Norepinephrine induces apoptosis in skin melanophores by attenuating cAMP-PKA signals via alpha2-adrenoceptors in the medaka, Oryzias latipes

The density of skin melanophores in many teleost fish decreases during long-term adaptation to a white background. Using the medaka, Oryzias latipes, we previously reported that apoptosis is responsible for the decrease in melanophores, and that a sympathetic neurotransmitter, norepinephrine (NE), induces their apoptosis in skin tissue cultures. In this study, we show that NE-induced apoptosis of melanophores is mediated by the activation of alpha2-adrenoceptors. Clonidine, an alpha2-adrenoceptor agonist, induced apoptotic melanophore death in skin organ culture, while phenylephrine, an alpha1-adrenoceptor agonist, had no effect. NE-induced apoptosis was diminished by an alpha2-adrenoceptor antagonist, yohimbine, but an alpha1-adrenoceptor antagonist, prazosin, did not abrogate the effect of NE. Furthermore, forskolin inhibited NE-induced apoptosis, while an inhibitor of PKA, H-89, mimicked the effect of NE. These results suggest that NE induces apoptosis in melanophores by attenuating cAMP-PKA signaling via alpha2-adrenoceptors.     

Norepinephrine Induces Apoptosis in Skin Melanophores by Attenuating cAMP‐PKA Signals Via α2‐Adrenoceptors in the Medaka,Oryzias Latipes

The density of skin melanophores in many teleost fish decreases during long‐term adaptation to a white background. Using the medaka, Oryzias latipes, we previously reported that apoptosis is responsible for the decrease in melanophores, and that a sympathetic neurotransmitter, norepinephrine (NE), induces their apoptosis in skin tissue cultures. In this study, we show that NE‐induced apoptosis of melanophores is mediated by the activation of α2‐adrenoceptors. Clonidine, an α2‐adrenoceptor agonist, induced apoptotic melanophore death in skin organ culture, while phenylephrine, an α1‐adrenoceptor agonist, had no effect. NE‐induced apoptosis was diminished by an α2‐adrenoceptor antagonist, yohimbine, but an α1‐adrenoceptor antagonist, prazosin, did not abrogate the effect of NE. Furthermore, forskolin inhibited NE‐induced apoptosis, while an inhibitor of PKA, H‐89, mimicked the effect of NE. These results suggest that NE induces apoptosis in melanophores by attenuating cAMP‐PKA signaling via α2‐adrenoceptors.     

The influence of long-term chromatic adaptation on pigment cells and striped pigment patterns in the skin of the zebrafish, Danio rerio

The striped pigment patterns in the flanks of zebrafish result from chromatophores deep within the dermis or hypodermis, while superficial melanophores associated with dermal scales add a dark tint to the dorsal coloration. The responses of these chromatophores were compared during the long-term adaptation of zebrafish to a white or a black background. In superficial skin, melanophores, xanthophores, and two types of iridophores are distributed in a gradient along the dorso-ventral axis independent of the hypodermal pigment patterns. Within one week the superficial melanophores and iridophores changed their density and/or areas of distribution, which adopted the dorsal skin color and the hue of the flank to the background, but did not affect the striped pattern. The increases or decreases in superficial melanophores are thought to be caused by apoptosis or by differentiation, respectively. When the adaptation period was prolonged for more than several months, the striped color pattern was also affected by changes in the width of the black stripes. Some black stripes disappeared and interstripe areas were emphasized with a yellow color within one year on a white background. Such long-term alteration in the pigment pattern was caused by a decrease in the distribution of melanophores and a concomitant increase in xanthophores in the hypodermis. These results indicate that morphological responses of superficial chromatophores contribute to the effective and rapid background adaptation of dorsal skin and while prolonged adaptation also affects hypodermal chromatophores in the flank to alter the striped pigment patterns.     

Changes in Adrenergic Innervation to Chromatophores During Prolonged Background Adaptation in the Medaka,Oryzias latipes

The pattern of adrenergic innervation to scale chromatophores of the wild-type medaka, Oryzias latipes, was examined by autoradiography with ³H-norepinephrine and found for the first time to be changed reversibly during prolonged background adaptation. In scales of the medaka, which was adapted to a black background for 10-15 days, a great number of melanophores and dense networks of varicose fibers were observed: many fibers built up a radial plexus around each melanophore. However, the dense distribution of varicose fibers disappeared with a decrease in the number of melanophores during long-term adaptation to a white background. As to the changes in the innervation pattern to amelanotic melanophores of the medaka, orange-red variety, a similar result was obtained. Although the increase in the number of leucophores was observed in the medaka adapted to a white background, no exact plexuses of labeled fibers were confirmed around leucophores. From these results, it is concluded that the density of chromatic nerve fibers changes in parallel with the variation of the number of melanophores during prolonged background adaptation.     

Role of the dermal tracts in the pigment pattern of the frog.

The pigment pattern of the ventral skin of the frog Rana esculenta is compared in skin fragments grown for 24 hr with or without antiserum directed to fibronectin (anti-FN). Melanocyte-stimulating hormone (MSH) was added to the medium during the last hour in culture in order to enhance visibility of melanophores in the ventral region of the frog skin. Comparison of these two treatments provides information regarding the precise localization of melanophores in the dermal tracts and their involvement in the pigment pattern of the ventral frog skin. In this regard, the whitish pigment pattern of skin fragments is compared to the tiny black spots found on anti-FN treated skin fragments and the abundant blotchy spots found on skin cultured alone. The distribution of melanophores in the dermal tracts observed in vertical semithin sections is found to be related to the three different levels of the dermal tracts. This report demonstrates the importance of fibronectin as a substrate for the melanophore migration, the importance of the tract level for the melanophore localization both involved in the pigment pattern of the ventral skin.     

Transgenic Medaka Fish Bearing the Mouse Tyrosinase Gene: Expression and Transmission of the Transgene Following Electroporation of the Orange-Colored Variant

Transgenic fish bearing the mouse tyrosinase gene (mg-Tyrs-J) were produced by transfection into fertilized eggs of the homozygous normal orange-colored variant of medaka fish, Oryzias latipes, by means of electroporation. Of 589 eggs transfected, 38 fish (6%) exhibited brownish wild-type skin pigmentation, which was discernible from control siblings. Light microscopy of the skin from the founders thus generated disclosed that 1) melanization occurred and was restricted to melanophores formed presumably from preexisting amelanotic melanophores, 2) there was a wide variation in the degree of melanization observed among melanophores, and 3) no melanin deposition was recognized in xanthophores or leucophores. Immunofluorescence using an antibody raised against mouse tyrosinase disclosed that melanophores at varying stages of maturation were reactive. Thus, it was shown that the transgene in medaka fish expressed its action in a cell type-specific manner. Crossing of transgenic founders with homozygous orange-colored variant fish yielded two groups of offspring expressing either the wild-type or the orange-colored skin pigmentation at an approximate ratio of 1:1. Crossing between founders exhibiting wild-type pigmentation yielded only offspring with melanized skin. Skin melanophores in these offspring formed vertical stripes, which are rare in this species. The hereditary basis of melanized skin was demonstrated in matings of Fl progenies, which resulted in similar degrees of melanization over whole skin melanophores. The sum of these findings implied that the transgene is expressed as a dominant character gene and is transmitted through germ cell lines according to the Mendelian law. PCR analysis combined with nested PCR technique strongly suggested that the transgene was integrated into the medaka genome, even though the copy number deduced from gel banding was largely diminished, possibly as a result of fragmentation or instability within the medaka genome.     

Evidence for a direct role of α-MSH in morphological background adaptation of the skin in Sarotherodon mossambicus

Summary The skin colour of the cichlid teleost Sarotherodon mossambicus adapted rapidly to changes in background colour. The physiological adaptation was associated with morphological changes in the dermis. Differences in the dermis were found between fish adapted to a black or white background for 14 days. Number and size of the melanophores as well as the amount of pigment in the cytoplasm of the melanophores were significantly increased in fish adapted to a black background. Changes in the dermis parallelled changes in the state of activity of the two endocrine cell types in the pars intermedia of the pituitary. Both the PAS positive cells and the MSH producing cells were more active when the fish were exposed to a black rather than a white background. Fish continuously infused with -MSH, using an osmotic minipump, had more melanophore cytoplasm and pigment per dermis surface unit area than untreated fish. The activity of the MSH cells in MSH-infused fish exposed to a black background was reduced to a level comparable to the MSH cell activity of untreated fish on a white background. -MSH treated fish that were exposed to a white background had many disintegrating MSH cells. These findings point to inactivation of these cells by exogenous -MSH. The activity of the PAS positive cells was not influenced by treatment with -MSH.     

In vitro pattern‐related effects of forskolin on melanophore adrenoceptor‐mediated responses of winter flounder

In vitro experiments were performed on melanophores on scales carrying small slips of skin (scale slips) sampled from the white spot, dark band and general background components of patterning in winter flounder Pseudopleuronectes americanus . In vitro , forskolin (10⁻⁴ to 10⁻⁷ M) was employed to increase melanophore intracellular cytosolic adenosine‐3'‐5'‐monophosphate (cAMP) levels by stimulating adenylate cyclase. Such increase shifted noradrenaline concentration‐response curves to the right, general background melanophores being least sensitive to the forskolin, indicating a relatively higher proportion of α₂‐adrenoceptors, and those from white spots the most sensitive. Melanosome dispersion, in balanced salt solution, was also enhanced by such raised levels of cAMP. Without forskolin, melanophores from white spots displayed the shortest pigment dispersion time in balanced salt solution, the slower melanophores from the other pattern components being those most affected by the addition of forskolin. The results demonstrate that differences in melanophore physiological responsiveness associated with flatfish patterns involve differential intracellular levels of cAMP, as well as differences in melanophore size and rates of melanosome movement.     

Expression and Transmission of Wild-Type Pigmentation in the Skin of Transgenic Orange-Colored Variants of Medaka (Oryzias latipes) Bearing the Gene for Mouse Tyrosinase

Transgenic fish carrying a reconstructed mouse tyrosinase gene, mg-Tyrs-J, were produced by microinjecting the gene into the oocyte nucleus of an orange-colored variant of medaka (Oryzias latipes). Of 64 oocytes microinjected and subsequently inseminated, 13 embryos developed normally beyond hatching and three of them exhibited brown skin pigmentation in the adult as was commonly observed in the wild type of this species. Light and electron microscopic examination disclosed a ubiquitous distribution of typical melanophores in the skin of these transgenic fish. Judging from their population density and distribution pattern, it was presumed that melanogenesis in these fish was elicited in amelanotic melanophores that resided in the skin of the orange-colored fish of this variant. Immunofluorescence with use of the anti-mouse tyrosinase antiserum lacking reactivity to medaka tyrosinase clearly disclosed that the gene introduced was expressed in the melanophores of transgenic fish. Crosses of female transgenic fish and males from an orange-colored variant yielded offspring exhibiting wild-type or orange-colored pigmentation in a ratio of 1:1, thus implying that mg-Tyrs-J integrated into the medaka genome behaves like a dominant gene. Little melanogenesis was observed in xanthophores, leucophores and iridophores in transgenic fish, suggesting possible specificity in recognition of teleostean cell types (i.e., melanophores) by the regulatory region of the mouse tyrosinase gene.     

Cyclical training enhances the melanophore responses of zebrafish to background colours

Zebrafish respond to visual stimuli to adapt their body colour to the background. If, rather than being a simple on/off reaction to visual stimulation, the colour change involves cognitive and memory-related processes, training fish with cyclical changes of the background would be expected to increase its ability to change colour. To test this, we developed a standardized procedure for quantifying the responses of melanophores to background changes in living adult specimens of leopard, a zebrafish mutant with spotted stripes. After training with 2-day cyclical alternation of white and black backgrounds for over 20 days, the proportion of the melanosome-filled area of dorsal melanophores, which was 20% on the black background before the training, increased up to 97%. In these trained fish, a rapid melanosome aggregation occurred within 10 s of the background change from black to white. The results indicate that the zebrafish melanophore responses can be modulated by learning, in which areal and speed control of melanosome movement are important for dispersion and aggregation, respectively.     

Developmental mechanisms of longitudinal stripes in the Japanese four‐lined snake

The developmental mechanisms of color patterns formation and its evolution remain unclear in reptilian sauropsids. We, therefore, studied the pigment cell mechanisms of stripe pattern formation during embryonic development of the snake Elaphe quadrivirgata. We identified 10 post‐ovipositional embryonic developmental stages based on external morphological characteristics. Examination for the temporal changes in differentiation, distribution, and density of pigment cells during embryonic development revealed that melanophores first appeared in myotome and body cavity but not in skin surface at Stage 5. Epidermal melanophores were first recognized at Stage 7, and dermal melanophores and iridophores appeared in Stage 9. Stripe pattern first appeared to establish at Stage 8 as a spatial density gradient of epidermal melanophores between the regions of future dark brown longitudinal stripes and light colored background. Our study, thus, provides a comprehensive pigment‐cell‐based understanding of stripe pattern formation during embryonic development. We briefly discuss the importance of the gene expression studies by considering the biologically relevant theoretical models with standard developmental staging for understanding reptilian color pattern evolution.     

Frog melanophores cultured on fluorescent microbeads: biomimic-based biosensing

Melanophores are pigmented cells in lower vertebrates capable of quick color changes and thereby suitable as whole cell biosensors. In the frog dermis skin layer, the large and dark pigmented melanophore surrounds a core of other pigmented cells. Upon hormonal stimulation the black-brown pigment organelles will redistribute within the melanophore, and thereby cover or uncover the core, making complex color changes possible in the dermis. Previously, melanophores have only been cultured on flat surfaces. Here we mimic the three dimensional biological geometry in the frog dermis by culturing melanophores on fluorescent plastic microbeads. To demonstrate biosensing we use the hormones melatonin and alpha-melanocyte stimulating hormone (alpha-MSH) as lightening or darkening stimuli, respectively. Cellular responses were successfully demonstrated on single cell level by fluorescence microscopy, and in cell suspension by a fluorescence microplate reader and a previously demonstrated computer screen photo-assisted technique. The demonstrated principle is the first step towards "single well/multiple read-out" biosensor arrays based on suspensions of different selective-responding melanophores, each cultured on microbeads with distinctive spectral characteristics. By applying small amount of a clinical sample, or a candidate substance in early drug screening, to a single well containing combinations of melanophores on beads, multiple parameter read-outs will be possible.     

Unusual development of light-reflecting pigment cells in intact and regenerating tail in the periodic albino mutant of Xenopus laevis

Unusual light-reflecting pigment cells, “white pigment cells”, specifically appear in the periodic albino mutant (a ^p /a ^p ) of Xenopus laevis and localize in the same place where melanophores normally differentiate in the wild-type. The mechanism responsible for the development of unusual pigment cells is unclear. In this study, white pigment cells in the periodic albino were compared with melanophores in the wild-type, using a cell culture system and a tail-regenerating system. Observations of both intact and cultured cells demonstrate that white pigment cells are unique in (1) showing characteristics of melanophore precursors at various stages of development, (2) accumulating reflecting platelets characteristic of iridophores, and (3) exhibiting pigment dispersion in response to α-melanocyte stimulating hormone (α-MSH) in the same way that melanophores do. When a tadpole tail is amputated, a functionally competent new tail is regenerated. White pigment cells appear in the mutant regenerating tail, whereas melanophores differentiate in the wild-type regenerating tail. White pigment cells in the mutant regenerating tail are essentially similar to melanophores in the wild-type regenerating tail with respect to their localization, number, and response to α-MSH. In addition to white pigment cells, iridophores which are never present in the intact tadpole tail appear specifically in the somites near the amputation level in the mutant regenerating tail. Iridophores are distinct from white pigment cells in size, shape, blue light-induced fluorescence, and response to α-MSH. These findings strongly suggest that white pigment cells in the mutant arise from melanophore precursors and accumulate reflecting platelets characteristic of iridophores.     

Measurements of Light Transmission Through Single Melanophores

A photometrical method has been developed that allows assessment of subcellular pigment migration in melanophores of the fish cockoo wrasse (Labrus ossifagus L.). The pigment migration was studied with local light spot transmission measurements. Depending on where the light beam is placed on the melanophores it is possible to study events within an area of approximately 75 μm². Measuring pigment translocation in different parts of a melanophore gives new possibilities to study how cell membrane receptor-mediated signals are spread within a single cell, which will increase our understanding of how receptor activating drugs exert their cellular effect. The technique can be used in pharmacological and biophysical studies and in biosensors, pharmaceutical screens, environmental detectors, etc. The method clearly has the ability to study local and small changes in light transmission due to displacement of melanophore pigment granules. Since one melanophore on the tip of an optical fibre would be enough to obtain a measurable effect, the presented technique provides the basis for future development of biosensors small enough for in vivo applications, e.g., to monitor the catecholamine levels of circulating blood.     

Pigment pattern in jaguar/obelix zebrafish is caused by a Kir7.1 mutation: implications for the regulation of melanosome movement

Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K⁺ channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the α₂-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for the pattern change in the jaguar/obelix mutant.     

Genetics of color of the ornamental carp: inheritance of underlying dark coloration]

In ornamental carps from the collection of the Experimental Station, All-Russia Research Institute of Fresh-Water Fisheries, fish with a dirty dark tint of body color among orange and white fish were found. This coloration was shown to be due to the presence of black pigment cells in the lower skin layer. In the outer skin layers these cells were almost entirely absent. This color type was found to be determined by the presence of at least one of alleles B1' or B2' of digenic system B1, B2, which is responsible for the development of melanophores in fish skin. Each of the genes of this digenic system is represented by three alleles with the following order of dominance: B > B' > b.     

Comparative analyses of the pigment-aggregating and -dispersing actions of MCH on fish chromatophores

In melanophores of the peppered catfish and the Nile tilapia, melanin-concentrating hormone (MCH) at low doses (<1 μM) induced pigment aggregation, and the aggregated state was maintained in the presence of MCH. However, at higher MCH concentrations (such as 1 and 10 μM), pigment aggregation was immediately followed by some re-dispersion, even in the continued presence of MCH, which led to an apparent decrease in aggregation. This pigment-dispersing activity at higher concentrations of MCH required extracellular Ca²⁺ ions. By contrast, medaka melanophores responded to MCH only by pigment aggregation, even at the highest concentration employed (10 μM). Since it is known that medaka melanophores possess specific receptors for α-melanophore-stimulating hormone (α-MSH), the possibility that interaction between MSH receptors and MCH at high doses in the presence of Ca²⁺ might cause pigment dispersion is ruled out. Cyclic MCH analogs, MCH (1–14) and MCH (5–17), failed to induce pigment dispersion, whereas they induced aggregation of melanin granules. These results suggest that another type of MCH receptor that mediates pigment dispersion is present in catfish and tilapia melanophores, and that intact MCH may be the only molecule that can bind to these receptors. Determinations of cAMP content in melanophores, which were isolated from the skin of three fish species and treated with 10 nM or 10 μM MCH, indicate that MCH receptors mediating aggregation may be coupled with Gi protein, whereas MCH receptors that mediate dispersion may be linked to Gs. The response of erythrophores, xanthophores and leucophores to MCH at various concentrations was also examined, and the results suggest that the distribution patterns of the two types of MCH receptors may differ among fish species and among types of chromatophore in the same fish.     

The effects of background colouration and α-MSH treatment on melanophore frequency in winter flounder,Pleuronectes americanus

Winter flounder, Pleuronectes americanus, adapting to black or white backgrounds display significant increase and decline respectively in the number of visible epidermal melanophores over periods up to 8 weeks or longer. This contrasts with a stability in the number of visible dermal melanophores during the same periods of exposure to each background. Flounders treated with -melanophore stimulating hormone exhibited an enhanced rate of increase in number of visible epidermal melanophores when the background was changed from white to black, whereas white-adapted flounder treated with -melanophore stimulating hormone without background change did not manifest any such increase in number of epidermal melanophores. Flounder treated with -melanophore stimulating hormone after transfer from black to white displayed a similar initial decline in visible epidermal melanophore number as in control fish, but the final decline was significantly attenuated. Thus -melanophore stimulating hormone, which has no apparent influence on melanosome dispersion in this species, may have a limited morphological melanophore regulatory role which is discussed in relation to possible antagonistic and synergistic factors that could influence melanogenesis and visible melanophore numbers.Abbreviations DMI dermal melanophore index - EMI epidermal melanophore index - LSD least significant difference - MCH melanosome concentrating hormone - MIF melanogenesis inhibiting factor - MSF melanogenesis stimulating factor - MSH melanophore stimulating hormone     

Deficiency of the Gene B Impairs Differentiation of Melanophores in the Medaka Fish,Oryzias latipes: Fine Structure Studies

In an orange-colored variant of the medaka fish, Oryzias latipes, which is homozygous for b allele, the melanophores represent a tissue-specific differentiation, manifesting an amelanotic appearance in the skin, an incomplete melanogenesis in the choroid and the peritoneum, and mosaic phenotype-like melano-iridophores in the peritoneum. In a wild-type strain of this species carrying the B gene, all melanophores are terminally differentiated irrespective of the tissues in which they are located. This indicates that the deficiency of B gene impairs the differentiation of melanophores in the medaka. Electron microscopy disclosed that the deficiency of B gene causes deterioration of melanogenesis to occur inside the melanosomes and that the manner of deterioration in the melanophores in the skin, the choroid and the peritoneum is different. The ubiquitous occurrence of reflecting platelet-laden melanophores in the peritoneum of this variant and the total absence of a mosaicism in pigment cells of the wild-type strain indicate that the deficiency of B gene predestines melanoblasts distributed in this tissue to an ambiguous state with regard to their differentiation. Little difference is observed between melanosomes maturation in pigment epithelial cells of the orange-colored variant and the wild-type strain, indicating an innocent role of the B gene in their differentiation.     

The movement of melanosomes in melanophore fragments obtained by laser microbeam irradiation

The melanophores of the teleost Gymnocorymbus ternetzi are filled with pigment granules, melanosomes, which in response to appropriate treatments, can disperse throughout the cytoplasm or form an aggregate in the cell center. Melanophores with the dispersed pigment were irradiated by a laser microbeam, focused on the cell center by the microscope objective. If the average energy of the microbeam was 6-7 microJ, either the center of the melanophore was damaged and a single ring-shaped fragment was formed, or the cell was broken into several fragments of smaller size. The fragments retained their ability to move the pigment granules. In ring-shaped fragments, after adrenaline treatment, the melanosomes formed a ring-shaped aggregate moving away from both outer and inner (irradiation-produced) margins of the fragment. The smaller fragments treated with adrenaline moved the pigment to their centers. Both small and ring-shaped fragments could aggregate melanosomes as soon as 5 minutes after irradiation.