Pigment cell mechanisms underlying dorsal color-pattern polymorphism in the Japanese four-lined snake.

To provide histological foundation for studying the genetic mechanisms of color-pattern polymorphisms, we examined light reflectance profiles and cellular architectures of pigment cells that produced striped, nonstriped, and melanistic color patterns in the snake Elaphe quadrivirgata. Both, striped and nonstriped morphs, possessed the same set of epidermal melanophores and three types of dermal pigment cells (yellow xanthophores, iridescent iridophores, and black melanophores), but spatial variations in the densities of epidermal and dermal melanophores produced individual variations in stripe vividness. The densities of epidermal and dermal melanophores were two or three times higher in the dark-brown-stripe region than in the yellow background in the striped morph. However, the densities of epidermal and dermal melanophores between the striped and background regions were similar in the nonstriped morph. The melanistic morph had only epidermal and dermal melanophores and neither xanthophores nor iridophores were detected. Ghost stripes in the shed skin of some melanistic morphs suggested that stripe pattern formation and melanism were controlled independently. We proposed complete- and incomplete-dominance heredity models for the stripe-melanistic variation and striped, pale-striped, and nonstriped polymorphisms, respectively, according to the differences in pigment-cell composition and its spatial architecture.

Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo.

Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin-myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 µm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and "purse string"-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified "face-to-face" scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin-myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.

Integumental reddish-violet coloration owing to novel dichromatic chromatophores in the teleost fish, Pseudochromis diadema.

In the reddish-violet parts of the skin of the diadema pseudochromis Pseudochromis diadema, we found novel dichromatic chromatophores with a reddish pigment and reflecting platelets. We named these novel cells 'erythro-iridophores'. In standard physiological solution, erythro-iridophores displayed two hues, red and dark violet when viewed with an optical microscope under ordinary transmission light and epi-illumination optics, respectively. Under transmission electron microscopy, however, we observed no typical red chromatosomes, i.e., erythrosomes, in the cytoplasm. High-performance thin-layer chromatography (HPTLC) analysis of the pigment eluted from the erythro-iridophores indicated that carotenoid is the main pigment generating the reddish color. Furthermore, when the irrigating medium was a K(+)-rich saline solution, the color reflected from the erythro-iridophores changed from dark violet to sky blue, but the red coloration remained. The motile activities of the erythro-iridophores may participate in the changes in the reddish-violet shades of the pseudochromis fish.

Effects of androgens on the development of nuptial coloration and chromatophores in the bitterling Rhodeus ocellatus ocellatus.

Sexually mature male bitterlings, Rhodeus ocellatus ocellatus, exhibit distinct nuptial color, whereas females maintain a body color similar to that of juveniles. In the present study, body color and chromatophores were compared between male and female bitterlings, and the effects of androgens on body color and chromatophore densities were examined in females to clarify the role of androgen in the development of nuptial coloration and chromatophores. Males showed green, blue, and red color in specific regions of their skin and red color on the dorsal and caudal fins; females showed a subdued silver body color. For chromatophores, small greenish-type iridophores were observed in the green color region in the skin of males, whereas females had large spindle-shaped silvery-type iridophores in corresponding regions. Many erythrophores were observed in males in blue and red color regions in the skin and red color regions in the fins, but females possessed xanthophores in corresponding regions. The melanophore density of the skin was not different between males and females, but the distribution of melanophores in the fins was different between them. Treatment with 11-ketotestosterone or methyltestosterone induced male-type nuptial coloration in the female skin and fins. The distribution of chromatophores in androgen-treated females was similar to that in sexually mature males: an increase in the number of greenish-type iridophores and erythrophores was also observed in the skin. These results indicate that androgen induces male-type nuptial coloration in the bitterling and that the responses of chromatophores to androgen differ with the type and distribution site of the chromatophores.

Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model.

We have established a new culture system to study re-epithelialization during fish epidermal wound healing. In this culture system, fetal bovine serum (FBS) stimulates the epidermal outgrowth of multi-cellular layers from scale skin mounted on a coverslip, even when cell proliferation is blocked. The rate of outgrowth is about 0.4 mm/h, and at 3 h after incubation, the area occupied by the epidermal sheet is nine times larger than the area of the original scale skin. Cells at the bottom of the outgrowth show a migratory phenotype with lamellipodia, and "purse string"-like actin bundles have been found over the leading-edge cells with polarized lamellipodia. In the superficial cells, re-development of adherens junctions and microridges has been detected, together with the appearance and translocation of phosphorylated p38 MAPK into nuclear areas. Thus, this culture system provides an excellent model to study the mechanisms of epidermal outgrowth accompanied by migration and re-differentiation. We have also examined the role of extracellular matrix proteins in the outgrowth. Type I collagen or fibronectin stimulates moderate outgrowth in the absence of FBS, but development of microridges and the distribution of phosphorylated p38 MAPK are attenuated in the superficial cells. In addition, the leading-edge cells do not have apparent "purse string"-like actin bundles. The outgrowth stimulated by FBS is inhibited by laminin. These results suggest that dermal substrates such as type I collagen and fibronectin are able to initiate epidermal outgrowth but require other factors to enhance such outgrowth, together with coordinated alterations in cellular phenotype.

Ultrastructure of the dermal chromatophores in a lizard (Scincidae: Plestiodon latiscutatus) with conspicuous body and tail coloration.

Microscopic observation of the skin of Plestiodon lizards, which have body stripes and blue tail coloration, identified epidermal melanophores and three types of dermal chromatophores: xanthophores, iridophores, and melanophores. There was a vertical combination of these pigment cells, with xanthophores in the uppermost layer, iridophores in the intermediate layer, and melanophores in the basal layer, which varied according to the skin coloration. Skin with yellowish-white or brown coloration had an identical vertical order of xanthophores, iridophores, and melanophores, but yellowish-white skin had a thicker layer of iridophores and a thinner layer of melanophores than did brown skin. The thickness of the iridophore layer was proportional to the number of reflecting platelets within each iridophore. Skin showing green coloration also had three layers of dermal chromatophores, but the vertical order of xanthophores and iridophores was frequently reversed. Skin showing blue color had iridophores above the melanophores. In addition, the thickness of reflecting platelets in the blue tail was less than in yellowish-white or brown areas of the body. Skin with black coloration had only melanophores.

Production and purification of recombinant somatolactin beta and its effects on melanosome aggregation in zebrafish.

A second form of somatolactin, somatolactin beta (SLbeta), was recently discovered in zebrafish (Danio rerio). This novel subtype of somatolactin is distantly related to somatolactin alpha (SLalpha) found in teleost species and is produced in a different region of the pituitary. To date, no physiological study of SLbeta has been reported. In order to study the physiological functions of SLbeta, recombinant SLbeta protein has been produced and purified. The cDNA of zebrafish SLbeta was cloned into a pET100 bacteria expression vector and His-tagged fusion proteins were produced in BL21 (DE3) Escherichia coli cells. The majority of recombinant somatolactins produced by E. coli were isolated in inclusion bodies although a small percentage of recombinant proteins (<1%) were also found in soluble form. Fusion proteins were solubilized from inclusion bodies using 6M guanidine hydrochloride. Pure recombinant somatolactins were obtained by affinity purification. The estimated molecular weight of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis correlates with the molecular mass calculated from the deduced amino acid sequence of SLbeta. Thereafter, specific polyclonal antibodies against the recombinant SLbeta were developed. These antibodies recognized specifically a group of cells located in the anterior pars intermedia of the pituitary. The antibodies did not react with SLalpha, growth hormone or prolactin cells in the zebrafish pituitary glands. Furthermore, recombinant SLbeta induced melanosome aggregation in a concentration-dependent manner in skin of zebrafish scales. Significant melanosome aggregation was observed in zebrafish melanophores at a concentration of 1 microg/ml. These results, combined with previous reports demonstrate that the recombinant SLbeta proteins produced here are bioactive. The function of inducing melanosome aggregation is conserved among the somatolactin functions.

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.

Molecular cloning and characterization of a new RGS protein of Medaka.

We identified eight genes of putative RGS proteins in skin of Medaka fish using PCR amplification with degenerate primers for the RGS domain of known RGS proteins. Then, we cloned a full-length cDNA for a new RGS protein. This RGS protein was similar to human RGS3 within the RGS domain, but other parts were unique among known RGS proteins. RT-PCR analysis demonstrated that this Medaka RGS3-like protein (MeRGS3L) is mainly expressed in skin and heart. When coexpressed in Xenopus oocytes, MeRGS3L accelerated the turning-on and -off of Gi/o-mediated modulation of GIRK channels without apparent desensitization in the presence of ligand. MeRGS3L also decreased the response of Gq signaling upon activation of m1 muscarinic receptor. This new RGS protein may play important roles in regulation of melanophore responses in Medaka skin.

Bhc-cNMPs as either water-soluble or membrane-permeant photoreleasable cyclic nucleotides for both one- and two-photon excitation.

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6-bromo-7-hydroxycoumarin-4-ylmethyl esters of cNMP (Bhc-cNMP) and their acetyl derivatives (Bhc-cNMP/Ac) as new caged second messengers. Irradiation of Bhc-cNMPs quantitatively produced the parent cNMPs with one-photon uncaging efficiencies (Phiepsilon) of up to one order of magnitude better than those of 2-nitrophenethyl (NPE) cNMPs. In addition, two-photon induced photochemical release of cNMP from Bhc-cNMPs (7 and 8) can be observed with the two-photon uncaging action cross-sections (delta(u)) of up to 2.28 GM (1 GM=10(-50) cm(4) s photon(-1)), which is the largest value among those of the reported Bhc-caged compounds. The wavelength dependence of the delta(u) values of 7 revealed that the peak wavelength was twice that of the one-photon absorption maximum. Bhc-cNMPs showed practically useful water solubility (nearly 500 microM), whereas 7-acetylated derivatives (Bhc-cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP-mediated transduction channels in newt olfactory receptor cells and cAMP-mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Somatolactin selectively regulates proliferation and morphogenesis of neural-crest derived pigment cells in medaka.

Species-specific colors and patterns on animal body surfaces are determined primarily by neural-crest-derived pigment cells in the skin (chromatophores). However, even closely related species display widely differing patterns. These contrasting aspects of chromatophores (i.e., the fixed developmental control within species and extreme diversity among species) seem to be a curious and suitable subject for understanding evolution and diversity of organisms. Here we identify a gene responsible for medaka "color interfere" mutants by positional cloning. These mutants do not show any obvious morphological and physiological defects other than defects in chromatophore proliferation and morphogenesis. The mutation has been identified as an 11-base deletion in somatolactin, which causes truncation 91 aa upstream of the C terminus of the protein's 230 aa. Somatolactin transcription changed dramatically during morphological body color adaptation to different backgrounds. This genetic evidence explains somatolactin function. Studying this mutant will provide further insights into the development and regulation of chromatophores and clues for reassessing other functions of somatolactin suggested in other fish.

Morphological color changes in fish: regulation of pigment cell density and morphology.

Pigment cells enable fish to change their coloration. It has been recognized that fish color changes can be divided into two categories; one is a physiological color change, which is attributed to rapid motile responses of chromatophores, and the other is a morphological color change, which results from changes in the morphology and density of chromatophores. Long-term adaptation of fish to a certain background can be a general cue to morphological color changes, and has been studied from the beginning of the 19th century. Although the motile mechanism and its control in fish chromatophores are now being elucidated, it is not yet clear how chromatophores change their density and what controls morphological color changes. In recent years, chromatophores, especially melanophores, have been shown to differentiate and to die by apoptosis under the influence of factors that regulate motile responses. Those factors are likely to utilize common intracellular signaling pathways used in part to regulate both types of color changes. In this article, after briefly reviewing the history of early studies, recent findings are discussed relevant to increases or decreases in chromatophores, and changes in their morphology. Finally, morphological color changes are discussed as physiological phenomena involved in the balance between differentiation and apoptosis of chromatophores.