The regulation of skin pigmentation in response to environmental light by pineal Type II opsins and skin melanophore melatonin receptors.

Coupling skin colour with the light/dark cycle helps regulate body temperature in ectotherms. In X. laevis, nocturnal release of melatonin from the pineal complex induces pigment aggregation and skin lightening. This nocturnal blanching is initiated by a sensor (type II opsin) that triggers melatonin release when light intensity falls below a minimum threshold, and an effector (melatonin receptor) in the skin which induces pigment aggregation. The sensor/s and effector/s belong to two families of G-protein coupled receptors that originated from a common ancestor, but diverged with subsequent evolution. The aim of this work was to identify candidate sensor/s and effector/s that regulate melatonin-mediated skin colour variation. In X. laevis, we identified a developmental time (stage 43/44) when skin lightening depends on pineal complex photosensitivity alone. At this stage, the pineal complex comprises the frontal organ and pineal gland. A total of 37 type II opsin (14 duplicated) and 6 melatonin receptor (3 duplicated) genes were identified through a full genome analysis of the allotetraploid, X. laevis. These genes were grouped into subfamilies based on their predicted amino acid sequences and the presence of specific amino acids essential for their function. The pineal complex expresses mainly blue light sensitive opsins [pinopsin, parietopsin, opn3, and melanopsins (opn4 and opn4b)] and UV-light sensitive opsins (opn5 and parapinopsin), while visual opsins and va-ancient opsin are absent, as determined by RT-PCR and in situ hybridization. The photoisomerase retinal G-protein coupled receptor, and an uncharacterized opn6b opsin, are also expressed. The spectral sensitivity that triggers melatonin secretion, and therefore melanophore aggregation, falls in the visible spectrum (470-650 Î·m) and peaks in the blue/green range, pointing to the involvement of opsins with sensitivities therein. The effector-melatonin receptors expressed in skin melanophores are mtnr1a and mtnr1c. Our data point to candidate proteins required in the neuroendocrine circuit that underlies the circadian regulation of skin pigmentation, and suggest that multiple initiators and effectors likely participate.

Melanin-concentrating hormone is a major substance mediating light wavelength-dependent skin color change in larval zebrafish.

Melanosome dispersion is important for protecting the internal organs of fish against ultraviolet light, especially in transparent larvae with underdeveloped skin. Melanosome dispersion leads to dark skin color in dim light. Melanosome aggregation, on the other hand, leads to pale skin color in bright light. Both of these mechanisms are therefore useful for camouflage. In this study, we investigated a hormone thought to be responsible for the light wavelength-dependent response of melanophores in zebrafish larvae. We irradiated larvae using light-emitting diode (LED) lights with peak wavelengths (λmax) of 355, 400, 476, 530, and 590 nm or fluorescent light (FL) 1-4 days post fertilization (dpf). Melanosomes in skin melanophores were more dispersed under short wavelength light (λmax ≤ 400 nm) than under FL. Conversely, melanosomes were more aggregated under mid-long wavelength light (λmax ≥ 476 nm) than under FL. In addition, long-term (1-12 dpf) irradiation of 400 nm light increased melanophores in the skin, whereas that of 530 nm light decreased them. In teleosts, melanin-concentrating hormone (MCH) aggregates melanosomes within chromatophores, whereas melanocyte-stimulating hormone, derived from proopiomelanocortin (POMC), disperses melanosomes. The expression of a gene for MCH was down-regulated by short wavelength light but up-regulated by mid-long wavelength light, whereas a gene for POMC was up-regulated under short wavelength light. Melanosomes in larvae (4 dpf) exposed to a black background aggregated when immersing the larvae in MCH solution. Yohimbine, an α2-adrenergic receptor antagonist, attenuated adrenaline-dependent aggregation in larvae exposed to a black background but did not induce melanosome dispersion in larvae exposed to a white background. These results suggest that MCH plays a key role in the light wavelength-dependent response of melanophores, flexibly mediating the transmission of light wavelength information between photoreceptors and melanophores.

Interaction and developmental activation of two neuroendocrine systems that regulate light-mediated skin pigmentation.

Lower vertebrates use rapid light-regulated changes in skin colour for camouflage (background adaptation) or during circadian variation in irradiance levels. Two neuroendocrine systems, the eye/alpha-melanocyte-stimulating hormone (α-MSH) and the pineal complex/melatonin circuits, regulate the process through their respective dispersion and aggregation of pigment granules (melanosomes) in skin melanophores. During development, Xenopus laevis tadpoles raised on a black background or in the dark perceive less light sensed by the eye and darken in response to increased α-MSH secretion. As embryogenesis proceeds, the pineal complex/melatonin circuit becomes the dominant regulator in the dark and induces lightening of the skin of larvae. The eye/α-MSH circuit continues to mediate darkening of embryos on a black background, but we propose the circuit is shut down in complete darkness in part by melatonin acting on receptors expressed by pituitary cells to inhibit the expression of pomc, the precursor of α-MSH.

Endothelin modulates the circadian expression of non-visual opsins.

The non-visual opsin, melanopsin, expressed in the mammalian retina, is considered a circadian photopigment because it is responsible to entrain the endogenous biological clock. This photopigment is also present in the melanophores of Xenopus laevis, where it was first described, but its role in these cells is not fully understood. X. laevis melanophores respond to light with melanin granule dispersion, the maximal response being achieved at the wavelength of melanopsin maximal excitation. Pigment dispersion can also be triggered by endothelin-3 (ET-3). Here we show that melanin translocation is greater when a blue light pulse was applied in the presence of ET-3. In addition, we demonstrated that mRNA levels of the melanopsins Opn4x and Opn4m exhibit temporal variation in melanophores under light/dark (LD) cycles or constant darkness, suggesting that this variation is clock-driven. Moreover, under LD cycles the oscillations of both melanopsins show a circadian profile suggesting a role for these opsins in the photoentrainment mechanism. Blue-light pulse decreased Opn4x expression, but had no effect on Opn4m. ET-3 abolishes the circadian rhythm of expression of both opsins; in addition the hormone increases Opn4x expression in a dose-, circadian time- and light-dependent way. ET-3 also increases the expression of its own receptor, in a dose-dependent manner. The variation of melanopsin levels may represent an adaptive mechanism to ensure greater melanophore sensitivity in response to environmental light conditions with ideal magnitude in terms of melanin granule dispersion, and consequently color change.

Effect of light on expression of clock genes in Xenopus laevis melanophores.

Light-dark cycles are considered important cues to entrain biological clocks. A feedback loop of clock gene transcription and translation is the molecular basis underlying the mechanism of both central and peripheral clocks. Xenopus laevis embryonic melanophores respond to light with melanin granule dispersion, response possibly mediated by the photopigment melanopsin. To test whether light modulates clock gene expression in Xenopus melanophores, we used qPCR to evaluate the relative mRNA levels of Per1, Per2, Clock and Bmal1 in cultured melanophores exposed to light-dark (LD) cycle or constant darkness (DD). LD cycles elicited temporal changes in the expression of Per1, Per2 and Bmal1. A 10-min pulse of blue light was able to increases the expression of Per1 and Per2. Red light had no effect on the expression of these clock genes. These data suggest the participation of a blue-wavelength sensitive pigment in the light-dark cycle-mediated oscillation of the endogenous clock. Our results add an important contribution to the emerging field of peripheral clocks, which in nonmammalian vertebrates have been mostly studied in Drosophila and Danio rerio. Within this context, we show that X. laevis melanophores, which have already led to melanopsin discovery, represent an ideal model to understanding circadian rhythms.

Light modulates the melanophore response to alpha-MSH in Xenopus laevis: an analysis of the signal transduction crosstalk mechanisms involved.

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X. laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane.

Participation of nitric oxide in the color change induced by UV radiation in the crab Chasmagnathus granulatus.

The ability of UV radiation to stimulate color change in vertebrates is well known; however, the signaling pathway involved is not fully explained. Since nitric oxide (NO) is among the candidates for this role, in this study the participation of NO signaling in the pigment migration induced by UV radiation in melanophores of the crab Chasmagnathus granulatus was investigated. When the NO donor, SIN-1, was incubated with pieces of epidermis, there was an induction of a dose-dependent pigment dispersion (in vitro assays). When male adults were exposed to different doses of UVA and UVB, N(G)-nitro-l-arginine-methyl-ester, an NO synthase (NOS) blocker produced a decrease of the pigment dispersion induced by UV (in vivo assays). However, in similar assays, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger, decreased only the pigment dispersion induced by UVA. Interestingly, buthionine sulfoximine did not produce any change in pigment dispersion induced by UVA (in vivo assays) and SIN-1 (in vitro assays). Our results using NADPH-diaphorase histochemistry and immunocytochemistry against nNOS indicated the production of NO by epidermal cells. In conclusion, we suggest that NO is a key molecule for the induction of pigment dispersion in the melanophores of Chasmagnthus granulatus, and also that NOS activation is a fundamental step for this process.

Light-sensitive response in melanophores of Xenopus laevis: I. Spectral characteristics of melanophore response in isolated tail fin of Xenopus tadpole.

Melanophores in the isolated tail from the amphibian larvae Xenopus laevis, Hyla japonicus, Rana pirica, and Hynobius retardatus aggregated melanin granules in response to light and dispersed them when placed in darkness. The spectral characteristics for the melanin-aggregation response were examined by irradiating the Xenopus tail-fin locally (diameter, 2.1 mm) with monochromatic light (380-1,020 nm). The spectral region of wave length which induced melanosome aggregation depended on the light intensity but was limited to the visible spectrum. At low light intensity (1.59 microW/cm2, delta lambda = 5 nm), the aggregation response occurred in the spectral region between 400 and 600 nm and the maximum response was observed at 500 nm. This range is very close to the absorption spectrum of rhodopsin in the visual rod cell. Hypodermic injection of cGMP into isolated tail-fin induced a marked melanin-dispersion in spite of light-stimuli. When the tail-fin was treated with isobutylmethylxanthine (IBMX; phosophodiesterase inhibitor) in darkness and then was re-exposed to light, the aggregation response was inhibited. The photo-sensitive melanin aggregation was independent of a requirement for Ca2+ ions but melanosome dispersion in darkness was Ca(2+)-dependent. K(+)-rich Hanks' solution, ouabain (inhibitor of Na(+)-K(+)-ATPase) or nonactin (cation ionophore), which induced a change of the membrane potential of melanophores, inhibited the aggregation response when the melanophores were re-exposed to light after a period in darkness. These results suggest that the molecular mechanism of photoreception in melanophores of amphibian tadpoles is similar to that in visual cells.

Transformation of an irreversible MSH antagonist into an irreversible MSH agonist by differential receptor crosslinking using the photo-affinity technique.

Photocrosslinking of receptors for alpha-melanocyte-stimulating hormone (alpha-MSH) on melanophores of frogs and lizards has been shown to induce long-lasting receptor stimulation whereby the photoreactive alpha-MSH may contain one or two photolabels in positions 1, 7, 9, or 13. The chemical synthesis and biological testing of an alpha-MSH analogue is now described which contains three photoreactive groups in positions 1, 9 and 13, one of which with a cleavable S-S disulphide bridge: [ApSSpr-Ser1, Trp(Naps)9 Pap13]-alpha-MSH. Photocrosslinking of MSH receptors on melanophores of Anolis carolinensis with this analogue led to almost complete receptor blockade which could be transformed into long-lasting receptor stimulation by exposure to a thiol reagent. By contrast, the analogue containing only two photoreactive groups in positions 9 and 13, [Trp(Naps)9, Pap13]-alpha-MSH, produced long-lasting receptor stimulation which was not altered by the thiol reagent. These results demonstrate that one and the same peptide ligand may contain structural information for both receptor activation and inhibition and that the receptor may become arrested in an activated or inhibited state by multiple photocrosslinking, depending on the relative positions of these crosslinks.

Tools for investigating functional interactions between ligands and G-protein-coupled receptors.

A general assay for evaluating functional interactions between ligands and G-protein-coupled receptors within minutes has been developed. The system uses the principles employed by animals such as reptiles, amphibians and fish to control their colors. In nature, activation of G-protein-coupled receptors expressed by skin cells called chromatophores effects pigment redistribution within the cells to change an animal's coloration. The in vitro 'chameleon in a dish' equivalent can use essentially any cloned G-protein-coupled receptor.

Pertussis toxin sensitive photoaggregation of pigment in isolated Xenopus tail-fin melanophores.

Direct illumination of Xenopus laevis tail-fin melanophores results in rapid, reversible translocation of intracellular pigment granules to a perinuclear location, an effect distinct from and opposite to the photodispersion of pigment found in melanophores isolated from Xenopus embryos. In this report we show that both pertussis toxin and dibutyryl-adenosine-3',5'-monophosphate block the ability of light to cause photoaggregation of pigment in cultured tail-fin melanophores, whereas dibutyryl-guanosine-3',5'-monophosphate is without effect.

Action of light on frog pigment cells in culture.

Solar radiation induces numerous biologic effects in skin but the mechanism underlying these responses is poorly understood. To study the etiology of these phenomena, we investigated the effect of light on cultured Xenopus laevis melanophores. Visible light stimulated a marked increase in intracellular cAMP levels within the first minute of irradiation. This light-induced elevation in cAMP was blocked by melatonin and was not seen in fibroblasts irradiated in a similar manner. These data show that the photoresponse of pigment cells from amphibian skin can be mediated by a cAMP-dependent mechanisms and suggest that a unique member of the rhodopsin family is involved in this process.

Light response of cultured melanophores of a freshwater teleost, Zacco temmincki.

Melanophores in the skin of the freshwater teleost Zacco temmincki are light sensitive: Melanin granules, melanosomes, in the melanophores aggregate in darkness and disperse in light. Cultured melanophores of Zacco temmincki exhibited light sensitivity in the same manner as the melanophores in isolated scales. The dark-induced aggregation response became conspicuous after 2 days in culture. The appearance of the light response was later than that of the response to norepinephrine or melatonin, which induced rapid melanosome aggregation at one day in culture. The light sensitivity of the melanophores in isolated scales differed between individuals. A high correlation was observed between the degree of dark-induced aggregation in scale melanophores and that in cultured ones.

A sensitive bioassay for melanotropic hormones using isolated medaka melanophores.

Melanophore-stimulating hormones (MSHs) from chum salmon cause pigment dispersion in isolated melanophores of medaka, a teleost. The in vitro medaka melanophore bioassay that responded to light with pigment dispersion and to the dark with pigment aggregation was utilized for measuring the activity of melanotropic hormones. alpha-MSH I was the most potent melanophore-dispersing agent tested. The minimal dose for the induction of pigment dispersion was 10(-15) M alpha-MSH I, 10(-13) M N-des-acetyl(Ac)-alpha-MSH, and 10(-11) M beta-MSH I, respectively. The melanosome-dispersing activity of beta-MSH I was enhanced about 40% by salmon N-acetyl-endorphin I (N-Ac-EP). The results suggest that N-Ac-EP may act as an enhancer for the activity of certain MSHs. The present bioassay provides a unique method for determining the biological activity of melanotropic peptides.

Spectral sensitivity of melanophores of a freshwater teleost, Zacco temmincki.

1. The melanophores of a freshwater teleost, Zacco temmincki, responded to changes in illumination: in darkness the melanophores induced a melanosome aggregation and when subjected to light they caused a melanosome dispersion. 2. Using monochromatic light, the spectral sensitivity of the melanophores was examined. 3. The melanophores showed a different sensitivity to light between 400 and 600 nm with a maximum at about 525 nm. 4. The action spectrum closely resembled a porphyropsin absorbance curve, suggesting a porphyropsin or similar photopigment is active in the melanophore light response of Zacco temmincki.

Local light stimulation of melanophores of a teleost, Zacco temmincki.

Responses of melanophores of the teleost, Zacco temmincki, to local light stimulation were examined in preparations of isolated scales. The melanophores induced the aggregation of melanosomes in darkness and their dispersion in light. Local illumination of a melanophore in the melanosome-dispersed state inhibited centripetal migration of melanosomes only in the stimulated area. Local illumination of a pigment-free branch of a melanophore with aggregated melanosomes generally brought about pigment dispersion into the stimulated area. However, when that area was at a significant distance from the edge of the central melanosome mass, the melanosomes never migrated into the irradiated area. Local illumination of the centrosphere of a cell inhibited the full aggregation of melanosomes in the dispersed and aggregated state. The degree of the inhibition depended on the size of the irradiated area. The results suggest that photoreceptive sites are distributed over the whole of a cell, and that the movements of melanosomes are regulated locally in a very precise manner.

Photoaffinity labelling of MSH receptors on Anolis melanophores: irradiation technique and MSH photolabels for irreversible stimulation.

Excised dorsal skin of Anolis carolinensis was exposed to high intensity UV-irradiation in the presence of different photoreactive alpha-MSH derivatives. The resulting covalent binding of the hormone to its receptor induced irreversible pigment dispersion. The duration of the longlasting response depended on the type and length of irradiation; it was maximal after two 5 min irradiation phases with a light intensity of approximately 180 mW/cm2 and a spectrum from 310 to 550 nm, fresh hormone being added after the first phase. [N alpha-(4-Azidophenylacetyl-serine1]-alpha-MSH (I), [2'-(2-nitro-4-azidophenylsulphenyl)-tryptophan9]-alpha-MSH (II) and [p-azidophenylalanine13]-alpha-MSH (III) all inserted into the receptor to about the same extent, as judged from the persistence of the longlasting signal. In contrast, [D-alanine1, p-azidophenylalanine2, norvaline4]-alpha-MSH (IV) and [N alpha-(4-azidophenylacetyl)-serine1, leucine9]-alpha-MSH (V) gave much less insertion and [leucine9, p-azidophenylalanine13]-alpha-MSH (VI) hardly any insertion when applied in the same relative excess (5-fold the concentration inducing a maximal response). Covalent attachment of the cleavable photolabel [N alpha-(4-azidophenyl)-1, 3'-dithio-propionyl-serine1]-alpha-MSH (VII) and subsequent washing of the skin in buffer containing 1% beta-mercaptoethanol released the peptide from the receptor. Insertion of the C-terminal photolabel [p-azidophenylalanine13]-alpha-MSH was reduced by the weak antagonist H-Phe-Ala-Trp-Gly-Gly-Pro-Val-NH2. These experiments prove that hormone receptors can be covalently labelled in tissue with very limited light transparency.