Color opponency with a single kind of bistable opsin in the zebrafish pineal organ

Seiji Wada; Baoguo Shen; Emi Kawano-Yamashita; Takashi Nagata; Masahiko Hibi; Satoshi Tamotsu; Mitsumasa Koyanagi; Akihisa Terakita

doi:10.1073/pnas.1802592115

Edited by King-Wai Yau, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 12, 2018 (received for review February 12, 2018)

Significance

Color discrimination in animals is considered to require opponent processing of signals from two or more opsins sensitive to different parts of the spectrum. We previously reported that lower vertebrate pineal organs, which discriminate between UV and visible light, employ a bistable opsin called parapinopsin that has two stable photointerconvertible states, a signaling-inactive state maximally sensitive to UV and a visible light-sensitive signaling-active photoproduct. Here, we present evidence that the photoequilibrium between these two states in the zebrafish pineal organ is dependent on the spectral composition of incident light, setting the opsin’s signaling activity according to color to allow parapinopsin alone to generate color opponency between UV and visible light at the level of single pineal photoreceptor cells.

Abstract

Lower vertebrate pineal organs discriminate UV and visible light. Such color discrimination is typically considered to arise from antagonism between two or more spectrally distinct opsins, as, e.g., human cone-based color vision relies on antagonistic relationships between signals produced by red-, green-, and blue-cone opsins. Photosensitive pineal organs contain a bistable opsin (parapinopsin) that forms a signaling-active photoproduct upon UV exposure that may itself be returned to the signaling-inactive “dark” state by longer-wavelength light. Here we show the spectrally distinct parapinopsin states (with antagonistic impacts on signaling) allow this opsin alone to provide the color sensitivity of this organ. By using calcium imaging, we show that single zebrafish pineal photoreceptors held under a background light show responses of opposite signs to UV and visible light. Both such responses are deficient in zebrafish lacking parapinopsin. Expressing a UV-sensitive cone opsin in place of parapinopsin recovers UV responses but not color opponency. Changes in the spectral composition of white light toward enhanced UV or visible wavelengths respectively increased vs. decreased calcium signal in parapinopsin-sufficient but not parapinopsin-deficient photoreceptors. These data reveal color opponency from a single kind of bistable opsin establishing an equilibrium-like mixture of the two states with different signaling abilities whose fractional concentrations are defined by the spectral composition of incident light. As vertebrate visual color opsins evolved from a bistable opsin, these findings suggest that color opponency involving a single kind of bistable opsin might have been a prototype of vertebrate color opponency.

Color discrimination is achieved through antagonistic responses to different wavelengths of light, known as color opponency. Such color opponency has been described in many animal species and has previously been considered to require antagonistic interaction between signals produced by two or more opsins each sensitive to a different portion of the spectrum. The best-known instance of color opponency originates in the retina and is well known as color vision. Thus, in humans, trichromatic vision arises from red-, green-, and blue-sensitive cone opsins, which are exclusively present in different photoreceptor cells (1 ⇓⇓–4). These photoreceptor cells transmit light information to the brain via a complex neural network involving neurons that subtract outputs from different photoreceptor cells to generate color opponency. Such operations enable humans to recognize an enormous number of colors. Many vertebrates also have extraocular photoreception, and color opponency in these systems has also been reported in the context of sensing dawn/dusk (5, 6). Mechanisms for such extraocular color opponency can be quite different from those of color vision. In lizard parietal eyes, blue vs. green discrimination is achieved at the level of single cells by comparing the activity of two opsins, blue-sensitive pinopsin and green-sensitive parietopsin, expressed in a single photoreceptor (5, 7). Interestingly, color opponency is generated through two distinct intracellular signal transduction cascades driven by pinopsin and parietopsin, respectively. Thus, in the lizard parietal eye, color opponency is a property of single cells rather than neural networks, as is the case for color vision.

Pineal organs of lower vertebrates such as lamprey and most teleosts also show color opponency, discriminating UV and visible light. This property has been recorded at the level of pineal ganglion cells (that receive light information from several pineal photoreceptor cells), with firing of these neurons suppressed by UV and enhanced by visible light (6, 8). We previously found that parapinopsin, an opsin first identified in catfish pineal and parapineal organs (9), is a UV-sensitive opsin and that it supports the UV reception involved in color opponency of the lamprey pineal organ (10). Interestingly, parapinopsin is a bistable opsin: exposure of dark-adapted parapinopsin to UV light produces a stable photoproduct that is itself maximally sensitive to visible light and, on light absorption, can regenerate the original dark state, showing interconvertibility between the dark state and its photoproduct. Therefore, parapinopsin has two stable “color states” with their absorption maxima at largely separated wavelengths, unlike other vertebrate cone opsins whose photoproduct is unstable (10, 11). However, it remains unclear how the stable photoproduct of parapinopsin is involved in the pineal color opponency.

Bistable opsins are also employed in visual cells of many invertebrates (12). Interestingly, under specific light conditions, the bistability can give rise to a form of “color opponent” behavior. Thus, for example, Drosophila visual cells contain a blue-sensitive rhodopsin whose photoproduct shows green/yellow sensitivity. The blue-sensitive opsin is not signaling-active, but its photoproduct is, driving depolarization of the photoreceptor cell. In white-eyed Drosophila, a bright blue light stimulus generates prolonged depolarization after light-off. This is known as prolonged depolarizing afterpotential (PDA), which occurs because the photoproduct (signaling active metarhodopsin) out-titrates the arrestin required to inactivate it. A subsequent long-wavelength (yellow or orange) light stimulus photoreisomerizes signaling-active metarhodopsin to signaling-inactive rhodopsin and results in suppression of the PDA, thereby generating an effective “hyperpolarization” (13). The opposite effects of blue and green/yellow light on photoreceptor polarization in this paradigm reveal the potential for bistable opsins to support a sort of color opponency but this does not translate to more physiological lighting conditions.

Recently, we found that parapinopsin in the zebrafish pineal organ exhibits a bistable nature identical to that observed for lamprey parapinopsin (SI Appendix, Fig. S1). We also obtained the promoter sequence of zebrafish parapinopsin (14). Here, we investigated the light response of parapinopsin-expressing cells (PP cells) by using genetic procedures. We found that parapinopsin alone can support color opponency at the single photoreceptor level. Color opponency in the zebrafish pineal organ has been considered to have little relevance to light regulation of pineal melatonin secretion (14, 15), and there is no evidence directly demonstrating its involvement in light entrainment of the circadian rhythms in zebrafish. Here, we also discuss physiological relevance of the zebrafish pineal wavelength discrimination on the basis of light conditions that enable parapinopsin-based color opponency.

Results

Antagonistic Chromatic Responses in a Single Pineal Photoreceptor Cell.

To investigate responses to light in PP cells, we performed calcium imaging in transgenic zebrafish PP cells expressing the calcium indicator GCaMP6s (16) by using a two-photon excitation microscope at 930 nm (Fig. 1 A and B). The excitation light for GCaMP6s is expected itself to activate photoreceptors and can therefore be considered equivalent to a continuous blue light stimulation (∼465 nm) throughout the experiment. After approximately 120 s of imaging, fluorescence reached a plateau (SI Appendix, Fig. S2), allowing us to investigate the calcium level changes in PP cells upon additional stimulation with different wavelengths of light. In each region of interest (ROI; ROIs 1–6 in Fig. 1B) containing part of a single PP cell, stimulation with 405-nm light caused a decrease in fluorescence intensity. This response is consistent with parapinopsins’ known ability to show light-dependent interaction with transducin, which is expected to lead to hyperpolarization of the PP cell and closure of voltage-sensitive calcium channels (17). In contrast, stimulation with 588-nm light elevated the fluorescence intensity, demonstrating the antagonistic effect of UV and visible light on the calcium in individual PP cells to UV and visible light (Fig. 1C). The fluorescence changes between the average of ROIs 1–6 and ROI 7 encompassing the whole pineal organ were almost identical, suggesting that the color opponency observed in the whole pineal organ was based on the antagonistic chromatic response of a single PP cell. Such antagonistic responses were reproducibly observed by repeated stimulation with UV and visible light (Fig. 1D). This represents color opponency in a single photoreceptor cell of the teleost pineal organ.

Fig. 1.

Color opponency in the calcium response of PP cells. (A) Construct used to express GCaMP6s in PP cells. (B) Fluorescence image of GCaMP6s in PP cells by two-photon excitation. ROIs 1–7 were used to calculate change rates in fluorescence intensity in C. (Scale bar: 20 µm.) (C) Color opponency in the calcium response of each ROI containing a part of single PP cells (ROIs 1–6) or all PP cells (ROI 7) to 405-nm (violet line) and 588-nm (yellow line) light stimuli. The averaged profile of ROIs 1–6 clearly shows color opponency. Error bars indicate SE. (D) Repeated antagonistic response to 405- and 588-nm light stimuli. The light intensities of 405- and 588-nm light stimuli in C and D were ∼3.2 × 10¹⁴ and ∼5.4 × 10¹⁷ photons per cm²·s, respectively. The durations of both stimuli were ∼450 ms. ∆F/F values are change rates of normalized fluorescence intensity with the averaged intensity of 10 points before initial light stimuli.

Color Opponency Depending on Molecular Property of Parapinopsin.

Color opponency in a single photoreceptor cell has previously been reported in the parietal eyes of the side-blotched lizard (5). In that photoreceptor cell, two different opsins, blue-sensitive pinopsin and green-sensitive parietopsin, drive gustducin- (Gt-type) and Go-mediated signal transduction cascades, leading to hyperpolarization and depolarization, respectively (7). Moreover, we previously found the coexpression of parapinopsin with parietopsin in photoreceptor cells of the parietal eye in iguana (18). These findings, together with the existence of a parietopsin gene in the zebrafish genome, encouraged us to investigate whether parietopsin is expressed in PP cells to mediate visible light sensitivity in color opponency, in combination with the UV-sensitive parapinopsin. RT-PCR and in situ hybridization analyses revealed that parietopsin was completely coexpressed with parapinopsin in the pineal organ (Fig. 2 A and B and SI Appendix, Fig. S3A). Like lizard and Xenopus parietopsins, zebrafish parietopsin is a green-sensitive opsin with an absorption maximum at ∼520 nm (7, 18, 19) (Fig. 2C). This observation and the localization of Go in PP cells (SI Appendix, Fig. S3B) suggested that parapinopsin and parietopsin mediate color opponency of the pineal single photoreceptor cell through a mechanism similar to that in parietal eye photoreceptor cells.

Fig. 2.

Contribution of opsins to color opponency in PP cells under two-photon imaging. (A) RT-PCR analyses of parietopsin and parapinopsin expression in the eye, brain, and pineal organ of zebrafish. (B) In situ hybridization analyses of two opsins in the zebrafish pineal organ using the double fluorescence method. Yellow arrowheads in B indicate coexpression of two opsins in the zebrafish pineal organ. Orientations marked with “d” and “r” indicate the dorsal and rostral sides, respectively. The white dotted traces indicate the landmarks of the pineal organ. (Scale bars: 100 µm.) (C) Relative difference absorption spectrum of zebrafish parietopsin before minus after light irradiation. The spectrum is shown as an average of three measurements. (D–H) Calcium level changes upon 405- or 588-nm light stimuli in PP cells of WT (D; n = 41), parietopsin-KO (E; PT^−/−, n = 28), parapinopsin-KO (F; PP^−/−, n = 40), double-KO (G; PP^−/−/PT^−/−, n = 27), and SWS1 opsin-expressing/parapinopsin-KO fish (H; SWS1/PP^−/−, n = 27). Error bars indicate SE. The light intensities of 405- and 588-nm light stimuli in D–H were ∼3.2 × 10¹⁴ and ∼5.4 × 10¹⁷ photons per cm²·s, respectively. The durations of both stimuli were ∼450 ms. ∆F/F values are change rates of normalized fluorescence intensity with the averaged intensity of 10 points before initial light stimuli. Statistical evaluation of the differences in amplitudes of calcium level changes among the WT and mutants are shown in SI Appendix, Fig. S5 A and B.

To investigate the contributions of the two opsins to the color opponency observed by calcium imaging, we established parapinopsin- and/or parietopsin-KO fish by CRISPR/Cas9-mediated genome editing (SI Appendix, Fig. S4). Surprisingly, fish lacking parietopsin (PT^−/−) still showed an antagonistic response to 405- and 588-nm light stimuli, similar to the response of the WT (Fig. 2 D and E). When the parapinopsin gene was disrupted (PP^−/−), the responses to 405- and 588-nm light stimuli largely disappeared (Fig. 2F and SI Appendix, Fig. S5). Double-KO fish showed a similar response (Fig. 2G, PP^−/−/PT^−/−). Slight residual calcium increases upon 588-nm light stimuli were observed in parapinopsin-deficient PP cells (PP^−/− and PP^−/−/PT^−/−) but were nearly negligible compared with the increase in PP cells containing parapinopsin. This suggests that, under the imaging conditions, parapinopsin alone mediated color opponency of the pineal photoreceptor cells, irrespective of the presence of parietopsin, enabling us to focus on the bistable nature of parapinopsin in further investigations.

To investigate the contribution of bistability, we generated transgenic fish expressing SWS1 opsin, a UV-sensitive cone opsin that shares parapinopsin’s transducin signaling cascade but is not bistable (17, 20), in PP cells (SWS1/PP^−/−). In the SWS1/PP^−/− mutant, SWS1-expressing PP cells clearly exhibited decrease of calcium levels in response to 405-nm light stimulation but did not significantly respond to 588-nm light (Fig. 2H), suggesting that the bistable nature of parapinopsin is responsible for the visible light-dependent calcium increase.

Color Opponency Based on Photoequilibrium of Parapinopsin Two States.

How the bistable nature underlies the antagonistic chromatic response in PP cells under imaging light conditions can be speculated as follows. As 930-nm light in two-photon excitation is considered to serve as blue light (∼465 nm) in one-photon excitation, the imaging conditions were considered to form a “photoequilibrium” or “semiphotoequilibrium” (i.e., photoequilibrium-like mixture) of the dark state and photoproduct of parapinopsin (Fig. 3A). For 10–20 s after starting imaging, fluorescence intensity decreased because of hyperpolarization of the PP cells, which demonstrates accumulation of the parapinopsin photoproduct under excitation light (SI Appendix, Fig. S2). The 405-nm light stimulus increases the short-wavelength component of incident light and thus increases the fractional concentration of the signaling active photoproduct from that produced by the imaging light alone, leading to hyperpolarization (Fig. 3 B and D, calcium decrease). Conversely, because the photoproduct is more sensitive to longer wavelengths, the 588-nm light stimulus decreases its fractional concentration, reducing the pool of signaling-active opsin, resulting in termination of hyperpolarization and increased calcium levels (Fig. 3 C and D, calcium increase). After the stimuli are turned off, the photoproduct amount is considered to revert to the original photoequilibrium level under “blue light” conditions (Fig. 3D). Therefore, the 405-nm and 588-nm light stimuli may transiently decrease and increase calcium levels by changing the fractional concentration of photoproduct from the photoequilibrium level.

Fig. 3.

Color opponency involving shift of photoequilibrium between the dark state and photoproduct of parapinopsin. (A–C) Schematic models of photoequilibrium between the dark state and photoproduct of parapinopsin and changes in the amount of its photoproduct upon different light stimuli under two-photon imaging. Circles filled with purple and green indicate molecules of the dark state and photoproduct of parapinopsin, respectively. Photoequilibrium of parapinopsin under two-photon excitation with 930-nm light (i.e., blue light) is shown with black arrows (A). The shifts in photoequilibrium caused by stimuli of 405- and 588-nm light are shown as purple (B) and orange (C) arrows, respectively. Red arrows indicate the amount of cone transducin (Gt2) activated by the photoproducts, which is equivalent to the hyperpolarization level. Note that the circles and the arrows schematically illustrated in A–D are not quantitatively drawn. The recovery of photoequilibrium to the original state after stimuli turn off under “blue light” condition could account for the antagonistic calcium response under two-photon imaging conditions. (D–F) Calcium level changes upon 405- and 588-nm light stimuli in WT PP cells under two-photon excitation with 930-nm (D; n = 26), 860-nm (E; n = 13), and 1,000-nm (F; n = 13) lights (i.e., blue, violet, and green lights, respectively). Schematic models for shifts in photoequilibrium by light stimuli are also shown in D. Error bars indicate SE. The light intensities of 405- and 588-nm light stimuli in D–F were ∼3.2 × 10¹⁴ and ∼5.4 × 10¹⁷ photons per cm²·s, respectively. The durations of both stimuli were ∼450 ms. ∆F/F values are change rates of normalized fluorescence intensity with the averaged intensity of 10 points before initial light stimuli.

To test this hypothesis, we analyzed calcium level changes under 860-, 930- and 1,000-nm two-photon excitations, which are considered equivalent to violet, blue, and green light (∼430, ∼465, and ∼500 nm) excitations, respectively, and are therefore expected to generate a different “basal photoequilibrium.” When imaging was performed with two-photon excitation with 860-nm light (i.e., “violet”), the 588-nm light stimulus increased the calcium levels, whereas the 405-nm light stimulus did not significantly decrease calcium levels (Fig. 3E). In contrast, in two-photon excitation with 1,000-nm light (i.e., “green”), the 405-nm light stimulus decreased the calcium level, whereas the 588-nm light stimulus did not evoke an obvious increase (Fig. 3F). These results, together with the response profile of two-photon excitation at 930 nm (Fig. 3D), strongly support that the photoequilibrium of parapinopsin is fundamentally important to generate color opponency to light stimuli.

We next confirmed whether color opponency based on a single parapinopsin is generated under environmental light conditions. In vitro spectroscopic analysis revealed that, under white light with spectral distribution similar to that of sunlight in the early afternoon, zebrafish parapinopsin formed a photoequilibrium similar to that under the sunlight conditions wherein the ratio of the dark state to the photoproduct was ∼4:1 (SI Appendix, Fig. S1, curve 4). The establishment of such a photoequilibrium allows the possibility that PP cells exhibit color opponency based on parapinopsin alone under sunlight. We investigated the calcium level changes in photoreceptor cells at 10-s intervals to reduce the effect of the two-photon imaging light, under continuous irradiation by white light with intensity ∼1% of that found in a sunny location in the early afternoon and with a similar spectral distribution (Fig. 4A). In WT and PT^−/−, a decrease followed by a steady state of calcium level was observed when the white light was turned on (SI Appendix, Fig. S6), similar to the case under imaging “blue” lights (SI Appendix, Fig. S2). In contrast, PP^−/− and PP^−/−/PT^−/− did not show large decreases in calcium levels in response to white light (SI Appendix, Fig. S6). After the calcium level reached a plateau (∼10 min), PP cells were exposed to “mixed white light” with UV or visible LED light, which has a broad spectral distribution (UV light- or visible light-mixed white light; Fig. 4A), to investigate whether color opponency based on parapinopsin is observed under natural light-like conditions. We found decreased and increased calcium levels following exposure to UV and visible light-mixed white light, respectively, in WT and PT^−/− but not in PP^−/− and PP^−/−/PT^−/− (Fig. 4B). Changes in calcium level were also observed in PP cells following exposure to different intensities and durations of UV and visible light-mixed white lights (SI Appendix, Fig. S7 A–C). These observations suggest that parapinopsin alone can allow pineal photoreceptors to respond to changes in the spectral distribution of incident light (i.e., color opponency) under physiological sunlight conditions.

Fig. 4.

White light spectra and color opponency of PP cells under white light conditions. (A) Spectra of the xenon white light (solid black trace), UV LED light-mixed white light (broken magenta trace), and visible LED light-mixed white light (broken green trace) are compared with those of 1% intensity of direct sunlight (solid gray trace) measured at 2:30 PM on April 16, 2018, in Osaka, Japan. The light intensities (360–780 nm) of white light, UV-mixed white light, visible light-mixed white light, and direct sunlight were ∼9.2 × 10¹⁴, ∼9.3 × 10¹⁴, ∼9.9 × 10¹⁴, and 1.1 × 10¹⁷ photons per cm²·s. The light intensities of UV and visible LED lights were ∼1.3 × 10¹³ and ∼7.6 × 10¹³ photons per cm²·s, respectively. (B) Changes in calcium level of PP cells in WT (red open circle; n = 24), PT^−/− fish (green closed circle; n = 23), PP^−/− fish (purple open triangle; n = 17), and PP^−/−/PT^−/− fish (black closed triangle; n = 16) under different light conditions shown in A. The shaded traces indicate calcium level changes of individual fish. PP cells of WT and mutants were exposed to the white light (Top, white bars), UV light-mixed white light (hatched magenta bars), and visible light-mixed white light (hatched green bars). Note that the calcium levels decreased and increased transiently in response to exposure to UV and visible light-mixed white lights, respectively, in WT and PT^−/−. Error bars indicate SE. The durations of both stimuli were ∼9.4 s. ∆F/F values are change rates of normalized fluorescence intensity with the averaged intensity of 10 points before UV-mixed white light exposures. Note that the relaxation of responses to UV and green LED light in WT and PT^−/− were not observed as a result of time-lapse recoding with low time resolution.

Discussion

In this study, we describe a chromatically antagonistic response of calcium in a single zebrafish pineal cell produced by a single kind of bistable opsin, parapinopsin. The mechanism relies upon parapinopsin’s ability to form two photointerconvertible opsin states with different spectral sensitivity and signaling capacity. Under extended light exposure, an equilibrium between these two states is formed in which the fractional concentration of the signaling active state is dependent on the spectral composition of incident light, with lights biased toward UV driving greater hyperpolarization than those biased toward visible light.

A previous study revealed a correlation of photoresponses between calcium dynamics and electrophysiological outputs in photoreceptor cells (i.e., cones) of the zebrafish retina (21). Therefore, the antagonistic chromatic changes in calcium levels measured in this study qualitatively suggest electrophysiological outputs of color opponency in the PP cells of zebrafish. Parapinopsin is found in a wide variety of lower vertebrates (9, 10, 14, 18), and its bistable nature might contribute to a single kind of opsin-based color opponency in pineal-related organs other than the teleost pineal organ.

Photoreceptor cells in many invertebrates such as insects, crustaceans, and cephalopods employ bistable opsins (12). Photoreceptor cells of white-eyed Drosophila show “opponent” responses to blue and orange light under specific artificial experimental conditions (13). Following intense blue light irradiation, fly photoreceptors generate a depolarizing PDA that persists in the dark as a result of insufficient shut-off of light-activated rhodopsin; orange light then causes “opponent” responses (i.e., hyperpolarizations) because of the reverse photoreaction of the accumulated “signaling-active metarhodopsin” (arrestin-free) to the inactive dark-state rhodopsin (13). Because, similarly to the mechanism of PDA, one might speculate that PP cells require abundant “active” or “not-inactivated” photoproducts to generate the reverse responses of photoreceptor cells (i.e., depolarization), it is important to consider the activity of biochemical deactivation mechanisms in this system. We therefore investigated the recovery kinetics of light response via shut-off of light-activated parapinopsin after turning off the white lights (i.e., in the dark). Interestingly, calcium levels recovered to that of the dark level by ∼20 s after turning off the white light (SI Appendix, Fig. S8), showing no prolongation of the light response, as in a typical PDA, which continues for hours in the dark. Therefore, it is of interest to discuss how the active photoproduct accumulates under white light.

The duration of light exposure for maximum changes in increase of calcium level in PP cells (10–20 s; SI Appendix, Fig. S7), which is considered to be correlated to accumulation of the photoproduct, is shorter than the duration (more than 100 s) estimated to be required for ∼99% level of the photoequilibrium formation (SI Appendix, Supplementary Information Text). To address this inconsistency, we roughly estimated a time evolution of the signaling active photoproduct after exposure to the white light, based on the very simplified reaction scheme of the dark state and photoproduct of parapinopsin according to that of bistable Drosophila rhodopsin and metarhodopsin (13) (SI Appendix, Supplementary Information Text and Fig. S9). The estimated profile suggests that the change in signaling active photoproduct amount in PP cells reaches maximum at approximately 20 s under exposure of PP cells to different white light (SI Appendix, Fig. S9C). The duration is similar to that for maximum changes in calcium level in PP cells under exposure to white light (SI Appendix, Fig. S7C). The profiles estimated with different values for reaction parameters suggest that the dark-inactivation of active photoproducts of parapinopsin under white light might contribute to accumulation of active photoproducts (SI Appendix, Fig. S9 C–E). Because the estimation is based on a very simplified model, investigation of a detailed mechanism(s) involved in the dark inactivation of the signaling-active photoproducts, such as phosphorylation of and arrestin binding to parapinopsin photoproducts and their kinetics is important to understand how a certain amount of the active photoproduct is kept in PP cells under white light (SI Appendix, Supplementary Information Text). In addition, this issue is also important to investigate if activities of proteins, such as arrestin (22), are regulated when the photoproducts revert to the dark state upon absorption of visible light.

We also observed slow elevation of calcium levels after these levels were greatly decreased under continuous blue light irradiation (i.e., two-photon excitation) or xenon white light (SI Appendix, Figs. S2 and S6). Such slow elevation of calcium may be related to the kinetics of signaling-active photoproduct (SI Appendix, Fig. S9C), which could constitute a mechanism of light adaptation. As calcium itself affects electrophysiological responses by regulating several signal transduction proteins (23, 24), it is important to investigate such parapinopsin-based light adaptation by comparing WT and SWS1/PP^−/− zebrafish.

We found coexpression of parapinopsin and green-sensitive parietopsin, which underlies depolarization in the lizard parietal eye, in the photoreceptor cells of the zebrafish pineal organ. Although parapinopsin-deficient PP cells exhibited a slight residual increase in calcium levels, suggesting depolarization (Fig. 2), the residual responses in PP^−/− and PP^−/−/PT^−/− were similar in amplitude (SI Appendix, Fig. S5B), suggesting that the residual increase in the calcium level did not involve a large contribution by parietopsin. Additionally, following exposure to white light, no significant difference in calcium changes was observed between PP^−/− and PP^−/−/PT^−/− (SI Appendix, Fig. S6). Taken together, these observations suggest a limited contribution of parietopsin in regulating calcium levels under the white light conditions used in this study. Such little involvement of parietopsin under these light conditions may be explained by its molecular property as a bleaching opsin (19). Under bright light conditions such as those in early to late afternoon (Fig. 4A and SI Appendix, Fig. S10A), the amount of functional parietopsin decreases because the photoproduct releases its retinal chromophore and loses its function after light absorption. In this circumstance, it remains possible that parietopsin contributes to color opponency by activating Go under dim light conditions (e.g., dusk and dawn), as suggested for the lizard parietal eye (5, 7).

In contrast to the two-opsin system involving parietopsin, the chromatic mechanism involving parapinopsin alone in the zebrafish pineal organ may not be suitable for detecting dawn and dusk. Light intensity at these times [e.g., irradiance of 0.1 W/m² or less (25), ∼3% brightness of the white light used in Fig. 4] is not sufficiently strong to form a photoequilibrium between the two states, which is essential for generating color opponency via this mechanism. The intensity and spectral distribution of natural sunlight varies depending on numerous factors, e.g., the time of day (e.g., early and late afternoon) and local conditions (e.g., sun or shade). Light intensity in the late afternoon is considered to be bright enough for parapinopsin-based color opponency (SI Appendix, Fig. S10A). Interestingly, the spectral distribution of light largely differs between the sunny and shady locations in the late afternoon; late-afternoon sunlight in a sunny location contains abundant longer-wavelength components whereas shorter-wavelength components are relatively increased in the shade. The photoequilibria of parapinopsin formed under sunny and shade sunlight conditions were different; the relative amounts of photoproduct in the photoequilibrium under the sunny and shady location conditions were ∼8% and ∼25%, respectively (SI Appendix, Fig. S10B). Therefore, such changes in wavelength components between sunny and shady locations in the late afternoon may be detected by the mechanism involving parapinopsin alone because late afternoon sunlight in the shade is approximately fivefold brighter than the white light used in this study, in which parapinopsin-based color opponency was generated (Fig. 4 A and B and SI Appendix, Fig. S10). Determining the intensity of white light that enables PP cells to generate color opponency would provide a clue to the physiological function of parapinopsin-based color opponency as well as how putative parapinopsin-based and parapinopsin/parietopsin-based opponent mechanisms are used for the color opponency of PP cells, depending on different situations.

It has been suggested that the ancestral animal opsin had a bistable nature (26). Therefore, a single kind of bistable opsin-based color opponency in a single photoreceptor cell might have been a prototype of color opponency. As shown in Fig. 3, the shift of the quantity ratio of signaling-inactive and -active (“dark” and “photoproduct,” respectively) states corresponding to the wavelengths of light is a key to generating color opponency. Bistable opsins in which dark and light states have largely overlapping absorption spectra do not clearly exhibit their photoequilibrium shift depending on the wavelengths of light, and therefore such a bistable opsin is not suitable for a single kind of opsin-based color opponency. In other words, the separation of spectral sensitivities between dark and light states could be critical to detect the color information. Accordingly, the color opponency with a single kind of bistable opsin could have simply developed from a brightness detection system with a single kind of bistable opsin through molecular evolution of the opsin, that is, amino acid mutations resulting in a bistable opsin having two spectroscopically separated states, like parapinopsin. Taken together, such a single kind of bistable opsin-based color opponency could be the first step for developing varied mechanisms of color opponency including color vision with multiple opsins.

Materials and Methods

Animals.

Zebrafish (Danio rerio) were obtained from the Zebrafish International Resource Center and National BioResource Project Zebrafish. Zebrafish were maintained on 14-h light/10-h dark cycles at 28.5 °C. Embryos (0–7 d postfertilization) were maintained in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄). The animal experiment procedures were approved by the Osaka City University Animal Experiment Committee (no. S0032) and complied with the regulations on animal experiments from Osaka City University.

Generation of Transgenic Fish.

The ∼5 kb upstream sequence of zebrafish parapinopsin (PP1; DDBJ/EMBL/GenBank accession no. AB626966) was previously isolated and inserted into the Tol2 vector pT2AL200R150G (14, 27), which contains the EGFP expression cassette. To introduce GCaMP6s into PP cells, the EGFP sequence was replaced with the GCaMP6s sequence obtained from pGP-CMV-GCaMP6s (no. 40753; Addgene). To introduce SWS1 opsin (DDBJ/EMBL/GenBank accession no. D85863) into zebrafish PP cells, the SWS1-P2A-mCherry sequence was used instead of the GCaMP6s sequence. Plasmid DNA (∼25 pg) and Tol2 mRNA (∼25 pg) were coinjected into embryos during the one-cell stage.

Generation of Gene-KO Fish.

Guide RNAs (SI Appendix, Fig. S4) were generated as described previously (28). The plasmids pT7-gRNA and pT3TS-nCas9n were obtained from Addgene (nos. 46759 and 46757). The oligonucleotide sets designed for guide RNAs were as follows: 5′-TAGGGTGGCGGTGTCCTGGGTC-3′ and 5′-AAACGACCCAGGACACCGCCAC-3′ for zebrafish parapinopsin guide RNA and 5′-GGCGTATGAGCGCTATAACG-3′ and 5′-AAACCGTTATAGCGCTCATACG-3′ for zebrafish parietopsin guide RNA. Annealed oligonucleotide sets were inserted into pT7-gRNA, which had been linearized by BsmB I. Guide RNAs were transcribed by using the MEGAscript T7 Transcription Kit. Cas9 mRNA was transcribed in vitro by using the mMESSAGE mMACHINE T3 Transcription Kit (Ambion). Guide RNA (∼50 pg) and Cas9 mRNA (∼150 pg) were coinjected into embryos during the one-cell stage. Mutations in the zebrafish parapinopsin and parietopsin nucleotide sequences were confirmed by a heteroduplex mobility assay (29). Stop codons appear after Val224 and Ser135 in the PP^−/− and PT^−/− mutants, respectively. Opsin(s)-deficient fish were obtained by mating the established KO strains.

Two-Photon Imaging.

Zebrafish larvae (7 d postfertilization) were mounted dorsal side up in low-melting agarose gel (1.5% in E3 medium) on 35-mm glass-bottom dishes (Iwaki). To prevent drying and immobilize the larvae, E3 medium containing 0.002% Tricaine (MS222; Sigma) was added to the dishes. Two-photon calcium imaging of transgenic fish was performed by using a multiphoton laser scanning microscope (FVMPE-RS; Olympus). The Mai Tai HP DeepSee IR laser (Spectra-Physics) was used for two-photon excitation of GCaMP6s in PP cells. The wavelength and intensity of the IR laser were controlled by using FLUOVIEW software (Olympus). The laser intensities at 860, 930, and 1,000 nm were 2.37, 1.49, and 0.74 W, and 2.5%, 4%, and 8% of each were used as experimental conditions, respectively. A Galvano scanner was used to acquire images for all procedures (∼450 ms in a 160 × 160-pixel image). Light stimulation using lasers was conducted by scanning with 405-nm (OBIS; Coherent) and 588-nm (Sapphire; Coherent) lasers with the same scanning areas as those of IR laser scanning. The light intensities of lasers were calculated based on the size of the scanning area (∼0.0064 mm²). In imaging under two-photon excitation, the intensities of light stimuli using 405- and 588-nm lasers were ∼3.2 × 10¹⁴ and ∼5.4 × 10¹⁷ photons per cm²·s, respectively, and the durations were ∼450 ms. Three types of white lights were used for white light experiments (Fig. 4A); the xenon lamp white light as a standard white light in this paper was mixed with UV (∼365 nm, ∼8.9 × 10¹² photons per cm²·s) or visible (∼580 nm, ∼4.7 × 10¹³ photons per cm²·s) LED light (pE-4000; CoolLED). White light was provided through the cold filter (CLDF-50S; Sigma Koki) from a xenon light source. White-light irradiation was blocked at the time of image acquisition by using a shutter synchronized with IR laser scanning. An XLPLN 25× WMP2 objective lens (NA, ∼1.05; Olympus) was used for all imaging procedures. The temporal changes in GCaMP6s fluorescence intensities in time-series images were analyzed by using FLUOVIEW. Subtraction of mean fluorescence intensity between each ROI containing PP cells and the nonfluorescent region outside PP cells (i.e., background) was carried out to calculate net fluorescence intensity. In Figs. 1D, 2 D–H, 3 D–F, and 4B and SI Appendix, Figs. S2 and S5–S8, the mean fluorescence intensity in an ROI containing all PP cells in one fish such as ROI 7 in Fig. 1B was used to eliminate arbitrariness in selecting the ROI. The fluorescence changes in time-series images were shown as the rates of change ΔF/F₀ (normalized with initial point; dark) or ΔF/F (defined in each figure legend).

RT-PCR.

Total RNA from the pineal organ, brain, and eye of zebrafish was purified with an RNeasy Mini kit (Qiagen) and reverse-transcribed into cDNA by using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). β-Actin was used as an internal reference gene. The primer sequences are shown in SI Appendix, Materials and Methods.

In Situ Hybridization.

Preparation of RNA probes and in situ hybridization were carried out as previously described (17). Digoxigenin (DIG)- and fluorescein-labeled antisense and sense RNA probes for zebrafish parapinopsin and parietopsin mRNAs were synthesized by using the DIG RNA labeling kit and fluorescein RNA labeling kit (Roche), respectively. Sections were pretreated with proteinase K and hybridized with each RNA probe in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). For double fluorescence labeling, sections hybridized with DIG-labeled probes were incubated with HRP-conjugated anti-DIG antibody (Roche) and subsequently treated with the TSA plus DNP (HRP) system (Perkin-Elmer), followed by incubation with Alexa 488-conjugated anti-DNP antibody. Fluorescein-labeled probes on the sections were detected by incubation with alkaline phosphatase-conjugated anti-fluorescein antibody (Roche) followed by a color reaction using the HNPP Fluorescent Detection Set (Roche).

Spectroscopic Analyses of Opsin-Based Pigment.

Zebrafish parapinopsin and parietopsin were expressed and purified as previously described (30, 31). Expressed proteins were reconstituted by adding an excess of 11-cis-retinal, extracted with 1% n-dodecyl β-d-maltoside (DM) in 50 mM Hepes buffer (pH 6.5) containing 140 mM NaCl (buffer A) and purified by using 1D4-agarose with buffer A containing 0.02% DM. Absorption spectra were measured with a UV2450 spectrophotometer (Shimadzu) at 0 °C. Full methods are described in SI Appendix, Materials and Methods.

Measurement of Natural Light Spectra.

A CL-500A illuminance spectrophotometer (Konica Minolta) was used for all measurements. Spectrum measurement in the early afternoon was carried out at 2:30 PM on April 16, 2018, in Osaka, Japan. Spectrum measurement of sunny and shady locations in the late afternoon was performed at 6:00 PM on June 16, 2018, in Osaka, Japan. The shade was made by shielding direct sunlight with a metallic case (height 30 cm × width 40 cm × depth 3 cm). Shade light, as indirect sunlight from the sky, was measured at a point 100 mm away from the metal case against the direction of the sun. The detector was oriented perpendicularly to the sky and measured at the ground level (∼5 cm).

Acknowledgments

We thank Robert J. Lucas (University of Manchester) for his valuable comments on this manuscript and Robert S. Molday (University of British Columbia) for supplying rho 1D4-producing hybridoma. This work was supported by Japanese Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research 15H05777 (to A.T.), 16KT0074 and 17H06015 (to M.K.), 14J40094 (to E.K.-Y.), and 13J05782 (to S.W.); Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) Grant JPMJCR1753 (to A.T.) and JST Precursory Research for Embryonic Science and Technology (PRESTO) Grant JPMJPR13A2 (to M.K.); and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to E.K.-Y. and S.W.).

Footnotes

↵¹To whom correspondence should be addressed. Email: terakita{at}sci.osaka-cu.ac.jp.

Author contributions: S.W., M.K., and A.T. designed research; S.W. and B.S. performed research; E.K.-Y., T.N., M.H., and S.T. contributed new reagents/analytic tools; and S.W., M.K., and A.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802592115/-/DCSupplemental.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

References

↵
1. Nathans J,
2. Thomas D,
3. Hogness DS
(1986) Molecular genetics of human color vision: The genes encoding blue, green, and red pigments. Science 232:193–202.
OpenUrl Abstract/FREE Full Text
↵
1. Schnapf JL,
2. Kraft TW,
3. Baylor DA
(1987) Spectral sensitivity of human cone photoreceptors. Nature 325:439–441.
OpenUrl CrossRef PubMed
↵
1. Merbs SL,
2. Nathans J
(1992) Absorption spectra of human cone pigments. Nature 356:433–435.
OpenUrl CrossRef PubMed
↵
1. Dacey DM,
2. Lee BB
(1994) The ‘blue-on’ opponent pathway in primate retina originates from a distinct bistratified ganglion cell type. Nature 367:731–735.
OpenUrl CrossRef PubMed
↵
1. Solessio E,
2. Engbretson GA
(1993) Antagonistic chromatic mechanisms in photoreceptors of the parietal eye of lizards. Nature 364:442–445.
OpenUrl CrossRef PubMed
↵
1. Uchida K,
2. Morita Y
(1994) Spectral sensitivity and mechanism of interaction between inhibitory and excitatory responses of photosensory pineal neurons. Pflugers Arch 427:373–377.
OpenUrl CrossRef PubMed
↵
1. Su CY, et al.
(2006) Parietal-eye phototransduction components and their potential evolutionary implications. Science 311:1617–1621.
OpenUrl Abstract/FREE Full Text
↵
1. Dodt E
(1962) [Comparative studies on the adaptive behavior of the pure cone retina. Citellus citellus, Sciurus vulgaris]. Pflugers Arch Gesamte Physiol Menschen Tiere 275:561–573. German.
OpenUrl PubMed
↵
1. Blackshaw S,
2. Snyder SH
(1997) Parapinopsin, a novel catfish opsin localized to the parapineal organ, defines a new gene family. J Neurosci 17:8083–8092.
OpenUrl Abstract/FREE Full Text
↵
1. Koyanagi M, et al.
(2004) Bistable UV pigment in the lamprey pineal. Proc Natl Acad Sci USA 101:6687–6691.
OpenUrl Abstract/FREE Full Text
↵
1. Terakita A, et al.
(2004) Counterion displacement in the molecular evolution of the rhodopsin family. Nat Struct Mol Biol 11:284–289, and erratum (2004) 11:384.
OpenUrl CrossRef PubMed
↵
1. Koyanagi M,
2. Terakita A
(2008) Gq-coupled rhodopsin subfamily composed of invertebrate visual pigment and melanopsin. Photochem Photobiol 84:1024–1030.
OpenUrl CrossRef PubMed
↵
1. Stavenga DG,
2. Hardie RC
(2011) Metarhodopsin control by arrestin, light-filtering screening pigments, and visual pigment turnover in invertebrate microvillar photoreceptors. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 197:227–241.
OpenUrl CrossRef PubMed
↵
1. Koyanagi M, et al.
(2015) Diversification of non-visual photopigment parapinopsin in spectral sensitivity for diverse pineal functions. BMC Biol 13:73.
OpenUrl CrossRef PubMed
↵
1. Ziv L,
2. Tovin A,
3. Strasser D,
4. Gothilf Y
(2007) Spectral sensitivity of melatonin suppression in the zebrafish pineal gland. Exp Eye Res 84:92–99.
OpenUrl CrossRef PubMed
↵
1. Chen TW, et al.
(2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499:295–300.
OpenUrl CrossRef PubMed
↵
1. Kawano-Yamashita E, et al.
(2015) Activation of transducin by bistable pigment parapinopsin in the pineal organ of lower vertebrates. PLoS One 10:e0141280.
OpenUrl
↵
1. Wada S,
2. Kawano-Yamashita E,
3. Koyanagi M,
4. Terakita A
(2012) Expression of UV-sensitive parapinopsin in the iguana parietal eyes and its implication in UV-sensitivity in vertebrate pineal-related organs. PLoS One 7:e39003.
OpenUrl CrossRef PubMed
↵
1. Sakai K, et al.
(2012) Photochemical nature of parietopsin. Biochemistry 51:1933–1941.
OpenUrl PubMed
↵
1. Lamb TD
(2013) Evolution of phototransduction, vertebrate photoreceptors and retina. Prog Retin Eye Res 36:52–119.
OpenUrl CrossRef PubMed
↵
1. Leung YT,
2. Fain GL,
3. Matthews HR
(2007) Simultaneous measurement of current and calcium in the ultraviolet-sensitive cones of zebrafish. J Physiol 579:15–27.
OpenUrl CrossRef PubMed
↵
1. Eichel K,
2. Jullié D,
3. von Zastrow M
(2016) β-arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat Cell Biol 18:303–310.
OpenUrl CrossRef PubMed
↵
1. Pugh EN Jr,
2. Nikonov S,
3. Lamb TD
(1999) Molecular mechanisms of vertebrate photoreceptor light adaptation. Curr Opin Neurobiol 9:410–418.
OpenUrl CrossRef PubMed
↵
1. Yau KW
(1991) Calcium and light adaptation in retinal photoreceptors. Curr Opin Neurobiol 1:252–257.
OpenUrl CrossRef PubMed
↵
1. Spitschan M,
2. Lucas RJ,
3. Brown TM
(2017) Chromatic clocks: Color opponency in non-image-forming visual function. Neurosci Biobehav Rev 78:24–33.
OpenUrl
↵
1. Koyanagi M,
2. Kawano-Yamashita E,
3. Wada S,
4. Terakita A
(2017) Vertebrate bistable pigment parapinopsin: Implications for emergence of visual signaling and neofunctionalization of non-visual pigment. Front Ecol Evol 5:23.
OpenUrl CrossRef
↵
1. Urasaki A,
2. Asakawa K,
3. Kawakami K
(2008) Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish. Proc Natl Acad Sci USA 105:19827–19832.
OpenUrl Abstract/FREE Full Text
↵
1. Jao LE,
2. Wente SR,
3. Chen W
(2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 110:13904–13909.
OpenUrl Abstract/FREE Full Text
↵
1. Ota S, et al.
(2013) Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells 18:450–458.
OpenUrl CrossRef PubMed
↵
1. Koyanagi M,
2. Takada E,
3. Nagata T,
4. Tsukamoto H,
5. Terakita A
(2013) Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue. Proc Natl Acad Sci USA 110:4998–5003.
OpenUrl Abstract/FREE Full Text
↵
1. Sugihara T,
2. Nagata T,
3. Mason B,
4. Koyanagi M,
5. Terakita A
(2016) Absorption characteristics of vertebrate non-visual opsin, Opn3. PLoS One 11:e0161215.
OpenUrl

Article Alerts

Email Article

Citation Tools

Request Permissions

Article Classifications

Biological Sciences
Neuroscience

Proceedings of the National Academy of Sciences: 115 (44)

Table of Contents

Submit

[1] ↵
Nathans J,
Thomas D,
Hogness DS
(1986) Molecular genetics of human color vision: The genes encoding blue, green, and red pigments. Science 232:193–202.
OpenUrl Abstract/FREE Full Text

[2] Nathans J,

[3] Thomas D,

[4] Hogness DS

[5] ↵
Schnapf JL,
Kraft TW,
Baylor DA
(1987) Spectral sensitivity of human cone photoreceptors. Nature 325:439–441.
OpenUrl CrossRef PubMed

[6] Schnapf JL,

[7] Kraft TW,

[8] Baylor DA

[9] ↵
Merbs SL,
Nathans J
(1992) Absorption spectra of human cone pigments. Nature 356:433–435.
OpenUrl CrossRef PubMed

[10] Merbs SL,

[11] Nathans J

[12] ↵
Dacey DM,
Lee BB
(1994) The ‘blue-on’ opponent pathway in primate retina originates from a distinct bistratified ganglion cell type. Nature 367:731–735.
OpenUrl CrossRef PubMed

[13] Dacey DM,

[14] Lee BB

[15] ↵
Solessio E,
Engbretson GA
(1993) Antagonistic chromatic mechanisms in photoreceptors of the parietal eye of lizards. Nature 364:442–445.
OpenUrl CrossRef PubMed

[16] Solessio E,

[17] Engbretson GA

[18] ↵
Uchida K,
Morita Y
(1994) Spectral sensitivity and mechanism of interaction between inhibitory and excitatory responses of photosensory pineal neurons. Pflugers Arch 427:373–377.
OpenUrl CrossRef PubMed

[19] Uchida K,

[20] Morita Y

[21] ↵
Su CY, et al.
(2006) Parietal-eye phototransduction components and their potential evolutionary implications. Science 311:1617–1621.
OpenUrl Abstract/FREE Full Text

[22] Su CY, et al.

[23] ↵
Dodt E
(1962) [Comparative studies on the adaptive behavior of the pure cone retina. Citellus citellus, Sciurus vulgaris]. Pflugers Arch Gesamte Physiol Menschen Tiere 275:561–573. German.
OpenUrl PubMed

[24] Dodt E

[25] ↵
Blackshaw S,
Snyder SH
(1997) Parapinopsin, a novel catfish opsin localized to the parapineal organ, defines a new gene family. J Neurosci 17:8083–8092.
OpenUrl Abstract/FREE Full Text

[26] Blackshaw S,

[27] Snyder SH

[28] ↵
Koyanagi M, et al.
(2004) Bistable UV pigment in the lamprey pineal. Proc Natl Acad Sci USA 101:6687–6691.
OpenUrl Abstract/FREE Full Text

[29] Koyanagi M, et al.

[30] ↵
Terakita A, et al.
(2004) Counterion displacement in the molecular evolution of the rhodopsin family. Nat Struct Mol Biol 11:284–289, and erratum (2004) 11:384.
OpenUrl CrossRef PubMed

[31] Terakita A, et al.

[32] ↵
Koyanagi M,
Terakita A
(2008) Gq-coupled rhodopsin subfamily composed of invertebrate visual pigment and melanopsin. Photochem Photobiol 84:1024–1030.
OpenUrl CrossRef PubMed

[33] Koyanagi M,

[34] Terakita A

[35] ↵
Stavenga DG,
Hardie RC
(2011) Metarhodopsin control by arrestin, light-filtering screening pigments, and visual pigment turnover in invertebrate microvillar photoreceptors. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 197:227–241.
OpenUrl CrossRef PubMed

[36] Stavenga DG,

[37] Hardie RC

[38] ↵
Koyanagi M, et al.
(2015) Diversification of non-visual photopigment parapinopsin in spectral sensitivity for diverse pineal functions. BMC Biol 13:73.
OpenUrl CrossRef PubMed

[39] Koyanagi M, et al.

[40] ↵
Ziv L,
Tovin A,
Strasser D,
Gothilf Y
(2007) Spectral sensitivity of melatonin suppression in the zebrafish pineal gland. Exp Eye Res 84:92–99.
OpenUrl CrossRef PubMed

[41] Ziv L,

[42] Tovin A,

[43] Strasser D,

[44] Gothilf Y

[45] ↵
Chen TW, et al.
(2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499:295–300.
OpenUrl CrossRef PubMed

[46] Chen TW, et al.

[47] ↵
Kawano-Yamashita E, et al.
(2015) Activation of transducin by bistable pigment parapinopsin in the pineal organ of lower vertebrates. PLoS One 10:e0141280.
OpenUrl

[48] Kawano-Yamashita E, et al.

[49] ↵
Wada S,
Kawano-Yamashita E,
Koyanagi M,
Terakita A
(2012) Expression of UV-sensitive parapinopsin in the iguana parietal eyes and its implication in UV-sensitivity in vertebrate pineal-related organs. PLoS One 7:e39003.
OpenUrl CrossRef PubMed

[50] Wada S,

[51] Kawano-Yamashita E,

[52] Koyanagi M,

[53] Terakita A

[54] ↵
Sakai K, et al.
(2012) Photochemical nature of parietopsin. Biochemistry 51:1933–1941.
OpenUrl PubMed

[55] Sakai K, et al.

[56] ↵
Lamb TD
(2013) Evolution of phototransduction, vertebrate photoreceptors and retina. Prog Retin Eye Res 36:52–119.
OpenUrl CrossRef PubMed

[57] Lamb TD

[58] ↵
Leung YT,
Fain GL,
Matthews HR
(2007) Simultaneous measurement of current and calcium in the ultraviolet-sensitive cones of zebrafish. J Physiol 579:15–27.
OpenUrl CrossRef PubMed

[59] Leung YT,

[60] Fain GL,

[61] Matthews HR

[62] ↵
Eichel K,
Jullié D,
von Zastrow M
(2016) β-arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat Cell Biol 18:303–310.
OpenUrl CrossRef PubMed

[63] Eichel K,

[64] Jullié D,

[65] von Zastrow M

[66] ↵
Pugh EN Jr,
Nikonov S,
Lamb TD
(1999) Molecular mechanisms of vertebrate photoreceptor light adaptation. Curr Opin Neurobiol 9:410–418.
OpenUrl CrossRef PubMed

[67] Pugh EN Jr,

[68] Nikonov S,

[69] Lamb TD

[70] ↵
Yau KW
(1991) Calcium and light adaptation in retinal photoreceptors. Curr Opin Neurobiol 1:252–257.
OpenUrl CrossRef PubMed

[71] Yau KW

[72] ↵
Spitschan M,
Lucas RJ,
Brown TM
(2017) Chromatic clocks: Color opponency in non-image-forming visual function. Neurosci Biobehav Rev 78:24–33.
OpenUrl

[73] Spitschan M,

[74] Lucas RJ,

[75] Brown TM

[76] ↵
Koyanagi M,
Kawano-Yamashita E,
Wada S,
Terakita A
(2017) Vertebrate bistable pigment parapinopsin: Implications for emergence of visual signaling and neofunctionalization of non-visual pigment. Front Ecol Evol 5:23.
OpenUrl CrossRef

[77] Koyanagi M,

[78] Kawano-Yamashita E,

[79] Wada S,

[80] Terakita A

[81] ↵
Urasaki A,
Asakawa K,
Kawakami K
(2008) Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish. Proc Natl Acad Sci USA 105:19827–19832.
OpenUrl Abstract/FREE Full Text

[82] Urasaki A,

[83] Asakawa K,

[84] Kawakami K

[85] ↵
Jao LE,
Wente SR,
Chen W
(2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 110:13904–13909.
OpenUrl Abstract/FREE Full Text

[86] Jao LE,

[87] Wente SR,

[88] Chen W

[89] ↵
Ota S, et al.
(2013) Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells 18:450–458.
OpenUrl CrossRef PubMed

[90] Ota S, et al.

[91] ↵
Koyanagi M,
Takada E,
Nagata T,
Tsukamoto H,
Terakita A
(2013) Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue. Proc Natl Acad Sci USA 110:4998–5003.
OpenUrl Abstract/FREE Full Text

[92] Koyanagi M,

[93] Takada E,

[94] Nagata T,

[95] Tsukamoto H,

[96] Terakita A

[97] ↵
Sugihara T,
Nagata T,
Mason B,
Koyanagi M,
Terakita A
(2016) Absorption characteristics of vertebrate non-visual opsin, Opn3. PLoS One 11:e0161215.
OpenUrl

[98] Sugihara T,

[99] Nagata T,

[100] Mason B,

[101] Koyanagi M,

[102] Terakita A

Main menu

User menu

Search