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.     

Berberine-induced pigment dispersion in Bufo melanostictus melanophores by stimulation of beta-2 adrenergic receptors

Abstract

Reduced production of melanin by decreased or the absence of melanocytes leads to various hypopigmentation disorders, and the development of melanogenetic agents for photoprotection and hypopigmentation disorders is one of the top priority areas of research. Hence, the present study was carried out to elucidate the ability of berberine, a principal active ingredient present in the roots of the herb Berberis vulgaris to stimulate pigment dispersion in the isolated skin melanophores of the toad Bufo melanostictus. In the present study, mean melanophore size index of the isolated skin melanophores of B. melanostictus was assayed after treating with various concentrations of berberine. A marked melanin dispersion response leading to skin darkening was observed in the isolated melanophores of toad in response to berberine, which was found to be mediated through beta-2 adrenergic receptors. The physiologically significant dose-related melanin dispersion effects of berberine per se were found to be completely abolished by propranolol, which is a specific beta-2 adrenergic receptor blocker. These per se melanin dispersal effects were also found to be markedly potentiated by isoprenaline, which is a specific beta-adrenoceptor agonist. The results indicate that berberine causes a tremendous, dose-dependent, physiologically significant pigment dispersing in the isolated skin melanophores of B. melanostictus.     

Melanin concentrating hormone. I. Influence of nerves and hormones on the control of trout melanophores

When melanophores on trout scales are cultured in vitro they show a transitory melanin concentration, which can be prevented by addition to the medium of the alpha adrenergic blocker, phentolamine. This indicates the release of endogenous nor-epinephrine from local nerve terminals. This initial phase of melanin aggregation is followed by redispersion and then by a second, more gradual melanin concentration over several days, which is not antagonized by phentolamine. A final melanophore index of between 2-2.5 is attained which may be the resting state of trout melanophores. Using short-term cultured melanophores which have passed the phase of endogenous nor-epinephrine release, it is shown that exogenous nor-epinephrine will interact synergistically with the melanin-concentrating hormone to achieve full melanin concentration. Evidence is discussed for believing that in the trout, such synergy is necessary to achieve maximum pallor in vivo.     

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.     

Melanophore multinucleation pathways in zebrafish

In zebrafish, apart from mononuclear melanophores, bi‐ and trinuclear melanophores are frequently observed; however, the manner in which multinucleation of these cells occurs during fish development remains unknown. Here, we analyzed the processes underlying multinucleation of zebrafish melanophores. Transgenic zebrafish in which melanophore nuclei were labeled with a histone H2B‐red fluorescent reporter protein were used to evaluate the distribution of mono‐, bi‐, and trinuclear melanophores in both the trunk and fin. Half of the melanophores examined were binuclear and approximately 1% were trinuclear. We compared cell size, cell motility, and survival rate between mono‐ and binuclear melanophores grown in a culture dish, and we found that cell size and survival rate were significantly larger in binuclear melanophores. We then analyzed the behavior of melanoblasts and melanophores from transgenic zebrafish using in vivo and in vitro live‐cell imaging. We detected division and differentiation of melanoblasts, as well as melanoblast nuclear division without subsequent cellular division. In addition, we observed cellular and nuclear division in melanophores, although these events were very infrequent in vitro. On the basis of our findings, we present a scheme for melanophore multinucleation in zebrafish.     

Fibronectin-induced migration of melanophores in vitro in scales of medaka,Oryzias latipes

Summary The effects of fibronectin on melanophores were examined in two mutant strains of medaka, Oryzias latipes: mm (BmmR), which has condensed melanophores and normal dendritic melanophores; and cm (BcmR), which has condensed melanophores. When medaka scales were cultured in the presence of fibronectin, melanophores of the wild type and dendritic melanophores of the mm mutant changed their shape and migrated, whereas melanophore migration was rarely seen in the absence of fibronectin. Melanophores of the cm mutant and condensed melanophores of the mm mutant did not migrate even in the presence of fibronectin. When melanophores of the wild type and mm mutant were condensed by adrenalin, they did not migrate. On the other hand, when melanophores of the cm mutant were dispersed by theophylline, they were able to migrate. These results indicate that fibronectin induces the migration of melanophores and that dispersion of melanin granules may be requisite for such migration.     

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.     

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.     

Maxadilan activates PAC1 receptors expressed in Xenopus laevis xelanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long-term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non-transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

Calcium Requirement for α-MSH Action on Melanophores: Studies with Forskolin

Abstract

α-MSH-induced pigment dispersion in melanophores shows an absolute requirement for extracellular Ca²⁺. To localize Ca²⁺ sites involved in the mechanism of action of α-MSH we studied the effects of Ca²⁺ deprivation on α-MSH and forskolin-induced melanophore responses. In an in vitro melanophore system employing ventral tailfins of Xenopus tadpoles, melanophore responses were assayed in terms of pigment dispersion and the phosphorylation state of a 53 kDa melanophore-specific protein. In the same melanophore system α-MSH has been shown to specifically increase the phosphorylation of this 53 kDa protein.

Forskolin induces a dose-dependent pigment dispersion (EC₅₀ 7 × 10^?7 M). In contrast to the dispersion induced by α-MSH forskolin-induced dispersion does not require extracellular Ca²⁺. Moreover, in a Ca²⁺-free medium melanophores with permanently activated MSH-receptors aggregate, but can be redispersed by the addition of forskolin. Forskolin increases 53 kDa phosphorylation in a dosedependent manner. Maximal stimulation with forskolin (10^?5 M) is four-fold and equals maximal 53 kDa phosphorylation obtainable with α-MSH. The MSH-induced increase in 53 kDa phosphorylation is inhibited by Ca²⁺ deprivation, whereas the forskolin-induced increase is unaffected. Our results suggest that α-MSH and forskolin stimulate melanophores through a common pathway and confirm that cAMP is a second messenger in α-MSH action in this system. We conclude that the Ca²⁺ sites in the mechanism of α-MSH action on melanophores precede adenylate cyclase activation.     

Serotoninergic activity stimulates melanin dispersion within dermal melanophores of newts

Systemic injections of 5-hydroxytryptophan (5-HTP), the precursor to serotonin, stimulates melanin dispersion within dermal melanophores of red-spotted newts (Notophthalmus viridescens). Injections of para-Chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, inhibited melanin dispersion, and hence darkening of the skin, when newts were transferred to a dark background. The results indicate a role for serotoninergic activity in the background adaptation response in this amphibian.     

Efficient, long-term transgene expression in Xenopus laevis dermal melanophores

Xenopus laevis dermal melanophores provide an excellent model system for the investigation of complex cellular processes. Specifically, the expression of exogenous genes in Xenopus melanophores is the basis of recombinant bioassays for the study of receptor-ligand interactions. However, due to their slow rate of cell division and to the relatively low efficiency of current transfection protocols, long-term expression of exogenous genes and the generation of stable melanophore cell lines remains problematic. In this report we demonstrate the efficient, long-term expression of two exogenous proteins, the enhanced green fluorescent protein (EGFP) and the human CD4 (hCD4) cell surface receptor, following stable introduction into Xenopus melanophores via an HIV-1 based vector. Transduction of melanophores with the EGFP expression vector resulted in up to 80% EGFP+ cells. After 1 year in continuous culture in the absence of antibiotic selection, more than 60% of the cells remained EGFP+. Furthermore, we demonstrate the expression of hCD4 melanophores for over 9 months in continuous culture in the absence of antibiotic selection. Our results indicate that lentivirus vectors provide an efficient means of introducing genetic information into Xenopus melanophores, resulting in sustained levels of gene expression. The significance of this gene transfer system for the study of cellular signal transduction pathways is discussed.     

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.     

The primate median eminence

A pressure device has been used to obtain information about the forces involved in the maintenance of the aggregated state of melanophores of the angelfish, Pterophyllum scalare. Single aggregated melanophores of isolated scales were submitted to mechanical compression with forces ranging from 50-320 mup. As a function of the gradually increasing force melanophores disperse their pigment, the degree of dispersion being proportional to the intensity of the force. When microtubules are destroyed by treatment with 0.3 mM vinblastine in KCl solution, pigment dispersion in response to the external force is similar to that observed in KCl alone. After changing the medium to NaCl solution, melanin granules remain concentrated in the cell center; the force-induced melanosome dispersion, however, is significantly enhanced. Distinctly lower forces are required to produce an expansion equivalent to that observed in KCl solution. When the medium is changed to vinblastine-KCl again, the dispersion in response to the external force resembles that obtained before NaCl treatment. Removal of Ca++ and Mg++ ions by treatment with 2 mM EDTA or EGTA in Ringer's solution containing 0.1 mM adrenalin produces a remarkable enhancement of melanosome dispersion in response to increasing external force. This effect of EDTA or EGTA is completely reversible. When the medium is changed to normal Ca++-Ringer's, the force/dispersion curve resembles that obtained before EDTA or EGTA treatment. It is concluded that a state of equilibrium exists between the external force and an opposing force produced by the melanophore. The differences in the opposing force under different experimental conditions may be due to a "contractile component". This component seems to be independent of microtubules, as indicated by vinblastine experiments. It "contracts" under aggregating stimulus and "relaxes" under dispersing stimulus. From the data presented in this paper, the order of magnitude of the pressure developed by the contractile component in the completely aggregated state was calculated as between 5-7 p/cm2 in the relaxed state and about 20 p/cm2 during contraction. These values are comparable to those observed in other nonmuscular cells.     

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.     

beta-Adrenergic receptor subtypes in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus.

The subtype of beta-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 microM phentolamine and 30-100 microM verapamil or 2-10 nM melatonin, and the responses of the melanophores to a beta-adrenergic agonist added to the incubating medium were recorded photoelectrically. The beta-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (beta 1-selective agonist) was inhibited by metoprolol (beta 1-selective antagonist), propranolol,- and butoxamine. Whereas, the effect of salbutamol (beta 2-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that beta 1- and beta 2-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the beta-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a beta-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of beta 2-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of beta 1-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.     

Maxadilan Activates PAC1 Receptors Expressed in Xenopus laevis Melanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long‐term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non‐transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

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.     

Biosensing of opioids using frog melanophores

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.     

Comparison of structural requirements of alpha-MSH and ACTH for inducing excessive grooming and pigment dispersion

alpha-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat. With the exception of ACTH1-24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle4,-D-Phe7]-alpha-MSH is extremely active in both assays. Weak agonists such as [Leu9]-alpha-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of alpha-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.