kcnj13 regulates pigment cell shapes in zebrafish and has diverged by cis-regulatory evolution between Danio species.

Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the divergence of the potassium channel gene kcnj13. Here, we show that kcnj13 is required only in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast in D. rerio. Cis-regulatory rather than protein coding changes underlie kcnj13 divergence between the two Danio species. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification.

Effects of monochromatic lights on the melanophores arrangement in the spotted snakehead fish Channa punctata (Bloch, 1793).

Some freshwater teleost fish have pigment cells whose arrangement and shape are affected by the environment. Natural light has a wide range of light intensity. Fish are sensitive to the background and exposed light colour. Fish body colour is a significant criterion in fixing its market value, whether it is ornamental or edible. By favourable light exposure, a culturist may get a good market value of fish on most ethical grounds. In this study, we recorded the changes in melanophore response with the changes in light colour on Channa punctata. Adult fish were treated with monochromatic lights (darkness, white, blue and red light) for 5 and 28 days. After treatment, their body colour and melanophore size, number, length and the number of dendrites were studied. The results showed a significant influence of monochromatic light on melanophore arrangement in fish skin. The data showed that blue light is appropriate for the overall species colour of photic C. punctata. Continuous black or white light caused severe damage to the fish's appearance.

Development and genetics of red coloration in the zebrafish relative Danio albolineatus.

Animal pigment patterns play important roles in behavior and, in many species, red coloration serves as an honest signal of individual quality in mate choice. Among Danio fishes, some species develop erythrophores, pigment cells that contain red ketocarotenoids, whereas other species, like zebrafish (D. rerio) only have yellow xanthophores. Here, we use pearl danio (D. albolineatus) to assess the developmental origin of erythrophores and their mechanisms of differentiation. We show that erythrophores in the fin of D. albolineatus share a common progenitor with xanthophores and maintain plasticity in cell fate even after differentiation. We further identify the predominant ketocarotenoids that confer red coloration to erythrophores and use reverse genetics to pinpoint genes required for the differentiation and maintenance of these cells. Our analyses are a first step toward defining the mechanisms underlying the development of erythrophore-mediated red coloration in Danio and reveal striking parallels with the mechanism of red coloration in birds.

Influence of survival, promotion, and growth on pattern formation in zebrafish skin.

The coloring of zebrafish skin is often used as a model system to study biological pattern formation. However, the small number and lack of movement of chromatophores defies traditional Turing-type pattern generating mechanisms. Recent models invoke discrete short-range competition and long-range promotion between different pigment cells as an alternative to a reaction-diffusion scheme. In this work, we propose a lattice-based "Survival model," which is inspired by recent experimental findings on the nature of long-range chromatophore interactions. The Survival model produces stationary patterns with diffuse stripes and undergoes a Turing instability. We also examine the effect that domain growth, ubiquitous in biological systems, has on the patterns in both the Survival model and an earlier "Promotion" model. In both cases, domain growth alone is capable of orienting Turing patterns above a threshold wavelength and can reorient the stripes in ablated cells, though the wavelength for which the patterns orient is much larger for the Survival model. While the Survival model is a simplified representation of the multifaceted interactions between pigment cells, it reveals complex organizational behavior and may help to guide future studies.

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.

Experimental manipulation of perceived predation risk and cortisol generates contrasting trait trajectories in plastic crucian carp.

Most animals constitute potential prey and must respond appropriately to predator-mediated stress in order to survive. Numerous prey also adaptively tailor their response to the prevailing level of risk and stress imposed by their natural enemies, i.e. they adopt an inducible defence strategy. Predator exposure may activate the stress axis, and drive the expression of anti-predator traits that facilitate survival in a high-risk environment (the predation-stress hypothesis). Here, we quantified two key morphological anti-predator traits, body morphology and coloration, in crucian carp reared in the presence or absence of a predator (pike) in addition to experimental manipulation of physiological stress via implants containing either cortisol or a cortisol inhibitor. We found that predator-exposed fish expressed a deeper-bodied phenotype and darker body coloration as compared with non-exposed individuals. Skin analyses revealed that an increase in the amount of melanophores caused the dramatic colour change in predator-exposed fish. Increased melanization is costly, and the darker body coloration may act as an inducible defence against predation, via a conspicuous signal of the morphological defence or by crypsis towards dark environments and a nocturnal lifestyle. By contrast, the phenotype of individuals carrying cortisol implants did not mirror the phenotype of predator-exposed fish but instead exhibited opposite trajectories of trait change: a shallow-bodied morphology with a lighter body coloration as compared with sham-treated fish. The cortisol inhibitor did not influence the phenotype of fish i.e. neither body depth nor body coloration differed between this group and predator-exposed fish with a sham implant. However, our results illuminate a potential link between stress physiology and morphological defence expression.

The minimal gap-junction network among melanophores and xanthophores required for stripe pattern formation in zebrafish.

Connexin 39.4 (Cx39.4) and connexin 41.8 (Cx41.8), two gap-junction proteins expressed in both melanophores and xanthophores, are crucial for the intercellular communication among pigment cells that is necessary for generating the stripe pigment pattern of zebrafish. We have previously characterized the gap-junction properties of Cx39.4 and Cx41.8, but how these proteins contribute to stripe formation remains unclear; this is because distinct types of connexins potentially form heteromeric gap junctions, which precludes accurate elucidation of individual connexin functions in vivo Here, by arranging Cx39.4 and Cx41.8 expression in pigment cells, we have identified the simplest gap-junction network required for stripe generation: Cx39.4 expression in melanophores is required but expression in xanthophores is not necessary for stripe patterning, whereas Cx41.8 expression in xanthophores is sufficient for the patterning, and Cx41.8 expression in melanophores might stabilize the stripes. Moreover, patch-clamp recordings revealed that Cx39.4 gap junctions exhibit spermidine-dependent rectification property. Our results suggest that Cx39.4 facilitates the crucial cell-cell interactions between melanophores and xanthophores that mediate a unidirectional activation-signal transfer from xanthophores to melanophores, which is essential for melanophore survival.

Zebrafish Pigment Pattern Formation: Insights into the Development and Evolution of Adult Form.

Vertebrate pigment patterns are diverse and fascinating adult traits that allow animals to recognize conspecifics, attract mates, and avoid predators. Pigment patterns in fish are among the most amenable traits for studying the cellular basis of adult form, as the cells that produce diverse patterns are readily visible in the skin during development. The genetic basis of pigment pattern development has been most studied in the zebrafish, Danio rerio. Zebrafish adults have alternating dark and light horizontal stripes, resulting from the precise arrangement of three main classes of pigment cells: black melanophores, yellow xanthophores, and iridescent iridophores. The coordination of adult pigment cell lineage specification and differentiation with specific cellular interactions and morphogenetic behaviors is necessary for stripe development. Besides providing a nice example of pattern formation responsible for an adult trait of zebrafish, stripe-forming mechanisms also provide a conceptual framework for posing testable hypotheses about pattern diversification more broadly. Here, we summarize what is known about lineages and molecular interactions required for pattern formation in zebrafish, we review some of what is known about pattern diversification in Danio, and we speculate on how patterns in more distant teleosts may have evolved to produce a stunningly diverse array of patterns in nature.

Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores.

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including the zebrafish Danio rerio, we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.

Distant Insulin Signaling Regulates Vertebrate Pigmentation through the Sheddase Bace2.

Patterning of vertebrate melanophores is essential for mate selection and protection from UV-induced damage. Patterning can be influenced by circulating long-range factors, such as hormones, but it is unclear how their activity is controlled in recipient cells to prevent excesses in cell number and migration. The zebrafish wanderlust mutant harbors a mutation in the sheddase bace2 and exhibits hyperdendritic and hyperproliferative melanophores that localize to aberrant sites. We performed a chemical screen to identify suppressors of the wanderlust phenotype and found that inhibition of insulin/PI3Kγ/mTOR signaling rescues the defect. In normal physiology, Bace2 cleaves the insulin receptor, whereas its loss results in hyperactive insulin/PI3K/mTOR signaling. Insulin B, an isoform enriched in the head, drives the melanophore defect. These results suggest that insulin signaling is negatively regulated by melanophore-specific expression of a sheddase, highlighting how long-distance factors can be regulated in a cell-type-specific manner.

Flexibility of pigment cell behavior permits the robustness of skin pattern formation.

The striped pigmentation pattern of zebrafish is determined by the interaction between pigment cells with different colors. Recent studies show the behaviors of pigment cells are substantially different according to the environment. Interestingly, the resulting patterns are almost identical, suggesting a robustness of the patterning mechanism. To know how this robustness originates, we investigated the behavior of melanophores in various environments including different developmental stages, different body positions, and different genetic backgrounds. Normally, when embryonic melanophores are excluded from the yellow stripe region in the body trunk, two different cellular behaviors are observed. Melanophores migrate to join the black stripe or disappear (die) in the position. In environments where melanophore migration was restricted, we observed that most melanophores disappeared in their position, resulting in the complete exclusion of melanophores from the yellow stripe. In environments where melanophore cell death was restricted, most melanophores migrated to join the black stripes, also resulting in complete exclusion. When both migration and cell death were restricted, melanophores remained alive in the yellow stripes. These results show that migration and cell death complement each other to achieve the exclusion of melanophores. This flexibility may be the basis of the mechanistic robustness of skin pattern formation.

A macrophage relay for long-distance signaling during postembryonic tissue remodeling.

Macrophages have diverse functions in immunity as well as in development and homeostasis. We identified a function for these cells in long-distance communication during postembryonic tissue remodeling. Ablation of macrophages in zebrafish prevented melanophores from coalescing into adult pigment stripes. Melanophore organization depends on signals provided by cells of the yellow xanthophore lineage via airinemes, long filamentous projections with vesicles at their tips. We show that airineme extension from originating cells, as well as vesicle deposition on target cells, depend on interactions with macrophages. These findings identify a role for macrophages in relaying long-range signals between nonimmune cells. This signaling modality may function in the remodeling and homeostasis of other tissues during normal development and disease.

How fish color their skin: A paradigm for development and evolution of adult patterns: Multipotency, plasticity, and cell competition regulate proliferation and spreading of pigment cells in Zebrafish coloration.

Pigment cells in zebrafish - melanophores, iridophores, and xanthophores - originate from neural crest-derived stem cells associated with the dorsal root ganglia of the peripheral nervous system. Clonal analysis indicates that these progenitors remain multipotent and plastic beyond embryogenesis well into metamorphosis, when the adult color pattern develops. Pigment cells share a lineage with neuronal cells of the peripheral nervous system; progenitors propagate along the spinal nerves. The proliferation of pigment cells is regulated by competitive interactions among cells of the same type. An even spacing involves collective migration and contact inhibition of locomotion of the three cell types distributed in superimposed monolayers in the skin. This mode of coloring the skin is probably common to fish, whereas different patterns emerge by species specific cell interactions among the different pigment cell types. These interactions are mediated by channels involved in direct cell contact between the pigment cells, as well as unknown cues provided by the tissue environment.

Pigment Cell Progenitors in Zebrafish Remain Multipotent through Metamorphosis.

The neural crest is a transient, multipotent embryonic cell population in vertebrates giving rise to diverse cell types in adults via intermediate progenitors. The in vivo cell-fate potential and lineage segregation of these postembryonic progenitors is poorly understood, and it is unknown if and when the progenitors become fate restricted. We investigate the fate restriction in the neural crest-derived stem cells and intermediate progenitors in zebrafish, which give rise to three distinct adult pigment cell types: melanophores, iridophores, and xanthophores. By inducing clones in sox10-expressing cells, we trace and quantitatively compare the pigment cell progenitors at four stages, from embryogenesis to metamorphosis. At all stages, a large fraction of the progenitors are multipotent. These multipotent progenitors have a high proliferation ability, which diminishes with fate restriction. We suggest that multipotency of the nerve-associated progenitors lasting into metamorphosis may have facilitated the evolution of adult-specific traits in vertebrates.

Zebrafish Melanophores: A Model for Teaching Second Messenger Systems.

A strong literature base supports the notion that active learning improves retention in the science classroom. To that end, a course was designed to allow students to develop their own experiments around a central biological question. The model system used in this particular course is control of melanosome dispersal via second messenger systems in zebrafish (Danio rerio) scales. Students start by applying agonists and antagonists to the cAMP and Ca(2+) second messenger systems, and then can progress to more refined questions with the model system. This project is advantageous because it could be easily adapted to fit the needs of many different courses and ability levels; it is relatively easy to perform; it is enjoyable to teach; and students can be largely given a free reign to decide upon the design of their experiments.

Long-distance communication by specialized cellular projections during pigment pattern development and evolution.

Changes in gene activity are essential for evolutionary diversification. Yet, elucidating the cellular behaviors that underlie modifications to adult form remains a profound challenge. We use neural crest-derived adult pigmentation of zebrafish and pearl danio to uncover cellular bases for alternative pattern states. We show that stripes in zebrafish require a novel class of thin, fast cellular projection to promote Delta-Notch signaling over long distances from cells of the xanthophore lineage to melanophores. Projections depended on microfilaments and microtubules, exhibited meandering trajectories, and stabilized on target cells to which they delivered membraneous vesicles. By contrast, the uniformly patterned pearl danio lacked such projections, concomitant with Colony stimulating factor 1-dependent changes in xanthophore differentiation that likely curtail signaling available to melanophores. Our study reveals a novel mechanism of cellular communication, roles for differentiation state heterogeneity in pigment cell interactions, and an unanticipated morphogenetic behavior contributing to a striking difference in adult form.

Volumetric tracking of migratory melanophores during zebrafish development by optoacoustic microscopy.

Unveiling mechanisms driving specification, recruitment and regeneration of melanophores is key in understanding melanin-related disorders. This study reports on the applicability of a hybrid focus optoacoustic microscope (HFOAM) for volumetric tracking of migratory melanophores in developing zebrafish. The excellent contrast from highly-absorbing melanin provided by the method is shown to be ideal for label-free dynamic visualization of melanophores in their unperturbed environment. We established safe laser energy levels that enable high-contrast longitudinal tracking of the cells over an extended period of developmental time without causing cell toxicity or pigment bleaching. Owing to its hybrid optical and acoustic resolution, the new imaging technique can be seamlessly applied for noninvasive studies of both optically-transparent larval as well as adult stages of the zebrafish model organism, which is not possible using other optical microscopy methods.

Cargo transport at microtubule crossings: evidence for prolonged tug-of-war between kinesin motors.

Intracellular transport of cargos along microtubules is often complicated by the topology of the underlying filament network. The fundamental building blocks for this complex arrangement are filament intersections. The navigation of cargos across microtubule intersections remains poorly understood. Here, we demonstrate that kinesin-driven cargos are engaged in a tug-of-war at microtubule intersections. Tug-of-war events result in long pauses that can last from a few seconds to several minutes. We demonstrate that the extent of the tug-of-war and the duration of pauses change with the number of motors on the cargo and can be regulated by ionic strength. We also show that dwell times at intersections depend on the angle between crossing microtubules. Our data suggest that local microtubule geometry can regulate microtubule-based transport.

Rotating pigment cells exhibit an intrinsic chirality.

In multicellular organisms, cell properties, such as shape, size and function are important in morphogenesis and physiological functions. Recently, 'cellular chirality' has attracted attention as a cellular property because it can cause asymmetry in the bodies of animals. In recent in vitro studies, the left-right bias of cellular migration and of autonomous arrangement of cells under some specific culture conditions were discovered. However, it is difficult to identify the molecular mechanism underlying their intrinsic chirality because the left-right bias observed to date is subtle or is manifested in the stable orientation of cells. Here, we report that zebrafish (Danio rerio) melanophores exhibit clear cellular chirality by unidirectional counterclockwise rotational movement under isolated conditions without any special settings. The chirality is intrinsic to melanophores because the direction of the cellular rotation was not affected by the type of extracellular matrix. We further found that the cellular rotation was generated as a counter action of the clockwise movement of actin cytoskeleton. It suggested that the mechanism that directs actin cytoskeleton in the clockwise direction is pivotal for determining cellular chirality.

The sensitivity of Turing self-organization to biological feedback delays: 2D models of fish pigmentation.

Turing morphogen models have been extensively explored in the context of large-scale self-organization in multicellular biological systems. However, reconciling the detailed biology of morphogen dynamics, while accounting for time delays associated with gene expression, reveals aberrant behaviours that are not consistent with early developmental self-organization, especially the requirement for exquisite temporal control. Attempts to reconcile the interpretation of Turing's ideas with an increasing understanding of the mechanisms driving zebrafish pigmentation suggests that one should reconsider Turing's model in terms of pigment cells rather than morphogens (Nakamasu et al., 2009, PNAS, 106: , 8429-8434; Yamaguchi et al., 2007, PNAS, 104: , 4790-4793). Here the dynamics of pigment cells is subject to response delays implicit in the cell cycle and apoptosis. Hence we explore simulations of fish skin patterning, focussing on the dynamical influence of gene expression delays in morphogen-based Turing models and response delays for cell-based Turing models. We find that reconciling the mechanisms driving the behaviour of Turing systems with observations of fish skin patterning remains a fundamental challenge.