Structural and ultrastructural aspects of the skin of large yellow croaker Larimichthys crocea.

The skin color of the large yellow croaker (Larimichthys crocea) is a crucial indicator to determine its economic value. However, the location of pigment cells in the skin structure is uncertain. To determine the pigment cell type in the skin, the vertical order and ultrastructure of pigment cells were examined using light microscopy and transmission electron microscopy. Both dorsal and ventral skins comprise the epidermis, dermis, and hypodermis. Xanthophores, melanophores, and iridophores were observed in the dermis of the dorsal skin, whereas the latter two were in the dermis of the ventral skin. Interestingly, the size of xanthophores in the dorsal skin was significantly smaller than that of xanthophores in the ventral skin; however, the density of dorsal xanthophores was significantly higher than that of ventral xanthophores. The type L-iridophores with large crystalline structures were observed in the uppermost area of the upper pigment layer, which contributed to the strikingly metallic luster shown by the ventral skin. The melanophores were exclusively found in the dorsal skin, offering the purpose of camouflage. Taken together, our results indicated that the pigment cells display different arrangement patterns between dorsal and ventral skin, and the golden color in the ventral skin results from the coexistence of light-reflecting iridophores and light-absorbing xanthophores.

Pigmentation formation and expression analysis of tyrosinase in Siniperca chuatsi.

Animal pigmentation primarily depends on the presence and mixing ratio of chromatophores, functioning in animal survival and communication. For the benthic and carnivorous Siniperca chuatsi, pigmentation pattern is key to concealment and predation. In this study, the formation, distribution, and main pattern of chromatophores were observed in the embryos, larvae, skins, and visceral tissues from S. chuatsi. Melanophores were firstly visualized in the yolk sac at segmentation stage, and then they were migrated to the whole body and further clustered into the black stripes, bands, and patches. In adult S. chuatsi, the head, black band, and body side skins mainly contained melanophores, showing as deep or light black. The abdomen skin mainly contained iridophores, showing as silvery. In the eye, the pigment layers were located in the epithelial layers of iris and retina and shown as black. Then, the pigmentation-related gene, tyrosinase gene from S. chuatsi (Sc-tyr) was analyzed by bioinformatics and quantitative methods. The Sc-tyr gene encoded a protein with 540 amino acids (Sc-TYR). The Sc-TYR contained two copper ion binding sites, which were coordinated by six conserved histidines (H182, H205, H214, H366, H370, H393) and necessary for catalytic activity. The Sc-TYR was well conserved compared with TYR of various species with higher degree of sequence similarity with other fishes (77.6-98.3%). The qRT-PCR test showed that the Sc-tyr mRNA reached the peak value at segmentation stage in the embryo development, the black skins displayed a higher expression level than that in silvery skin, and the eye had the highest expression level compared with other tissues. Further research on enzyme activity showed that the expression patterns of tyrosinase activity were similar to that of the Sc-tyr mRNA. Comparing with the results of molecular and phenotype, it was found that the temporal and spatial distributions of tyrosinase corresponded well with changes in pigmentation patterns and the intensity of skin melanization. This study initially explored the pigmentation formation and tyrosinase expression, which served as a foundation for further insight into the genetics mechanism of body color formation in S. chuatsi.

Different morphologic formation patterns of dark patches in the black-spotted frog (Pelophylax nigromaculata) and the Asiatic toad (Bufo gargarizans).

The black-spotted frog (Pelophylax nigromaculata) and Asiatic toad (Bufo gargarizans), two relatively distantly related species, live in different habitats with different adaptive dark patches. To explain the formation of dark patches, the distribution patterns of melanin granules were examined with light microscopy and transmission electron microscopy. Melanin granules were produced and gathered into the "cap" structures on top of the nuclei in most epidermal cells. The "cap" structures may play a role in forming the dorsal dark patches coupled with three-layer melanophores, which can give rise to three layers of interconnected melanin networks in the dorsal dermis in P. nigromaculata. Epidermal melanocytes are rare and do not have a definitive role in forming dorsal dark patches in either P. nigromaculata or B. gargarizans. In B. gargarizans, the dermal melanophores only give rise to a single-layered melanin network, which hardly results in dark patches in the dorsal skin. However, the dermal melanophores migrate twice and form into pseudostratified networks, leading to dark patch formation in the ventral skin in B. gargarizans. The melanin granules precisely coregulate dark patches in the dermis and/or epidermis in P. nigromaculata and B. gargarizans. The dark patch formation depends on melanin granules in the epidermis or/and dermis in P. nigromaculata and B. gargarizans.

Multiple pigment cell types contribute to the black, blue, and orange ornaments of male guppies (Poecilia reticulata).

The fitness of male guppies (Poecilia reticulata) highly depends on the size and number of their black, blue, and orange ornaments. Recently, progress has been made regarding the genetic mechanisms underlying male guppy pigment pattern formation, but we still know little about the pigment cell organization within these ornaments. Here, we investigate the pigment cell distribution within the black, blue, and orange trunk spots and selected fin color patterns of guppy males from three genetically divergent strains using transmission electron microscopy. We identified three types of pigment cells and found that at least two of these contribute to each color trait. Further, two pigment cell layers, one in the dermis and the other in the hypodermis, contribute to each trunk spot. The pigment cell organization within the black and orange trunk spots was similar between strains. The presence of iridophores in each of the investigated color traits is consistent with a key role for this pigment cell type in guppy color pattern formation.

oca2 Regulation of chromatophore differentiation and number is cell type specific in zebrafish.

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410-kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2-deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar-type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore-specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post-fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.

Effects of Roundup and glyphosate formulations on intracellular transport, microtubules and actin filaments in Xenopus laevis melanophores.

Glyphosate containing herbicides, such as Roundup, are commonly used and generally considered to be safe. However, some toxic effects are found on amphibians in vivo and human and mouse cells in vitro. In this study the effects of Roundup, glyphosate, glyphosateisopropylamine and isopropylamine were studied on intracellular transport by measuring aggregation capacity in Xenopus laevis melanophores. The chemicals inhibited retrograde transport of melanosomes in the range of 0.5-5mM. Cellular morphology and localization of microtubules and actin filaments were affected as determined by immunocytochemistry. Both glyphosate and Roundup decreased pH in the media. Acidic pH inhibited melanosome transport and altered microtubule and actin morphology in the absence of chemicals, while transport inhibiting concentrations of glyphosate, Roundup and glyphosateisopropylamine disassembled both microtubules and actin filaments. At physiological pH the effects of Roundup decreased whereas glyphosate failed to inhibit transport. Physiological pH decreases glyphosate lipophilicity and its diffusion into the cytoplasm. The Roundup formulation contains surfactants, such as POEA (polyetylated tallow amine) that increases membrane permeability allowing cellular uptake at physiological pH. Our results show that the effects of glyphosate containing compounds are pH-dependent and that they inhibit intracellular transport through disassembly of the cytoskeleton possibly by interfering with intracellular Ca(2+)-balance.

The dynamic properties of intermediate filaments during organelle transport.

Intermediate filament (IF) dynamics during organelle transport and their role in organelle movement were studied using Xenopus laevis melanophores. In these cells, pigment granules (melanosomes) move along microtubules and microfilaments, toward and away from the cell periphery in response to alpha-melanocyte stimulating hormone (alpha-MSH) and melatonin, respectively. In this study we show that melanophores possess a complex network of vimentin IFs which interact with melanosomes. IFs form an intricate, honeycomb-like network that form cages surrounding individual and small clusters of melanosomes, both when they are aggregated and dispersed. Purified melanosome preparations contain a substantial amount of vimentin, suggesting that melanosomes bind to IFs. Analyses of individual melanosome movements in cells with disrupted IF networks show increased movement of granules in both anterograde and retrograde directions, further supporting the notion of a melanosome-IF interaction. Live imaging reveals that IFs, in turn, become highly flexible as melanosomes disperse in response to alpha-MSH. During the height of dispersion there is a marked increase in the rate of fluorescence recovery after photobleaching of GFP-vimentin IFs and an increase in vimentin solubility. These results reveal a dynamic interaction between membrane bound pigment granules and IFs and suggest a role for IFs as modulators of granule movement.

Organelle transport: dynamic actin tracks for myosin motors.

Transport of cargo by molecular motors on microtubule and actin filament tracks is a fundamental property of eukaryotic cells. A new study reports that actin dynamics are required in cells for myosin I and V motor proteins to transport their organelle cargos on actin tracks.

Addressing variability in a Xenopus laevis melanophore cell line.

Xenopus laevis melanophores can be used in high-throughput screens for guanine nucleotide binding protein coupled receptor ligands and have potential as biosensors. Inherent in this immortal cell line is a substantial variability, which macroscopic evaluations disregard. Here we demonstrate a systematic way to incorporate this natural variability in the evaluations. Clusters of similar cells from a sparsely seeded cell culture are examined by imaging changes in cell appearance, pigment motility, and cumulative displacements. The time evolution of the image intensity distributions of clusters upon a pigment-dispersing stimulus is used as a signature of the cell clusters, and their behaviors are classified by multivariate analysis. Conventional image subtraction procedures are used to highlight cumulative and transitory changes in the pigment dynamics, enabling characterization of multiple aspects of the cell response from a single experiment. Additionally, a simple way to accomplish standard optical density changes at the single-cell group level is shown. The present results also provide evidence that natural cell variability arising from a cell culture can enrich the diversity of responses from pigment-containing cells assays and underscore that in conventional macroscopic evaluations these aspects are overlooked and can lead to spurious results.

Multiple splice variants within the bovine silver homologue (SILV) gene affecting coat color in cattle indicate a function additional to fibril formation in melanophores.

BACKGROUND: The silver homologue(SILV) gene plays a major role in melanosome development. SILV is a target for studies concerning melanoma diagnostics and therapy in humans as well as on skin and coat color pigmentation in many species ranging from zebra fish to mammals. However, the precise functional cellular mechanisms, in which SILV is involved, are still not completely understood. While there are many studies addressing SILV function upon a eumelaneic pigment background, there is a substantial lack of information regarding the further relevance of SILV, e.g. for phaeomelanosome development. RESULTS: In contrast to previous results in other species reporting SILV expression exclusively in pigmented tissues, our experiments provide evidence that the bovine SILV gene is expressed in a variety of tissues independent of pigmentation. Our data show that the bovine SILV gene generates an unexpectedly large number of different transcripts occurring in skin as well as in non-pigmented tissues, e.g. liver or mammary gland. The alternative splice sites are generated by internal splicing and primarily remove complete exons. Alternative splicing predominantly affects the repeat domain of the protein, which has a functional key role in fibril formation during eumelanosome development. CONCLUSION: The expression of the bovine SILV gene independent of pigmentation suggests SILV functions exceeding melanosome development in cattle. This hypothesis is further supported by transcript variants lacking functional key elements of the SILV protein relevant for eumelanosome development. Thus, the bovine SILV gene can serve as a model for the investigation of the putative additional functions of SILV. Furthermore, the splice variants of the bovine SILV gene represent a comprehensive natural model to refine the knowledge about functional domains in the SILV protein. Our study exemplifies that the extent of alternative splicing is presumably much higher than previously estimated and that alternatively spliced transcripts presumably can generate molecules of deviating function compared to their constitutive counterpart.

Ultrastructure of the skin melanophores and iridophores in paddlefish, Polyodon spathula.

The ultrastructure of melanophores and iridophores of Polyodon spathula has been examined by transmission electron microscopy. In the skin, two types of chromatophores, melanophores and iridophores were founded. Melanophores were localized both in epidermis and dermis. Epidermal melanophores were present on the dorsal region of the trunk, sides, outer surface of the operculum and rostrum. Iridophores were founded in the dermis from ventral skin. The cytoplasm of iridophores is filled with reflecting platelets with variable orientation. The length of the long axis of the platelets varies from 1 to 2.10 microm.

Nonlocal mechanism of self-organization and centering of microtubule asters.

Fragments of fish melanophore cells can form and center aggregates of pigment granules by dynein-motor-driven transport along a self-organized radial array of microtubules (MTs). We present a quantitative model that describes pigment aggregation and MT-aster self-organization and the subsequent centering of both structures. The model is based on the observations that MTs are immobile and treadmill, while dynein-motor-covered granules have the ability to nucleate MTs. From assumptions based on experimental observations, we derive partial integro-differential equations describing the coupled granule-MT interaction. We use scaling arguments and perturbation theory to study the model in two limiting cases. The model analysis explains the mechanism of aster self-organization as a positive feedback loop between motor aggregation at the MT minus ends and MT nucleation by motors. Furthermore, the centering mechanism is explained by the spontaneous nucleation of MTs throughout the cytosol which acts as a volume sensing tool. Numerical simulations lend additional support to the analysis. The model sheds light on role of polymer dynamics and polymer-motor interactions in cytoskeletal organization.

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.

Identification of novel hexapeptide agonists at the Xenopus laevis melanophore melanocortin receptor.

We used a combinatorial chemical approach to identify novel agonists for the endogenous melanocortin receptor expressed in Xenopus laevis melanophores. A random one-bead one-compound hexapeptide library was screened to detect new molecules able to induce pigment dispersion in melanophores. Our approach led to the discovery of seven related novel peptides able to stimulate pigment dispersion with EC50 in the range of 0.1-10 microM. Their action was inhibited by the amphibian melanocortin receptor antagonist dWRL. These novel peptides share no significant sequence homology with known melanocortins. This study may aid in the understanding of the chemical interaction between the melanocortin receptors and their ligands.

Centering of a radial microtubule array by translocation along microtubules spontaneously nucleated in the cytoplasm.

Positioning of a radial array of microtubules (MTs) in the cell centre is crucial for cytoplasmic organization, but the mechanisms of such centering are difficult to study in intact cells that have pre-formed radial arrays. Here, we use cytoplasmic fragments of melanophores, and cytoplasts of BS-C-1 cells to study MT centering mechanisms. Using live imaging and computer modelling, we show that the MT aster finds a central location in the cytoplasm by moving along spontaneously nucleated non-astral MTs towards a point at which MT nucleation events occur equally on all sides. We hypothesize that similar mechanisms, in the presence of the centrosome, contribute to this centering mechanism and ensure the robustness of cytoplasmic organization.

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.

A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell.

The p24alpha, -beta, -gamma, and -delta proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24delta2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24delta2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing.

Height changes associated with pigment aggregation in Xenopus laevis melanophores.

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgCl2-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

Differential regulation of dynein-driven melanosome movement.

Cytoplasmic dyneins are multisubunit minus-end-directed microtubule motors. Different isoforms of dynein are thought to provide a means for independent movement of different organelles. We investigated the differential regulation of dynein-driven transport of pigment organelles (melanosomes) in Xenopus melanophores. Aggregation of melanosomes to the cell center does not change the localization of mitochondria, nor does dispersion of melanosomes cause a change in the perinuclear localization of the Golgi complex, indicating that melanosomes bear a dedicated form of dynein. We examined the subcellular fractionation behavior of dynein light intermediate chains (LIC) and identified at least three forms immunologically, only one of which fractionated with melanosomes. Melanosome aggregation was specifically blocked after injection of an antibody recognizing this LIC. Our data indicate that melanosome-associated dynein is regulated independently of bulk cytoplasmic dynein and involves a subfraction of dynein with a distinct subunit composition.

Enigmas of pterorhodin, a red melanosomal pigment of tree frogs.

Melanosomes observed in dermal melanophores of adult leaf frogs contain a unique wine red pigment identified as pterorhodin, a pteridine dimer never before found in any vertebrate. This type of melanosome, almost twice as large as the typical eumelanin melanosome, contains a small electron dense core of eumelanin surrounded by a concentric fibrous mass of pterorhodin. Dermal melanophores of larval leaf frogs contain small eumelanin melanosomes that transform at metamorphosis through the gradual accumulation of pterorhodin on the eumelanin surface to form the compound melanosomes of adults. This process may be mediated by thyroxine. No explanation for the unique presence of pterorhodin in leaf frogs has yet surfaced. A variety of tree frog species from Australia and Papua New Guinea also possess pterorhodin and the large melanosome suggesting that tree frogs from the New World and those from Australia are closely related and may have been separated during continental drift. Several of the unsolved problems posed by the emergence of pterorhodin in a unique melanosome are discussed.