Effect of x-ray irradiation on the physiological reactions of dermal melanophores in tadpoles of the common frog

The effect of X-ray irradiation (700 R) on the physiological reactions of the dermal melanophores in Rana temporaria L. tadpoles of 20-22d stages of development has been studied. It has been shown that the irradiation of the aggregated dermal melanophores does not change the physiological state. As compared these data with previous ones a conclusion of high selective susceptibility to the X-ray irradiation of the tadpoles' epidermal melanophores has been drawn.     

Ultrastructure of the integumental melanophores of the coelacanth,Latimeria chalumnae

The integumental melanophores of Latimeria chalumnae were studied by light and electron microscopy. The epidermal melanophore located in the mid-epidermis consists of a round perikaryon with long slender dendrites extending into epidermal cells and intercellular spaces. The dermal melanophores occur in the loose dermal matrix underlying a relatively thick layer of collagen fibers. The dermal melanophores are usually flattened and their dendrites lie parallel to the collagen layer. Both epidermal and dermal melanophores contain oval, electron-opaque melanosomes, large mitochondria, agranular vacuoles of endoplasmic reticulum and microtubules. Microfilaments and RNP particles are less conspicuous. While the peripheral cytoplasm of both dermal and epidermal melanophores is filled with a large number of melanosomes, the perinuclear cytoplasm of many dermal melanophores is occupied by premelanosomes in various stages of differentiation, and that of the epidermal melanophore contains numerous large vacuoles. Despite the scarcity of epidermal melanophores, the epidermal melanin unit is present in the form of melanosome complexes. In addition, the melanophores of Latimeria possess the basic characteristics common to other vertebrates, but they more closely resemble those of lungfish and other aquatic vertebrates.     

Migration of epidermal melanophores to the dermis through the basement membrane during metamorphosis in the frog, Rana japonica

The number of epidermal melanophores of the skin decreases dramatically during metamorphosis in the frog, Rana japonica. This decrease may represent an adaptation for rapid color change, a property which the animal acquires after metamorphosis. We concluded that the decrease was due to the migration of epidermal melanophores to the dermis. Epidermal melanophores and epidermal cells are tightly associated with each other in the young tadpole. The association becomes looser at the metamorphic stage and, occasionally, small breaks in the basement membrane are seen. These breaks may facilitate the migration. The migration was observed exclusively at the metamorphic stage, in spite of careful observation of other stages under the electron microscope. The migration of epidermal melanophores was induced by treatment with thyroxine of cultured skin from tadpoles at stage 15, and this hormone may act directly on epidermal melanophores. Until now, the increase in the number of dermal melanophores during metamorphosis has been explained by the differentiation of dermal melanophores from melanoblasts and by their mitotic division. Our results show that the migration of epidermal melanophores to the dermis may be a factor which accounts for the increase in the number of dermal melanophores.     

Ultrastructural changes in dermal melanophores in the process of pigment granule migration

The results of electron microscope investigations on dermal melanophores of Rana temporaria L. during migration of pigment granules are presented. It was shown that in comparison to the previous observations dermal melanophores are flat cells without branches. Ultrastructural differences have been demonstrated in dermal melanophores during migration of pigment granules. During melanosome dispersion membrane vesicle bodies are seen in the cytoplasm to be inserted in the melanophore membrane.     

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

Ultrastructure of the integumental melanophores of the South American lungfish (Lepidosiren paradoxa) and the African lungfish (Protopterus sp.)

The integumental melanophores of two genera of lungfish, Lepidosiren paradoxa and Protopterus sp. were examined by light and electron microscopy. Both species possess both epidermal and dermal melanophores with fine structural characteristics basically similar to those of other vertebrates. The epidermal melanophores of both species are located in the intermediate epidermis, and possess thin perikarya containing round nuclei, and slender dendrites extending into the nearby intercellular spaces. The dermal melanophores occur immediately beneath the basement membrane, and possess flat perikarya and dendrites running horizontally between the collagen fibers of the dermis. The integument of both species does not possess an epidermal melanin unit or a dermal chromatophore unit. As in other vertebrates, each melanophore contains numerous oval, electron-opaque melanosomes, relatively large mitochondria, vacuolar endoplasmic reticula, and groups of RNP particles. Although micro filaments running randomly between other organelles occur regularly, microtubules were not demonstrated. Premelanosomes at various stages of differentiation were best illustrated in the dermal melanophores of Protopterus, and it is concluded from the observation of their fine structure that the morphological development of lungfish melanosomes closely parallels that of higher vertebrates. On the basis of melanophore morphology, Lepidosiren and Protopterus appear to be more closely related to each other than to Neoceratodus.     

Identification of Microtubule-Organizing Centers in Interphase Melanophores of Xenopus Laevis Larvae In Vivo

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immuno-stained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

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.     

Formation of the Dermal Chromatophore Unit (DCU) in the Tree Frog Hyla Arborea

In the tadpole of the tree frog Hyla arborea, the color of the dorsal skin was dark brown. Dermal melanophores, xanthophores, and iridophores were scattered randomly under the subepidermal collagen layer (SCL). After metamorphosis, the dorsal color of the animal changed to green and the animal acquired the ability of dramatic color change, demonstrating that the dermal chromatophore unit (DCU) was formed at metamorphosis. Fibroblasts invaded the SCL and divided it into two parts: the stratum spongiosum (SS) and the stratum compactum (SC). The activity of collagenase increased at metamorphosis. The fibroblasts appeared to dissolve the collagen matrix as they invaded the SCL. Then, three types of chromatophores migrated through the SCL and the DCU was formed in the SS. The mechanism how the three types of chromatophores were organized into a DCU is uncertain, but different migration rates of the three chromatophore types may be a factor that determines the position of the chromatophores in the DCU. Almost an equal number of each chromatophore type is necessary to form the DCUs. However, the number of dermal melanophores in the tadpoles was less than the number of xanthophores and iridophores. It was suggested that epidermal melanophores migrated to the dermis at metamorphosis and developed into dermal melanophores. This change may account for smaller number of dermal melanophores available to form the DCU_s.     

Adrenergic nerves to the dermal melanophores of the rainbow trout,Salmo Gairdneri

Summary The stellate processes and cell bodies of the dermal melanophores in the rainbow trout are intimately enclosed by a plexus of thin varicose nerves which display a specific catecholamine fluorescence. The nerves contain probably small amounts of noradrenaline and have the ability to take up and concentrate this amine. Denervation of the skin leading to dispersion of the melanophores causes the nerves to disappear. The findings leave little doubt that the dermal chromatic motor nerves are adrenergic.This study was supported by a grant from the Swedish Research Council for Natural Sciences (99-35) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B69-14X-56-05C and No. B69-14X-712-04C).     

Response of the chromatophores in relation to the healing of skin wounds in an Indian Major Carp, Labeo rohita (Hamilton)

Chromatophores show significant changes during healing of skin wounds in Labeo rohita (Common Name - Rohu). Wound area can be divided into regions I, II and III. After infliction of wound, skin colour becomes significantly dark by 2 h that is gradually restored by 2 d. In regions II and III at 5 min, epidermal melanophores appear with beaded dendrites. In these regions at 2 h and in region I at 6 h, epidermal melanophores appear small, rounded or irregular shaped having dendritic processes with aggregated melanosomes. Subsequently, melanophores appear having elongated dendrites with dispersed or aggregated melanosomes. At 24 h, clusters of pigmented bodies appear in regions I and II. These bodies increase up to 2 d, and then diminish gradually and disappear by 8 d. Changes in dermal melanophores in region II at 5 min indicate the onset of degeneration. Degenerating melanophores increase up to 12 h, then gradually decline, and disappear by 4 d. Simultaneously, stellate melanophore reappear, gradually increase and appear like control by 8 d. Dermal melanophores in region III at different intervals appear stellate. In region I stellate dermal melanophores appear at 4 d. Stellate melanophores in all regions show different distribution of dispersed or aggregated melanosomes. With the appearance of dermal melanophores, highly refractive, crystalline structures, possibly the refractive platelets of the iridophores, are visualized around them. At subsequent intervals, these are frequently observed. This study provides interesting insights in injury induced changes in chromatophores in fish. The findings could be considered useful in perception of intriguing features in the development of pigment research in future.     

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. J. Morphol. 274:1353–1364, 2013. © 2013 Wiley Periodicals, Inc.     

Mechanisms of Melanophore Induction in Amphibian Development

Induction of melanophores was examined by the sandwich method of explantation with embryonic tissues of Xenopus laevis +/+ and the white mutant, a^P/a^P. Interspecific combinations of tissues of Triturus taeniatus and Xenopus borealis were also used. The ectoderm used as the reacting system was taken from embriyos at various stages and combined with various tissues known to be melanogenic inductors. The following results were obtained: 1) The sources of melanophore induction in both +/+ and a^p/a^p studied by sandwich explantation were the same in both retinal pigmented epithelium and dermal melanophores: 2) Melanophores were induced in epidermal material from embryos at stages from the early gastrula to the late tail bud stage: 3) The presence of melanoblasts together with other ectomesenchymal cells in the neural crest is not sine qua non for their determination and differentiation: 4) On isolation of reacting material from the late gastrula, melanophores appeared in all cases. This shows that two hours contact between inductor tissues and the ectoderm is necessary and sufficient for melanophore induction: 5) Melanophore induction is not species-specific, but occurred in Xenopus ectoderm under the action of endomesoderm of Tr. taeniatus or X. borealis , and vice versa. The shapes and structures of melanophores induced were typical for the species from which the ectoderm was taken: 6) Melanogenic activity in the late gastrula stage has a gradient of distribution with a maximum in the prechordal plate: 7) In the mutant only the primary source of melanogenic inductors, the prechordal plate (PrP1), was active in stages both before and after its invagination: 8) Despite the fact that skin melanophores and retinal melanocytes have different genesis in development, all the present data suggest the identity of the mechanisms of melanin synthesizing machinery in the two.     

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.     

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.     

Cell surface carbohydrate involvement in controlling the adhesion and morphology of neural crest cells and melanophores of Xenopus laevis

Pieces of dorsal neural tube (stages 22-23) or late neural crest tissue (stages 24-26) of Xenopus laevis were cultured. Migratory cells moved out of explants to form an outgrowth of multipolar melanophores on the substratum. Treatment with beta-galactosidase (0.1-0.4 U/ml) to remove cell surface galactose was correlated with detachment of melanophores. In the presence of lower concentrations of this enzyme the shapes of these cells were converted to arborized, spidery morphologies and cell movement was inhibited. Unpigmented cells were affected more slowly. Neuraminidase treatment, to remove cell surface sialic acid and expose more galactose, only affected melanophores. These became increasingly spread on the substratum and cell overlap was observed. These results suggest that the relative amounts of galactose and sialic acid at the cell surface become increasingly important in controlling cell adhesion as X. laevis neural crest cells migrate and differentiate into melanophores.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long-term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore-fibroblast co-culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast-derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane-enclosed melanosomes in a process that was upregulated by alpha-melanocyte-stimulating hormone (alpha-MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane-proteins seemed to be of importance, as the cluster-like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long-term colour changes and for studies of factors that affect pigmentation.     

Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians

Alibardi, L. 2011. Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians. —Acta Zoologica (Stockholm) 00 :1–11. The cytology and distribution of chromatophores responsible for skin pigmentation in chelonians is analyzed. Epidermal melanocytes are involved in the formation of dark spots or stripes in growing shelled and non‐shelled skin. Melanocytes rest in the basal layer of the epidermis and transfer melanosomes into keratinocytes during epidermal growth. Dermal melanophores and other chromatophores instead remain in the dermis and form the gray background of the skin. When dermal melanophores condense, they give origin to the dense spots or stripes in areas where no epidermal melanocytes are present. In the latter case, the epidermis and the corneous layer are transparent and reveal the dermal distribution of melanophores and other chromatophores underneath. As a result of this basic process of distribution of pigment cells, the dark areas visible in scales can have a double origin (epidermal and dermal) or a single origin (epidermal or dermal). Xanthophores, lipophores, and a cell containing both pterinosomes and lipid droplets are sparse in the loose dermis while iridophores are rarely seen in the skin of chelonians analyzed in the present study. Xanthophores and lipophores contribute to form the pale, yellow or oranges hues present among the dark areas of the skin in turtles.     

Localization of pigment cells in cultured frog skin

The pigmentation pattern of ventral skin of the frog Rana esculenta consists mainly of melanophores and iridophores, rather than the three pigment cells (xanthophores, iridophores, and melanophores) which form typical dermal chromatophore units in dorsal skin. The present study deals with the precise localization and identification of the types of pigment cells in relation to their position in the dermal tracts of uncultured or cultured frog skins. Iridophores were observed by dark-field microscopy; both melanophores and iridophores were observed by transmission electron microscopy. In uncultured skins, three levels were distinguished in the dermal tracts connecting the subcutaneous tissue to the upper dermis. Melanophores and iridophores were localized in the upper openings of the tracts directed towards the superficial dermis (level 1). The tracts themselves formed level 2 and contained melanophores and a few iridophores. The inner openings of the tracts made up level 3 in which mainly iridophores were present. These latter openings faced the subcutaneous tissue In cultured skins, such pigment-cell distribution remained unchanged, except at level 2 of the tracts, where pigment cells were statistically more numerous; among these, mosaic pigment cells were sometimes observed.     

Cytoskeletal Architecture of Dermal Chromatophores of the Freshwater Teleost Oryzias latipes

Cytoskeletal construction of dermal chromatophores of Orgzias latipes was studied by immunofluorescence microscopy. A microtubule system was most prominent in melanophores where a large number of microtubules emanated from the center of the cell. Xanthophores had an arrangement basically similar to that of melanophores, though the radial pattern became more irregular in the peripheral region where intersecting wavy microtubules were quite frequent. Oval-shaped leucophores exhibited the least-developed microtubule system, where the limited number of microtubules formed a loose basket-like architecture. Intermediate filaments were ubiquitously present in all types of chromatophores and were found to be vimentin-immunoreactive. Examination of doubly-labeled cells indicated that vimentin filaments had similar distribution patterns with microtubules. Orderly arranged bundles of actin filaments were found only in xanthophores, while in melanophores and xanthophores, actin expression was diffuse without displaying a conspicuous filamentous organization. Colchicine treatment induced depolymerization of microtubules and retraction of dendrites in varying degrees in cells in culture and in situ. Melanophores in culture are very sensitive to the treatment while xanthophores appeared to be more resistant in respect to the maintenance of cell morphology.