 57-nm red-shift in the λof wild-type fish to 618 nm, whereas the λof TH-treated cyp27c1mutant fish was unchanged ( Figures 4 D and 4E). Thus, knockout of cyp27c1 completely eliminates the red-shift in red cone spectral sensitivity induced by TH treatment.

If Cyp27c1 mediates the rhodopsin-porphyropsin switch, zebrafish lacking this enzyme should not produce vitamin Ain response to TH treatment. To test this hypothesis, we generated several mutant alleles of cyp27c1 by using transcription activator-like effector nucleases (TALENs) to introduce small insertions/deletions within the first and fourth exons []. We obtained multiple alleles carrying frameshift mutations predicted to result in premature stop codons ( Figures 4 A and S3 ). Two independent lines were crossed to produce transheterozygous mutant fish (cyp27c1), which survived to adulthood without overt developmental abnormalities. Upon treatment with TH, the mutant fish failed to produce Cyp27c1 protein, in contrast to their wild-type siblings ( Figure 4 B). We then used HPLC to assess retinoid content in the eyes of TH-treated cyp27c1mutants and their wild-type siblings. Whereas a conversion to vitamin Awas observed in TH-treated wild-type fish, TH-treated cyp27c1mutants failed to produce any vitamin A Figure 4 C), indicating that Cyp27c1 is necessary for vitamin Aproduction in vivo. We also observed loss of vitamin Aproduction in fish carrying two additional heteroallelic combinations, cyp27c1and cyp27c1 Figure S3 ), indicating that this effect is attributable to the loss of Cyp27c1 rather than off-target mutations caused by the TALENs.

(E) Physiological parameters of the red cones are indicated ± SEM. These include wavelength of peak sensitivity (λ max ), dark current (I dark ), estimated half-saturation light intensity at λ max (I o ), dim flash response time to peak (T peak ), recovery time constant (τ rec ; estimated from a single-exponential fit to the tail of the response), and integration time (T int ; estimated as the time integral of the normalized flash response).

(D) The spectral sensitivity of red cones from TH-treated cyp27c1 Δ1Δ2 fish and their wild-type siblings was assessed using single-cell suction electrode recording. The photoreceptors were exposed to a series of dim flashes at 560, 600, 660, and 700 nm, and their response amplitudes were used to calculate flash sensitivity (y axis) as a function of wavelength (x axis). No significant difference was observed between vehicle-treated cyp27c1 Δ/1Δ2 and wild-type red cones. However, TH-treated wild-type red cones were significantly more sensitive to 660- and 700-nm light and less sensitive to 560-nm light relative to cyp27c1 Δ/1Δ2 red cones (Student’s t test; ∗ p < 0.0005; n = 12–21; error bars = SEM). Fitted curves were calculated using a model that incorporates both vitamin A 1 - and A 2 -based pigment. This model predicts 100% vitamin A 1 in vehicle-treated wild-type and cyp27c1 Δ/1Δ2 fish, 99.7% vitamin A 1 in TH-treated cyp27c1 Δ/1Δ2 fish, and 95.5% vitamin A 2 in TH-treated wild-type siblings.

(C) Wild-type and cyp27c1 Δ1/Δ2 siblings were treated with TH or vehicle control for 3 weeks, and retinas and RPE from three animals were pooled and analyzed for retinoid content using HPLC. A TH-driven switch to vitamin A 2 was observed in wild-type fish, but not in cyp27c1 Δ/1Δ2 mutants.

(B) Western blot with rabbit polyclonal anti-Cyp27c1 antibody shows that Cyp27c1 protein is detected in the RPE of wild-type fish, but not in the RPE of cyp27c1 Δ1/Δ2 fish, after 3 weeks of TH treatment. β-actin was used as a loading control.

(A) TALENs were used to generate cyp27c1 mutant zebrafish. Two independent alleles are shown, cyp27c1 Δ1 and cyp27c1 Δ2 . Both alleles contain frame-shift mutations resulting in premature stop codons at amino acids 151 and 150, respectively.

To test whether Cyp27c1 is sufficient to produce vitamin A, we expressed Cyp27c1 in HEK293 cells, which were then incubated with vitamin Afor 24 hr and subjected to HPLC to assess retinoid content. Cells transfected with an empty vector contained only the vitamin Asubstrate, whereas cells transfected with the Cyp27c1 expression construct contained vitamin Ain addition to vitamin A Figure 3 A). To further characterize enzyme kinetics, we expressed and purified Cyp27c1 and determined its catalytic efficiency with three different retinoid substrates: all-trans retinol, retinal, and retinoic acid. Cyp27c1 can act on all three substrates but most efficiently catalyzes the conversion of retinol (vitamin A) to 3,4-didehydroretinol (vitamin A Figures 3 B and S2 ). Furthermore, the catalytic efficiency of Cyp27c1 with vitamin A(k/K= 1.4 ± 0.5 × 10min) is among the higher values reported for animal cytochrome P450 family members []. These results confirm that Cyp27c1 has the requisite biochemical activity to mediate the rhodopsin-porphyropsin switch.

(B) A cell-free assay system was used to assess the catalytic efficiency of Cyp27c1 for all-trans retinol, retinal, and retinoic acid. Purified Cyp27c1 was incubated with bovine adrenodoxin (Adx), NADPH-adrenodoxin reductase (ADR), and substrate (with concentrations ranging from 0.1 to 10 μM). The reaction was then initiated using an NADPH-generating system, run for 60 s, and quenched. HPLC was used to quantify the products in order to calculate k cat and K m . A high catalytic efficiency (k cat /K m ) was observed for all three substrates. Error bars represent SEM.

(A) HEK293 cells were transfected with a cyp27c1 expression construct or an empty vector and incubated with vitamin A 1 for 24 hr, after which cells and media were harvested for HPLC. Cells transfected with empty vector did not produce vitamin A 2 , but cells transfected with cyp27c1 converted a substantial fraction of the vitamin A 1 to vitamin A 2 .

To assay the expression levels of Cyp27c1 protein in zebrafish and bullfrog RPE, we raised a polyclonal anti-Cyp27c1 antibody in rabbit and confirmed its activity against Cyp27c1 produced in HEK293 cell culture ( Figure S1 ). We then used this antibody in a western blot to confirm the induction of Cyp27c1 protein in TH-treated zebrafish RPE and its enrichment in dorsal bullfrog RPE ( Figure 2 E). In addition, using immunohistochemistry, we determined that Cyp27c1 protein is localized specifically to the RPE of TH-treated, but not control, albino zebrafish, in a pattern consistent with the cyp27c1 transcript ( Figures 2 D and 2F). Overall, the distribution of both the cyp27c1 transcript and protein correspond precisely to the pattern of vitamin Aproduction in zebrafish and bullfrog.

To determine whether the presence of cyp27c1 correlates with the distribution of vitamin Aand its derivatives, we analyzed cyp27c1 expression in zebrafish and bullfrog eyes. We first confirmed that the cyp27c1 transcript is significantly increased in TH-treated zebrafish RPE and dorsal bullfrog RPE by quantitative real-time PCR ( Figure 2 C). We then determined the cellular expression pattern of cyp27c1, by performing in situ hybridization on eyes from TH-treated albino zebrafish and vehicle controls []. The albino strain was used to improve visualization of staining in the RPE, and we confirmed that it undergoes a TH-dependent switch to vitamin Asimilar to wild-type strains (data not shown). The cyp27c1 antisense probe localized exclusively to the RPE after TH treatment, with no signal observed in the vehicle-treated control eyes ( Figure 2 D). In contrast, the RPE-specific transcript, rpe65a, was detected in both TH- and vehicle-treated eyes, confirming the presence of RPE in the sections ( Figure 2 D). Taken together, these results indicate a specific upregulation of cyp27c1 in the RPE of TH-treated fish and are consistent with a previous report suggesting that the RPE is the primary locus of vitamin Aproduction in some species [].

The RPE plays a critical role in regenerating 11-cis retinal and is the locus of vitamin Ato Aconversion in some species []. Therefore, to identify candidate genes that might encode the vitamin A3,4-dehydrogenase, we profiled zebrafish and bullfrog RPE by RNA-seq. We compared the transcriptomes of RPE from TH-treated zebrafish with that of vehicle-treated controls, identifying a total of 35 TH-upregulated genes ( Figure 2 A; Table S1 ). We also compared dorsal and ventral RPE from adult bullfrogs, identifying a total of 40 dorsally enriched genes ( Figure 2 B; Table S2 ). In both analyses, one candidate gene stood out as being among the most highly upregulated and enriched, the cytochrome P450 family member, cyp27c1 ( Figures 2 A and 2B). Members of the P450 family are involved in the metabolism of a variety of xenobiotics and endogenous small molecules including retinoids, although a functional role for cyp27c1 has not previously been reported []. Thus, Cyp27c1 is an excellent candidate for the vitamin A3,4-dehydrogenase.

(F) Immunohistochemistry of albino TH- and vehicle-treated zebrafish indicates induction of Cyp27c1 expression in the RPE of TH-treated fish (green), with DAPI used to counter-stain nuclei (blue). The scale bar represents 50 μm.

(E) Western blot with a rabbit polyclonal anti-Cyp27c1 antibody confirmed enrichment of Cyp27c1 protein in TH-treated zebrafish and dorsal bullfrog RPE. β-actin was used as a loading control.

(D) In situ hybridization of albino zebrafish treated with TH or vehicle control for 3 weeks. Top panels show cross-sections of the whole eye. Bottom panels show high-magnification images of the boxed regions from the top panels. The antisense probe for cyp27c1 localized exclusively to the RPE in TH-treated fish. No signal was observed with the cyp27c1 sense probe, but strong signal was observed in the RPE of TH-treated and control fish with an antisense probe against rpe65a, a gene that is expressed at high levels in RPE. The scale bars represent 200 μm low power and 50 μm high power.

(C) Enrichment of the cyp27c1 transcript in cDNA samples used for RNA-seq was confirmed by quantitative real-time PCR (qRT-PCR). Expression was normalized to ribosomal protein rpl13a for zebrafish and rpl7a for bullfrog (two-sided Student’s t test; n = 2–3; ∗ p < 0.005; error bars = SEM).

(B) Dorsal and ventral thirds of bullfrog RPE were isolated and used to construct a cDNA library for transcriptome profiling by RNA-seq. Expression levels of individual transcripts from dorsal RPE (y axis) and ventral RPE (x axis) are shown as dots, with significantly differentially expressed genes in black (qCML test; FDR < 0.05; n = 3).

(A) Zebrafish were treated with TH or a vehicle control for 3 weeks, after which RPE was isolated and used to construct a cDNA library for transcriptome profiling by RNA-seq. Expression levels (in RPKM [reads per kilobase of transcript per million mapped reads]) of individual transcripts from TH-treated RPE (y axis) and vehicle-treated RPE (x axis) are shown as dots, with significantly differentially expressed genes in black (quantile-adjusted conditional maximum likelihood [qCML] test; FDR < 0.05; n = 3).

Next, we analyzed a specialized pattern of regional chromophore localization within the American bullfrog retina. Most amphibians employ A-based visual pigments during the aquatic phase of their life cycle, switching to predominantly A-based pigments after metamorphosis []. However, American bullfrogs retain A-based pigments in the dorsal third of their retinas and RPE, even as adults ( Figure 1 G) []. Because adult bullfrogs spend a considerable amount of time with their eyes just above the water’s surface [], the presence of vitamin A-based pigments in the dorsal retina has been speculated to facilitate downward vision into the murky, red-shifted aquatic environment []. We dissected dorsal and ventral RPE from adult American bullfrogs, extracted retinoids, and analyzed them by HPLC. Whereas the ventral RPE contained exclusively vitamin Aand its derivatives, the dorsal RPE contained a mixture of vitamin Aand A Figures 1 H and 1I). Thus, both TH-treated zebrafish and American bullfrog represent suitable model systems for analyzing the mechanistic basis of the rhodopsin-porphyropsin switch.

To identify the enzyme responsible for converting vitamin Ainto vitamin A, we used zebrafish (Danio rerio) and American bullfrog (Lithobates catesbeianus) models. Zebrafish photoreceptors predominantly contain vitamin A-based visual pigments under normal laboratory conditions, but treatment with thyroid hormone (TH) induces a conversion to vitamin A-based pigments []. To corroborate this result, we treated zebrafish with TH and then analyzed the retinoid content of retinas and retinal pigment epithelium (RPE) by high-performance liquid chromatography (HPLC). We found a nearly complete conversion of vitamin Ainto vitamin Ain the TH-treated animals ( Figures 1 C–1F).

Discussion

We have shown that the action of a single enzyme, Cyp27c1, is necessary and sufficient for the production of vitamin A 2 and its derivatives and for the resultant red-shift in photoreceptor spectral sensitivity. Furthermore, we have demonstrated that this change in sensitivity at the cellular level can be translated into altered behavioral responsiveness to long-wavelength light. These findings assign a biochemical function to a previously orphan member of the cytochrome P450 family of enzymes. In addition, the generation of cyp27c1 mutants offers an opportunity to study the behavioral consequences of switching from vitamin A 1 - to vitamin A 2 -based photopigments.

20 Saari J.C. Vitamin A metabolism in rod and cone visual cycles. 33 Babino D.

Perkins B.D.

Kindermann A.

Oberhauser V.

von Lintig J. The role of 11-cis-retinyl esters in vertebrate cone vision. 20 Saari J.C. Vitamin A metabolism in rod and cone visual cycles. 34 Saarem K.

Bergseth S.

Oftebro H.

Pedersen J.I. Subcellular localization of vitamin D3 25-hydroxylase in human liver. 35 Saarem K.

Pedersen J.I.

Tollersrud S. Soluble 25-hydroxyvitamin D3-1 alpha-hydroxylase from kidney mitochondria of rachitic pigs. Our work raises a number of intriguing questions about the role of Cyp27c1 in vivo. For example, the preferred substrate(s) of Cyp27c1 in vivo is currently unknown. A number of potential substrates are present in the RPE, including all-trans retinol, all-trans retinyl esters, 11-cis retinol, 11-cis retinal, or even 11-cis retinyl esters []. Our cell-free assays suggest that all-trans retinol could be the in vivo substrate, but future studies will be needed to measure the activity of Cyp27c1 on 11-cis retinoids and characterize the retinoids metabolized by Cyp27c1 in vivo. Interestingly, whereas some enzymes involved in the visual cycle (LRAT, RPE65, and 11cRDH) are localized to the smooth ER, prediction algorithms suggest that Cyp27c1 may be located in mitochondria, as are Cyp27a1 and Cyp27b1 []. The subcellular localization of Cyp27c1 and the pathways by which retinoids might be trafficked between cellular organelles also remain open questions.

1 - and A 2 -based photopigments in these animals [ 36 Beatty D.D. Visual pigment changes in juvenile kokanee salmon in response to thyroid hormones. 37 Temple S.E.

Ramsden S.D.

Haimberger T.J.

Veldhoen K.M.

Veldhoen N.J.

Carter N.L.

Roth W.M.

Hawryshyn C.W. Effects of exogenous thyroid hormones on visual pigment composition in coho salmon (Oncorhynchus kisutch). 37 Temple S.E.

Ramsden S.D.

Haimberger T.J.

Veldhoen K.M.

Veldhoen N.J.

Carter N.L.

Roth W.M.

Hawryshyn C.W. Effects of exogenous thyroid hormones on visual pigment composition in coho salmon (Oncorhynchus kisutch). 38 Ng L.

Hurley J.B.

Dierks B.

Srinivas M.

Saltó C.

Vennström B.

Reh T.A.

Forrest D. A thyroid hormone receptor that is required for the development of green cone photoreceptors. 39 Suzuki S.C.

Bleckert A.

Williams P.R.

Takechi M.

Kawamura S.

Wong R.O. Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors. The ability of TH to induce the expression of cyp27c1 raises the possibility that endogenous TH might play a general role in the regulation of responsiveness to long-wavelength light under natural conditions. For instance, TH is thought to play a key role in coordinating the physiologic changes that salmonids undergo during migration, suggesting that TH may mediate the switch between vitamin A- and A-based photopigments in these animals []. In addition, the salmon genome contains multiple paralogous red and green cone opsin genes and TH treatment induces a shift in the expression of these opsins in favor of the red-shifted paralogs []. Lastly, both zebrafish and mice require thyroid hormone receptor β2 for normal expression of long wavelength-sensitive cone opsin []. These findings suggest that TH signaling might represent an ancient pathway for the regulation of sensitivity to long-wavelength light, both at the level of chromophore modification and opsin gene expression.

2 -based photopigments have never been documented in the eyes of birds or mammals. One possible explanation for this absence is that A 2 -based chromophores are less thermally stable than those based on A 1 . Thus, increased thermal noise may limit the usefulness of A 2 chromophores in the visual systems of warm-blooded species. Nevertheless, orthologs of cyp27c1 are present in nearly all sequenced avian genomes as well as most mammalian genomes, including the human genome. The function of cyp27c1 in these species is currently unknown, but several studies suggest diverse roles for this gene outside of the eye. For example, 3,4-didehydroretinoic acid (a derivative of vitamin A 2 ) has been reported to have biological activity similar to retinoic acid in chicken embryos and is the predominant form of “retinoic acid” in the developing chick [ 40 Maden M.

Sonneveld E.

van der Saag P.T.

Gale E. The distribution of endogenous retinoic acid in the chick embryo: implications for developmental mechanisms. 41 Thaller C.

Eichele G. Isolation of 3,4-didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud. 42 Rollman O.

Vahlquist A. Vitamin A in skin and serum--studies of acne vulgaris, atopic dermatitis, ichthyosis vulgaris and lichen planus. 43 Vahlquist A. The identification of dehydroretinol (vitamin A2) in human skin. Despite the widespread use of the rhodopsin-porphyropsin system among cold-blooded vertebrates, vitamin A-based photopigments have never been documented in the eyes of birds or mammals. One possible explanation for this absence is that A-based chromophores are less thermally stable than those based on A. Thus, increased thermal noise may limit the usefulness of Achromophores in the visual systems of warm-blooded species. Nevertheless, orthologs of cyp27c1 are present in nearly all sequenced avian genomes as well as most mammalian genomes, including the human genome. The function of cyp27c1 in these species is currently unknown, but several studies suggest diverse roles for this gene outside of the eye. For example, 3,4-didehydroretinoic acid (a derivative of vitamin A) has been reported to have biological activity similar to retinoic acid in chicken embryos and is the predominant form of “retinoic acid” in the developing chick []. In addition, the presence of 3,4-didehydroretinoids has been documented in normal human skin (and at elevated levels in hyperkeratotic lesions and some skin neoplasms), suggesting a potential role for CYP27C1 in retinoid biosynthesis in this organ []. Future studies will be needed to address the role of cyp27c1 in these contexts.

1 to A 2 can vary between rods and cones in the same retina [ 44 Saarinen P.

Pahlberg J.

Herczeg G.

Viljanen M.

Karjalainen M.

Shikano T.

Merilä J.

Donner K. Spectral tuning by selective chromophore uptake in rods and cones of eight populations of nine-spined stickleback (Pungitius pungitius). 21 Wang J.S.

Kefalov V.J. The cone-specific visual cycle. 2 -based chromophores are known to be used in some invertebrate species. This fact suggests that functional homologs of Cyp27c1 could be widespread in the animal kingdom. Further work may explore the extent of Cyp27c1 expression and function in the visual systems of different species. A recent study demonstrated that the ratio of vitamin Ato Acan vary between rods and cones in the same retina []. This finding raises the possibility that Cyp27c1 might be differentially expressed between these two cell types in some species. Alternatively, these species might express Cyp27c1 in Müller glia, a cell type that supports cone, but not rod, pigment regeneration []. It is also worth noting that vitamin A-based chromophores are known to be used in some invertebrate species. This fact suggests that functional homologs of Cyp27c1 could be widespread in the animal kingdom.