Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.     

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.     

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.     

Molecular Evidence that Only Two Opsin Subfamilies,the Blue Light- (SWS2) and Green Light-Sensitive (RH2), Drive Color Vision in Atlantic Cod (Gadus morhua)

Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (Gadus morhua) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.     

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.     

Chemistry and biology of vision

Visual perception in humans occurs through absorption of electromagnetic radiation from 400 to 780 nm by photoreceptors in the retina. A photon of visible light carries a sufficient amount of energy to cause, when absorbed, a cis,trans-geometric isomerization of the 11-cis-retinal chromophore, a vitamin A derivative bound to rhodopsin and cone opsins of retinal photoreceptors. The unique biochemistry of these complexes allows us to reliably and reproducibly collect continuous visual information about our environment. Moreover, other nonconventional retinal opsins such as the circadian rhythm regulator melanopsin also initiate light-activated signaling based on similar photochemistry.     

Chimeric nature of pinopsin between rod and cone visual pigments.

Chicken pineal pinopsin is the first example of extra-retinal opsins, but little is known about its molecular properties as compared with retinal rod and cone opsins. For characterization of extra-retinal photon signaling, we have developed an overexpression system providing a sufficient amount of purified pinopsin. The recombinant pinopsin, together with similarly prepared chicken rhodopsin and green-sensitive cone pigment, was subjected to photochemical and biochemical analyses by using low-temperature spectroscopy and the transducin activation assay. At liquid nitrogen temperature (-196 degrees C), we detected two kinds of photoproducts, bathopinopsin and isopinopsin, having their absorption maxima (lambda(max)) at 527 and approximately 440 nm, respectively, and we observed complete photoreversibility among pinopsin, bathopinopsin, and isopinopsin. A close parallel of the photoreversibility to the rhodopsin system strongly suggests that light absorbed by pinopsin triggers the initial event of cis-trans isomerization of the 11-cis-retinylidene chromophore. Upon warming, bathopinopsin decayed through a series of photobleaching intermediates: lumipinopsin (lambda(max) 461 nm), metapinopsin I (460 nm), metapinopsin II (385 nm), and metapinopsin III (460 nm). Biochemical and kinetic analyses showed that metapinopsin II is a physiologically important photoproduct activating transducin. Detailed kinetic analyses revealed that the formation of metapinopsin II is as fast as that of a chicken cone pigment, green, but that the decay process of metapinopsin II is as slow as that of the rod pigment, rhodopsin. These results indicate that pinopsin is a new type of pigment with a chimeric nature between rod and cone visual pigments in terms of the thermal behaviors of the meta II intermediate. Such a long-lived active state of pinopsin may play a role in the pineal-specific phototransduction process.     

Molecular cloning and characterization of rhodopsin in a teleost (Plecoglossus altivelis,Osmeridae)

Amplified fragments encoding exon-4 of opsin cDNAs were cloned from the retina of landlocked ayu (Plecoglossus altivelis), and sequenced. On the basis of the sequence homology to previously characterized fish visual pigments, one clone was identified as rod opsin (AYU-Rh), and two clones as green (AYU-G1, -G2), one as red (AYU-R) and two as ultraviolet (AYU-UV1, -UV2) cone opsins. The 335-amino acid sequence deduced from the full-length cDNA of AYU-Rh included residues highly conserved in vertebrate rhodopsins and showed the greatest degree (88%) of similarity with salmon rhodopsin. Southern blotting analysis indicated that ayu possess two rhodopsin genes, one encoding visual rhodopsin (AYU-Rh) and the other non-visual extra-ocular rhodopsin (AYU-ExoRh). RT-PCR experiments revealed that AYU-Rh was expressed in the retina and AYU-ExoRh in the pineal gland. In situ hybridization experiments showed that the mRNA of AYU-Rh was localized only in rod cells not in cone cells. Lake and river type landlocked ayu having different amounts of retinal and 3-hydroxyretinal in their retinas expressed a rhodopsin (AYU-Rh) of identical amino acid sequence.     

Cone visual pigments

Cone visual pigments are visual opsins that are present in vertebrate cone photoreceptor cells and act as photoreceptor molecules responsible for photopic vision. Like the rod visual pigment rhodopsin, which is responsible for scotopic vision, cone visual pigments contain the chromophore 11-cis-retinal, which undergoes cis–trans isomerization resulting in the induction of conformational changes of the protein moiety to form a G protein-activating state. There are multiple types of cone visual pigments with different absorption maxima, which are the molecular basis of color discrimination in animals. Cone visual pigments form a phylogenetic sister group with non-visual opsin groups such as pinopsin, VA opsin, parapinopsin and parietopsin groups. Cone visual pigments diverged into four groups with different absorption maxima, and the rhodopsin group diverged from one of the four groups of cone visual pigments. The photochemical behavior of cone visual pigments is similar to that of pinopsin but considerably different from those of other non-visual opsins. G protein activation efficiency of cone visual pigments is also comparable to that of pinopsin but higher than that of the other non-visual opsins. Recent measurements with sufficient time-resolution demonstrated that G protein activation efficiency of cone visual pigments is lower than that of rhodopsin, which is one of the molecular bases for the lower amplification of cones compared to rods. In this review, the uniqueness of cone visual pigments is shown by comparison of their molecular properties with those of non-visual opsins and rhodopsin. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Neural control of motile activity of light-sensitive iridophores in the neon tetra

Experiments with skin pieces revealed that the sympathetic nervous system controls the activity of the light-sensitive iridophores in the stripes of the neon tetra. The spectral peak reflected from the cells was shifted toward longer wavelengths as a result of a direct interaction between norepinephrine and alpha-adrenoceptors present on the cell membrane. Adenosine accelerated the recovery from the effects of the amine. Such regulation seems to operate when fish are in an excited state or under stress. Since alpha-melanophore-stimulating hormone (alpha-MSH), melanin-concentrating hormone (MCH) and melatonin caused the responses only at high concentrations, it is possible that these peptides and amine do not affect the properties of the light-reflecting cells in vivo.     

Comparative retinal morphology of the platypus

The purpose of this study is to identify evolutionary origin and fate of anatomic features of the duck‐billed platypus eye. Eyes from the duck‐billed platypus and four key evolutionary basal vertebrates (Pacific hagfish, north hemisphere sea lamprey, and Australian and South American lungfishes) were prepared for light microscopy. In addition to a standard panel of stains, tissues were immunostained against a variety of rod and cone opsins. Finally, published opsin sequences of platypus and several other vertebrate species were aligned and compared with immunohistochemical results. A complete scleral cartilage similar to that seen in birds, reptiles and amphibians encloses the platypus eye. This feature is present in sharks and rays, and in extant relatives of tetrapods, the lungfishes. The choroid lacks a tapetum. The retina is largely avascular and is rod‐dominated, with a minority of red‐ and blue‐ cone immunoreactive photoreceptors. Like marsupials and many nonmammalian vertebrates, cones contain clear inner segment droplets. Double cones were present, a feature not found in eutherian mammals or marsupials. Evaluation of opsins indicates that red and blue immunoreactive cone opsins, but not rhodopsin, are present in the most basal of the extant species examined, the Pacific hagfish. Rhodopsin appears in the Australian and South American lungfishes, establishing emergence of this pigment in an extant relative of tetrapods. Unlike eyes of eutherian mammals, the platypus eye has retained morphologic features present in early tetrapods such as amphibians and their evolutionarily basal sister group, the lungfishes. These include scleral cartilage, double cones and cone droplets. In the platypus, as in other mammals, rod rhodopsin is the predominant photoreceptor pigment, at expense of the cone system. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Functional characterization of visual opsin repertoire in Medaka (Oryzias latipes)

A variety of visual pigment repertoires present in fish species is believed due to the great variation under the water of light environment. A complete set of visual opsin genes has been isolated and characterized for absorption spectra and expression in the retina only in zebrafish. Medaka (Oryzias latipes) is a fish species phylogenetically distant from zebrafish and has served as an important vertebrate model system in molecular and developmental genetics. We previously isolated a medaka rod opsin gene (RH1). In the present study we isolated all the cone opsin genes of medaka by genome screening of a lambda-phage and bacterial artificial chromosome (BAC) libraries. The medaka genome contains two red, LWS-A and LWS-B, three green, RH2-A, RH2-B and RH2-C, and two blue, SWS2-A and SWS2-B, subtype opsin genes as well as a single-copy of the ultraviolet, SWS1, opsin gene. Previously only one gene was believed present for each opsin type as reported in a cDNA-based study. These subtype opsin genes are closely linked and must be the products of local gene duplications but not of a genome-wide duplication. Peak absorption spectra (lambda(max)) of the reconstituted photopigments with 11-cis retinal varied greatly among the three green opsins, 452 nm for RH2-A, 516 nm for RH2-B and 492 nm for RH2-C, and between the two blue opsins, 439 nm for SWS2-A and 405 nm for SWS2-B. Zebrafish also has multiple opsin subtypes, but phylogenetic analysis revealed that medaka and zebrafish gained the subtype opsins independently. The lambda and BAC DNA clones isolated in this study could be useful for investigating the regulatory mechanisms and evolutionary diversity of fish opsin genes.     

Low-frequency vibrational modes and infrared absorbance of red, blue and green opsin

Vibrational excitations of low-frequency collective modes are essential for functionally important conformational transitions in proteins. We carried out an analysis of the low-frequency modes in the G protein coupled receptors (GPCR) family of cone opsins based on both normal-mode analysis and molecular dynamics (MD) simulations. Power spectra obtained by MD can be compared directly with normal modes. In agreement with existing experimental evidence related to transmembrane proteins, cone opsins have functionally important transitions that correspond to approximately 950 modes and are found below 80 cm⁻¹. This is in contrast to bacteriorhodopsin and rhodopsin, where the important low-frequency transition modes are below 50 cm⁻¹. We find that the density of states (DOS) profile of blue opsin in a solvent (e.g. water) has increased populations in the very lowest frequency modes (<15 cm⁻¹); this is indicative of the increased thermostability of blue opsin. From our work we found that, although light absorption behaves differently in blue, green and red opsins, their low-frequency vibrational motions are similar. The similarities and differences in the domain motions of blue, red and green opsins are discussed for several representative modes. In addition, the influence of the presence of a solvent is reported and compared with vacuum spectra. We thus demonstrate that terahertz spectroscopy of low-frequency modes might be relevant for identifying those vibrational degrees of freedom that correlate to known conformational changes in opsins. An erratum to this article can be found at     

<Emphasis Type="Italic">In silico</Emphasis> characterisation and chromosomal localisation of human <Emphasis Type="Italic">RRH</Emphasis> (peropsin) – implications for opsin evolution

Background  

The vertebrate opsins are proteins which utilise a retinaldehyde chromophore in their photosensory or photoisomerase roles in the visual/irradiance detection cycle. The majority of the opsins, such as rod and cone opsins, have a very highly conserved gene structure suggesting a common lineage. Exceptions to this are RGR-opsin and melanopsin, whose genes have very different intron insertion positions. The gene structure of another opsin, peropsin (retinal pigment epithelium-derived rhodopsin homologue,RRH) is unknown.     

Coexpression of spectrally distinct rhodopsins in Aedes aegypti R7 photoreceptors

The retina of the mosquito Aedes aegypti can be divided into four regions based on the non-overlapping expression of a UV sensitive Aaop8 rhodopsin and a long wavelength sensitive Aaop2 type rhodopsin in the R7 photoreceptors. We show here that another rhodopsin, Aaop9, is expressed in all R7 photoreceptors and a subset of R8 photoreceptors. In the dorsal region, Aaop9 is expressed in both the cell body and rhabdomere of R7 and R8 cells. In other retinal regions Aaop9 is expressed only in R7 cells, being localized to the R7 rhabdomere in the central and ventral regions and in both the cell body and rhabdomere within the ventral stripe. Within the dorsal-central transition area ommatidia do not show a strict pairing of R7-R8 cell types. Thus, Aaop9 is coexpressed in the two classes of R7 photoreceptors previously distinguished by the non-overlapping expression of Aaop8 and Aaop2 rhodopsins. Electroretinogram analysis of transgenic Drosophila shows that Aaop9 is a short wavelength rhodopsin with an optimal response to 400-450 nm light. The coexpressed Aaop2 rhodopsin has dual wavelength sensitivity of 500-550 nm and near 350 nm in the UV region. As predicted by the spectral properties of each rhodopsin, Drosophila photoreceptors expressing both Aaop9 and Aaop2 rhodopsins exhibit a uniform sensitivity across the broad 350-550 nm light range. We propose that rhodopsin coexpression is an adaptation within the R7 cells to improve visual function in the low-light environments in which Ae. aegypti is active.     

Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase

Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.     

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

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

The putative brain photoperiodic photoreceptors in the vetch aphid,Megoura viciae

In an attempt to identify the brain photoreceptors that mediate the photoperiodic response of the vetch aphid, Megoura viciae, we utilised immunocytochemical techniques and employed 20 antibodies directed against invertebrate and vertebrate opsins and phototransduction proteins. A sub-set of these antibodies (to Drosophila rhodopsin 1: RH1-1; vertebrate cone opsins: COS-1; CERN-874; CERN-933; vertebrate rod opsin: CERN-901; vertebrate arrestin: AB-Arr; vertebrate transducin+arrestin+rhodopsin kinase+cGMP phosphodiesterase: CERN-911; and vertebrate cellular retinoid binding protein: CRALBP) consistently labelled an anterior ventral neuropile region of the protocerebrum. These anatomical findings, coupled with previous localised illumination and micro-lesion studies, provide strong evidence that this region of the aphid brain houses the photoperiodic photoreceptors. The present study also confirms that the medial (Group I) neurosecretory cells are not the photoperiodic photoreceptors.     

The involvement of a rhodopsin-like photopigment in the photoperiodic response of the Japanese quail

Summary A technique has been developed for the investigation of the photopigment involved in the photoperiodic control of reproduction in Japanese quail,Coturnix coturnix. When these photoreceptors were exposed to white or monochromatic light a clear relationship was found between light intensity and the extent of photo-induced luteinizing hormone (LH) secretion. A spectroradiometric investigation of the passage of light through the skull and brain enabled us to illuminate the hypothalamic region with equal numbers of photons at a range of wavelengths. Action spectra were then conducted and showed a photopigment with a peak sensitivity at wavelengths near 500 nm. An excellent match was obtained when the standard absorption spectrum for a rhodopsin was fitted to the action spectrum, suggesting a rhodopsin maximally sensitive at 492 nm. The absolute sensitivity of the photoreceptors was calculated at a range of wavelengths: with light at 500 nm, 2.85×10^–12 E·cm^–2·s^–1 triggered the photoperiodic response. This level of sensitivity is matched only by the rhodopsin visual pigments.Abbreviations LH luteinizing hormone - T transmission     

Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system

Animals have evolved specialized photoreceptors in the retina and in extraocular tissues that allow them to measure light changes in their environment. In mammals, the retina is the only structure that detects light and relays this information to the brain. The classical photoreceptors, rods and cones, are responsible for vision through activation of rhodopsin and cone opsins. Melanopsin, another photopigment first discovered in Xenopus melanophores (Opn4x), is expressed in a small subset of retinal ganglion cells (RGCs) in the mammalian retina, where it mediates non-image forming functions such as circadian photoentrainment and sleep. While mammals have a single melanopsin gene (opn4), zebrafish show remarkable diversity with two opn4x-related and three opn4-related genes expressed in distinct patterns in multiple neuronal cell types of the developing retina, including bipolar interneurons. The intronless opn4.1 gene is transcribed in photoreceptors as well as in horizontal cells and produces functional photopigment. Four genes are also expressed in the zebrafish embryonic brain, but not in the photoreceptive pineal gland. We discovered that photoperiod length influences expression of two of the opn4-related genes in retinal layers involved in signaling light information to RGCs. Moreover, both genes are expressed in a robust diurnal rhythm but with different phases in relation to the light-dark cycle. The results suggest that melanopsin has an expanded role in modulating the retinal circuitry of fish.