Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

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 α‐melanocyte‐stimulating hormone (α‐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.     

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.     

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.     

Receptor mechanisms in fish chromatophores--VI. Adenosine receptors mediate pigment dispersion in guppy and catfish melanophores

Using the guppy, Lebistes reticulatus, and the siluroid catfish, Parasilurus asotus , the effects of purine and pyrimidine derivatives on the movement of melanophores were studied. All the substances tested did not aggregate pigment within melanophores. Adenosine and adenine nucleotides were very effective in dispersing melanosomes within the cell, although adenine itself lacked such action. Derivatives of other purines than adenine and of pyrimidines did not disperse melanosomes. The pigment dispersion induced by adenine derivatives was specifically antagonized by methylxanthines. It was concluded that adenosine receptors are present on the melanophore membrane, which take part in the darkening reaction of fishes.     

Calcium-dependent irreversible effect of ionophore A23187 on melanophores

The calcium ionophore, A231187, induces a Ca2+ -dependent movement (dispersion) of melanosomes within skin melanophores of the lizard, Anolis carolinensis, in vitro. The effects of A23187 are irreversible, since after repeated rinsing of the skins in the absence of the ionophore they will always darken in Ringer containing Ca2+ but will immediately lighten when transferred to Ca2+ -free Ringer. These results suggest that the ionophore is irreversibly localized to the melanophore membrane and that its melanosome-dispersing effect is continuously dependent upon extracellular calcium.     

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

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.     

A melanin-dispersing effect of cyclic adenosine monophosphate on Fundulus melanophores

Treatment of Fundulus melanophores with adenosine 3′,5′-monophosphate (cyclic AMP) is followed by reversible melanin dispersion in these cells. Adenosine 3′-monophosphate and adenosine 5′-monophosphate both have a similar, but weaker dispersing action. In addition, adenosine 5′-monophosphate also has a melanin aggregating effect. These results are interpreted to mean that nerve transmitters may act by controlling the level of cyclic AMP within the Fundulus melanophore.     

Ultrastructure of the integumental melanophores of the Australian lungfish,Neoceratodus forsteri

The integumental melanophores of Australina lungfish, Neoceratodus forsteri, were examined by light and electron microscopy and found to possess essentially the same structural characteristics observed in other vertebrates. The epidermal melanophores are located in the intermediate epidermis and possess round perikarya and slender dendrites extending into nearby intercellular spaces. The dermal melanophores are found immediately below the basement membrane as well as in the deeper dermis. These cells possess flattened nuclei and dendrites running parallel to the basement membrane. Each melanophore contains numerous oval or elliptical, intensely electron-dense melanosomes, relatively large mitochondria, systems of vacuolar endoplasmic reticulum, groups of free RNP particles, and some microfilaments. Only a few, short microtubules could be demonstrated in the perinuclear cytoplasm of the dermal melanophore, while a relatively large number of late premelanosomes are found both in perikarya and dendritic processes of epidermal melanophores. These premelanosomes exhibit a particulate internal structure in cross section. Both melanosomes and premelanosomes occur singly in the cytoplasm of epidermal cells, thereby confirming the existence of the epidermal melanin unit in the lowest vertebrates thus far examined electron microscopically.     

Control of melanosome movement in intact and cultured melanophores in the bitterling, Acheilognathus lanceolatus

The melanophores in the dermis on scales in the bitterling, Acheilognathus lanceolatus were studies to obtain information about the control mechanism of aggregation and dispersion using intact, membrane-permeabilized and cultured cells. The cultured melanophores showed supersensitivity, namely, they responded to norepinephrine with much higher sensitivity than intact cells. The cultured melanophores failed to respond to high KCl. Melatonin aggregated and adenosine dispersed melanosomes within a cell. Digitonin permeabilized cells showed aggregation with Ca ions and dispersion by cyclic adenosine 3',5'-monophosphate (cAMP) in the presence of ATP. Movement of melanosomes was observed under the high magnification of light microscope and the tracks of each pigment granule were followed. The granules moved fast and linearly during aggregation, whereas they showed to-and-fro movement during dispersion.     

The morphology of cultured melanophores from tadpoles of Xenopus laevis: Scanning electron microscopical observations

Summary Tail-fin melanophores of tadpoles of Xenopus laevis (Daudin) in primary culture were examined scanning electron microscopically in the aggregated and in the dispersed state. After isolation, the melanophores are spherical, but within 24 h they develop thin filopodia for attachment to the substratum. Subsequently, cylinder-like as well as flat sheet-like processes are formed, which adhere to the substratum with terminal pseudopodia and filopodia. The processes of adjacent melanophores contact each other, thus forming an interconnecting network between the melanophores.In the aggregated state the central part of the melanophore is spherical and voluminous. Both the central part and the processes bear microvilli. In melanophores with dispersed melanosomes the central part is much flatter; the distal parts have a thickness that equals a monolayer of melanosomes. The surface of the cell bears only scarce microvilli.These features indicate that melanophores do not have a fixed shape and that pigment migration is accompanied by reciprocal volume transformation between the cell body and its processes.     

The localisation of lead in the skin of light- and dark-adapted Xenopus laevis

Toads pretreated for 2 months on either a dark or a light background were then exposed to lead nitrate at 50 ppm lead for 21 days, the illumination regimes being maintained. Metal analysis of dorsal skin showed significantly higher lead levels (p less than 0.01) in dark-adapted toads. No precipitated lead deposits were observed at the ultrastructural level, necessitating X-ray microanalysis of sections containing melanophores, gland cells and general (non-melanophore) cytoplasm. Analysis showed the lead to be concentrated within the melanosomes of the melanophores, and to be significantly higher (p less than 0.01) in individual melanosomes of dark-adapted toads than in light adapted ones. Copper was also found to be concentrated in the melanosomes and was higher (p less than 0.01) in the melanosomes of the dark-adapted toads. The results are consistent with the known affinity of melanin for heavy metals and the documented increase in melanophore number under prolonged dark background regimes. Since all toads received the same lead exposure, the melanosome results give rise to speculation that higher melanin levels might occur in individual melanosomes of dark-adapted skin.     

Different distribution of melanophores and xanthophores in early tailbud and larval stages inTriturus alpestris

Summary The subepidermal distribution of xanthophores and melanophores is investigated in embryos ofTriturus alpestris with a uniform (stage 28+) and a banded melanophore pattern (stage 35/36). In ultrathin head and trunk sections from stage 35/36 embryos which externally show longitudinal dorsal and lateral melanophore bands in the trunk and less compact continuations of the dorsal bands in the head, xanthophores were discovered in addition to melanophores. Melanophores contain melanosomes while xanthophores which are not externally visible, are recognized by their pterinosomes. Both chromatophore cell types are mutually exclusively distributed on the epidermal basement membrane (bm). Mesenchymal cells seemed not to be able to replace them, except on the bm of the corneal epithelium where there were only mesenchymal cells. In head and trunk sections from stage 28+ embryos which externally show a distribution of uniformly scattered melanophores on the dorsolateral halves, melanophores were found on the dorsolateral neural crest migration route. No epidermal bm was present and xanthophores were undetectable. In ventrolateral and ventral portions of embryos of both stages no chromatophores occurred. This investigation defines the histological localization of melanophores and xanthophores in embryos with a typical uniform and banded melanophore arrangement; a subsequent study analyzes when xanthophores appear and how they arrange with melanophores in alternating zones.     

The localisation of lead in the skin of light- and dark-adapted Xenopus laevis

Summary Toads pretreated for 2 months on either a dark or a light background were then exposed to lead nitrate at 50 ppm lead for 21 days, the illumination regimes being maintained. Metal analysis of dorsal skin showed significantly higher lead levels (p<0.01) in dark-adapted toads. No precipitated lead deposits were observed at the ultrastructural level, necessitating X-ray microanalysis of sections containing melanophores, gland cells and general (non-melanophore) cytoplasm. Analysis showed the lead to be concentrated within the melanosomes of the melanophores, and to be significantly higher (p<0.01) in individual melanosomes of dark-adapted toads than in light-adapted ones. Copper was also found to be concentrated in the melanosomes and was higher (p<0.01) in the melanosomes of the dark-adapted toads.The results are consistent with the known affinity of melanin for heavy metals and the documented increase in melanophore number under prolonged dark background regimes. Since all toads received the same lead exposure, the melanosome results give rise to speculation that higher melanin levels might occur in individual melanosomes of dark-adapted skin.     

Unusual leucophore-like cells specifically appear in the lineage of melanophores in the periodic albino mutant of Xenopus laevis

In the periodic albino mutant (a(p)/a(p)) of Xenopus laevis, peculiar leucophore-like cells appear in the skins of tadpoles and froglets, whereas no such cells are observed in the wild-type (+/+). These leucophore-like cells are unusual in (1) appearing white, but not iridescent, under incident light, (2) emitting green fluorescence under blue light, (3) exhibiting pigment dispersion in the presence of alpha-melanocyte stimulating hormone (alphaMSH), and (4) containing an abundance of bizarre-shaped, reflecting platelet-like organelles. In this study, the developmental and ultrastructural characteristics of these leucophore-like cells were compared with melanophores, iridophores and xanthophores, utilizing fluorescence stereomicroscopy, and light and electron microscopy. Staining with methylene blue, exposure to alphaMSH, and culture of neural crest cells were also performed to clarify the pigment cell type. The results obtained clearly indicate that: (1) the leucophore-like cells in the mutant are different from melanophores, iridophores and xanthophores, (2) the leucophore-like cells are essentially similar to melanophores of the wild-type with respect to their localization in the skin and manner of response to alphaMSH, (3) the leucophore-like cells contain many premelanosomes that are observed in developing melanophores, and (4) mosaic pigment cells containing both melanosomes specific to mutant melanophores and peculiar reflecting platelet-like organelles are observed in the mutant tadpoles. These findings strongly suggest that the leucophore-like cells in the periodic albino mutant are derived from the melanophore lineage, which provides some insight into the origin of brightly colored pigment cells in lower vertebrates.     

Long-term cultivation of amphibian melanophores. In vitro ageing and spontaneous transformation to a continuous cell line

Pure melanophore populations isolated from the tail skin of the tadpole, Rana catesbeiana, were mass cultured for a period of 2-3 years. All cell lines of amphibian melanophores studied exhibited growth crisis (in vitro ageing) followed by spontaneous transformation to a continuous cell line, as shown by changes in growth characteristics in mass culture and in clone culture, by the appearance of the cells, and by measurements of cell volumes. Even after becoming a continuous cell line, amphibian melanophores continued to have a diploid chromosome number (2n = 26) in three of four cell lines examined. The chromosome mode in one cell line, however, changed to thirty. Measurement of melanin dispersion after the addition of alpha-melanocyte-stimulating hormone suggested that the mechanism for melanin dispersion in melanophores changed during in vitro ageing.     

Regulatory control of both microtubule- and actin-dependent fish melanosome movement

In fish melanophores, melanosomes can either aggregate around the cell centre or disperse uniformly throughout the cell. This organelle transport involves microtubule- and actin-dependent motors and is regulated by extracellular stimuli that modulate levels of intracellular cyclic adenosine 3-phosphate (cAMP). We analysed melanosome dynamics in Atlantic cod melanophores under different experimental conditions in order to increase the understanding of the regulation and relative contribution of the transport systems involved. By inhibiting dynein function via injection of inhibitory antidynein IgGs, and modulating cAMP levels using forskolin, we present cellular evidence that dynein is inactivated by increased cAMP during dispersion and that the kinesin-related motor is inactivated by low cAMP levels during aggregation. Inhibition of dynein further resulted in hyperdispersed melanosomes, which subsequently reversed movement towards a more normal dispersed state, pointing towards a peripheral feedback regulation in maintaining the evenly dispersed state. This reversal was blocked by noradrenaline. Analysis of actin-mediated melanosome movements shows that actin suppresses aggregation and dispersion, and indicates the possibility of down-regulating actin-dependent melanosome movement by noradrenaline. Data from immuno-electron microscopy indicate that myosinV is associated with fish melanosomes. Taken together, our study presents evidence that points towards a model where both microtubule- and actin-mediated melanosome transport are synchronously regulated during aggregation and dispersion, and this provides a cell physiological explanation behind the exceptionally fast rate of background adaptation in fish.     

Melatonin, melatonin receptors and melanophores: a moving story

Melatonin (5-methoxy N-acetyltryptamine) is a hormone synthesized and released from the pineal gland at night, which acts on specific high affinity G-protein coupled receptors to regulate various aspects of physiology and behaviour, including circadian and seasonal responses, and some retinal, cardiovascular and immunological functions. In amphibians, such as Xenopus laevis, another role of melatonin is in the control of skin coloration through an action on melanin-containing pigment granules (melanosomes) in melanophores. In these cells, very low concentrations of melatonin activate the Mel(1c) receptor subtype triggering movement of granules toward the cell centre thus lightening skin colour. Mel(1c) receptor activation reduces intracellular cAMP via a pertussis toxin-sensitive inhibitory G-protein (Gi), but how this and other intracellular signals regulate pigment movement is not yet fully understood. However, melanophores have proven an excellent model for the study of the molecular mechanisms which coordinate intracellular transport. Melanosome transport is reversible and involves both actin- (myosin V) and microtubule-dependent (kinesin II and dynein) motors. Melanosomes retain both kinesin and dynein during anterograde and retrograde transport, but the myosin V motor seems to be recruited to melanosomes during dispersion, where it assists kinesin II in dominating dynein thus driving net dispersion. Recent work suggests an important role for dynactin in coordinating the activity of the opposing microtubule motors. The melanophore pigment aggregation response has also played a vital role in the ongoing effort to devise specific melatonin receptor antagonists. Much of what has been learnt about the parts of the melatonin molecule required for receptor binding and activation has come from detailed structure-activity data using novel melatonin ligands. Work aiming to devise ligands specific for the distinct melatonin receptor subtypes stands poised to deliver selective agonists and antagonists which will be valuable tools in understanding the role of this enigmatic hormone in health and disease.     

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.     

Effects of lithium on pigmentation in the embryonic zebrafish (Brachydanio rerio)

Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores.