A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest.

Multipotent neural crest (NC) progenitors generate an astonishing array of derivatives, including neuronal, skeletal components and pigment cells (chromatophores), but the molecular mechanisms allowing balanced selection of each fate remain unknown. In zebrafish, melanocytes, iridophores and xanthophores, the three chromatophore lineages, are thought to share progenitors and so lend themselves to investigating the complex gene regulatory networks (GRNs) underlying fate segregation of NC progenitors. Although the core GRN governing melanocyte specification has been previously established, those guiding iridophore and xanthophore development remain elusive. Here we focus on the iridophore GRN, where mutant phenotypes identify the transcription factors Sox10, Tfec and Mitfa and the receptor tyrosine kinase, Ltk, as key players. Here we present expression data, as well as loss and gain of function results, guiding the derivation of an initial iridophore specification GRN. Moreover, we use an iterative process of mathematical modelling, supplemented with a Monte Carlo screening algorithm suited to the qualitative nature of the experimental data, to allow for rigorous predictive exploration of the GRN dynamics. Predictions were experimentally evaluated and testable hypotheses were derived to construct an improved version of the GRN, which we showed produced outputs consistent with experimentally observed gene expression dynamics. Our study reveals multiple important regulatory features, notably a sox10-dependent positive feedback loop between tfec and ltk driving iridophore specification; the molecular basis of sox10 maintenance throughout iridophore development; and the cooperation between sox10 and tfec in driving expression of pnp4a, a key differentiation gene. We also assess a candidate repressor of mitfa, a melanocyte-specific target of sox10. Surprisingly, our data challenge the reported role of Foxd3, an established mitfa repressor, in iridophore regulation. Our study builds upon our previous systems biology approach, by incorporating physiologically-relevant parameter values and rigorous evaluation of parameter values within a qualitative data framework, to establish for the first time the core GRN guiding specification of the iridophore lineage.

The N-terminal domain of gastrulation brain homeobox 2 (Gbx2) is required for iridophore specification in zebrafish.

Although body color pattern formation by pigment cells plays critical roles in animals, pigment cell specification has not yet been fully elucidated. In zebrafish, there are three chromatophores: melanophore, iridophore, and xanthophore, that are derived from neural crest cells (NCCs). A recent study has reported the differentially expressed genes between melanophores and iridophores. Based on transcriptome data, we identified that Gbx2 is required for iridophore specification during development. In support of this, iridophore formation is suppressed by gbx2 knockdown by morpholino antisense oligonucleotide, at 72 h post fertilization (hpf) in zebrafish. Moreover, gbx2 is expressed in sox10-expressing NCCs and guanine crystal plates-containing iridophores during development at 24 and 48 hpf, respectively. In gbx2 knockdown zebrafish embryos, apoptosis of sox10-expressing NCCs was detected at 24 hpf without any effect on the formation of melanophores and xanthophores at 48 hpf. We further observed that the N-terminal domain of Gbx2 is able to rescue the iridophore formation defect caused by gbx2 knockdown. Our study provides insights into the requirement of N-terminal domain of Gbx2 for iridophore specification in zebrafish.

Development of Betta splendens embryos and larvae reveals variation in pigmentation patterns.

Vertebrate pigmentation provides an ideal system for studying the intersections between evolution, genetics, and developmental biology. Teleost fish, with their accessible developmental stages and intense and diverse colours produced by chromatophores, are an ideal group for study. We set out to test whether Betta splendens is a good model organism for studying the evolution and development of diverse pigmentation. Our results demonstrate that B. splendens can be bred to produce large numbers of offspring with easily visualized pigment cells. Depending on the colour of the parents, there was variation in larval pigmentation patterns both within and between breeding events. In juveniles the developing adult pigmentation patterns showed even greater variation. These results suggest that B. splendens has great potential as a model organism for pigmentation studies.

Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout, and first description of erythrophore ultrastructure in salmonids.

Skin pigmentation in animals is an important trait with many functions. The present study focused on two closely related salmonid species, marble trout (Salmo marmoratus) and brown trout (S. trutta), which display an uncommon labyrinthine (marble-like) and spot skin pattern, respectively. To determine the role of chromatophore type in the different formation of skin pigment patterns in the two species, the distribution and ultrastructure of chromatophores was examined with light microscopy and transmission electron microscopy. The presence of three types of chromatophores in trout skin was confirmed: melanophores; xanthophores; and iridophores. In addition, using correlative microscopy, erythrophore ultrastructure in salmonids was described for the first time. Two types of erythrophores are distinguished, both located exclusively in the skin of brown trout: type 1 in black spot skin sections similar to xanthophores; and type 2 with a unique ultrastructure, located only in red spot skin sections. Morphologically, the difference between the light and dark pigmentation of trout skin depends primarily on the position and density of melanophores, in the dark region covering other chromatophores, and in the light region with the iridophores and xanthophores usually exposed. With larger amounts of melanophores, absence of xanthophores and presence of erythrophores type 1 and type L iridophores in the black spot compared with the light regions and the presence of erythrophores type 2 in the red spot, a higher level of pigment cell organisation in the skin of brown trout compared with that of marble trout was demonstrated. Even though the skin regions with chromatophores were well defined, not all the chromatophores were in direct contact, either homophilically or heterophilically, with each other. In addition to short-range interactions, an important role of the cellular environment and long-range interactions between chromatophores in promoting adult pigment pattern formation of trout are proposed.

Expression of squid iridescence depends on environmental luminance and peripheral ganglion control.

Squid display impressive changes in body coloration that are afforded by two types of dynamic skin elements: structural iridophores (which produce iridescence) and pigmented chromatophores. Both color elements are neurally controlled, but nothing is known about the iridescence circuit, or the environmental cues, that elicit iridescence expression. To tackle this knowledge gap, we performed denervation, electrical stimulation and behavioral experiments using the long-fin squid, Doryteuthis pealeii. We show that while the pigmentary and iridescence circuits originate in the brain, they are wired differently in the periphery: (1) the iridescence signals are routed through a peripheral center called the stellate ganglion and (2) the iridescence motor neurons likely originate within this ganglion (as revealed by nerve fluorescence dye fills). Cutting the inputs to the stellate ganglion that descend from the brain shifts highly reflective iridophores into a transparent state. Taken together, these findings suggest that although brain commands are necessary for expression of iridescence, integration with peripheral information in the stellate ganglion could modulate the final output. We also demonstrate that squid change their iridescence brightness in response to environmental luminance; such changes are robust but slow (minutes to hours). The squid's ability to alter its iridescence levels may improve camouflage under different lighting intensities.

Ocular-side lateralization of adult-type chromatophore precursors: development of pigment asymmetry in metamorphosing flounder larvae.

The adult-type chromatophores of flounder differentiate at metamorphosis in the skin of ocular side to establish asymmetric pigmentation. In young larva and before metamorphosis, adult-type melanophores that migrate to the ocular side during metamorphosis reside at the base of the dorsal fin as latent precursors. However, the migration route taken by these precursor cells and the mechanisms by which lateralization and asymmetric pigmentation develop on the ocular side are unknown. To further investigate this migration and lateralization, we used in situ hybridization with gch2 probe, a marker for melanoblasts and xanthoblasts (precursors of adult type chromatophores), to examine the distribution of chromatophore precursors in metamorphosing larvae. The gch2-positive precursors were present in the myoseptum as well as in the skin. This finding indicated that these precursors migrated from the dorsal part of the fin to the skin via the myoseptum. Additionally, there were much fewer gch2-positive cells in the myoseptum of the blind side than in the skin and myoseptum of the ocular side, and this finding indicated either that migration of the precursor cells into the myoseptum of blind side was inhibited or that the precursors were eliminated from the myoseptum of the blind side. Therefore, we propose that the signals responsible for development of asymmetric pigmentation in flounder reside not only in the skin but on a larger scale and in multiple tissues throughout the lateral half of the trunk.

Genetic and correlative light and electron microscopy evidence for the unique differentiation pathway of erythrophores in brown trout skin.

Based on their cell ultrastructure, two types of erythrophores in the spotted skin regions of brown trout (Salmo trutta) were previously described. To test the hypothesis regarding the origin of a new cell type following genome duplication, we analysed the gene and paralogue gene expression patterns of erythrophores in brown trout skin. In addition, the ultrastructure of both erythrophore types was precisely examined using transmission electron microscopy (TEM) and correlative light microscopy and electron microscopy (CLEM). Ultrastructural differences between the sizes of erythrophore inclusions were confirmed; however, the overlapping inclusion sizes blur the distinction between erythrophore types, which we have instead defined as cell subtypes. Nevertheless, the red spots of brown trout skin with subtype 2 erythrophores, exhibited unique gene expression patterns. Many of the upregulated genes are involved in melanogenesis or xanthophore differentiation. In addition, sox10, related to progenitor cells, was also upregulated in the red spots. The expressions of paralogues derived from two genome duplication events were also analysed. Multiple paralogues were overexpressed in the red spots compared with other skin regions, suggesting that the duplicated gene copies adopted new functions and contributed to the origin of a new cell subtype that is characteristic for red spot. Possible mechanisms regarding erythrophore origin are proposed and discussed. To the best of our knowledge, this is the first study to evaluate pigment cell types in the black and red spots of brown trout skin using the advanced CLEM approach together with gene expression profiling.

Imitation of variable structural color in Paracheirodon innesi using colloidal crystal films.

Spacing variation of adjoining reflecting thin films in iridophore is responsible for the variable interference color in the paracheirodon innesi. On the basis of this phenomenon, colloidal crystal thin films with different structures are fabricated from monodisperse poly(styrene-methyl methacrylate-acrylic acid) (PSMA) colloids. The relationship between the colors and structures of the films is investigated and discussed according to the principle of light interference. A two-layer colloidal film having uniform color is researched and it displays diverse colors before and after swelling by styrene (St), which can be used to mimic the variable structural color of the paracheirodon innesi.

Principles underlying chromatophore addition during maturation in the European cuttlefish, Sepia officinalis.

The goal of this work was to identify some of the principles underlying chromatophore growth and development in the European cuttlefish, Sepia officinalis. One set of experiments used a regeneration model to follow the re-growth of black chromatophores for 30 days following excision of a small piece of fin tissue. A separate set of experiments tracked and analyzed the addition of new fin chromatophores during a month of normal, undisturbed growth. We also followed the development of individual chromatophores from their initial appearance to full maturation to determine whether their color type was fixed. Based on the results of these studies, we propose five guiding principles for chromatophore growth and maturation. (1) The three chromatophore cell types--black, reddish-brown and yellow--are present at different spatial frequencies in the cuttlefish fin. (2) During normal growth, new chromatophores are inserted at a higher spatial frequency than existing (control) chromatophores of the same color type. (3) In regenerating tissue, new black chromatophores are initially added at low spatial frequencies. As regeneration continues, new black chromatophores appear at increasing spatial frequencies until they are inserted at a spatial frequency higher than that observed in control tissue, similar to the way in which chromatophores were observed to be added in normally growing tissue. (4) All chromatophores first appear as pale orange cells and slowly darken into their respective color types without passing through intermediate color stages. (5) New black chromatophores undergo a doubling in size as they mature, while reddish-brown and yellow chromatophores do not grow at all after they are inserted in the dermis.

Highly Efficient Knockout of a Squid Pigmentation Gene.

Seminal studies using squid as a model led to breakthroughs in neurobiology. The squid giant axon and synapse, for example, laid the foundation for our current understanding of the action potential [1], ionic gradients across cells [2], voltage-dependent ion channels [3], molecular motors [4-7], and synaptic transmission [8-11]. Despite their anatomical advantages, the use of squid as a model receded over the past several decades as investigators turned to genetically tractable systems. Recently, however, two key advances have made it possible to develop techniques for the genetic manipulation of squid. The first is the CRISPR-Cas9 system for targeted gene disruption, a largely species-agnostic method [12, 13]. The second is the sequencing of genomes for several cephalopod species [14-16]. If made genetically tractable, squid and other cephalopods offer a wealth of biological novelties that could spur discovery. Within invertebrates, not only do they possess by far the largest brains, they also express the most sophisticated behaviors [17]. In this paper, we demonstrate efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9. Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by Tryptophan 2,3 Dioxygenase (TDO [18-20]). Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. This study represents a critical advancement toward making squid genetically tractable.

The dermal chromatophore unit.

Rapid color changes of amphibians are mediated by three types of dermal chromatophores, xanthophores, iridophores, and melanophores, which comprise a morphologically and physiologically distinct structure, the dermal chromatophore unit. Xanthophores, the outermost element, are located immediately below the basal lamella. Iridophores, containing light-reflecting organelles, are found just beneath the xanthophores. Under each iridophore is found a melanophore from which processes extend upward around the iridophore. Finger-like structures project from these processes and occupy fixed spaces between the xanthophores and iridophores. When a frog darkens, melanosomes move upward from the body of the melanophore to fill the fingers which then obscure the overlying iridophore. Rapid blanching is accomplished by the evacuation of melanosomes from these fingers. Pale coloration ranging from tan to green is provided by the overlying xanthophores and iridophores. Details of chromatophore structure are presented, and the nature of the intimate contact between the chromatophore types is discussed.

Morphological and biochemical characterization of goldfish erythrophores and their pterinosomes.

The fine structure of integumental erythrophores and the intracellular location of pteridine and carotenoid pigments in adult goldfish, Carassius auratus, were studied by means of cytochemistry, paper and thin-layer chromatography, ionophoresis, density-gradient centrifugal fractionation, and electron microscopy. The ultrastructure of erythrophores is characterized by large numbers of somewhat ellipsoidal pigment granules and a well-developed system of tubules which resembles endoplasmic reticulum. The combined morphological and biochemical approaches show that pteridine pigments of erythrophores are located characteristically in pigment granules and are the primary yellow pigments of these organelles. Accordingly, this organelle is considered to be the "pterinosome" which was originally found in swordtail erythrophores. Major pteridines obtainable from goldfish pterinosomes are sepiapterin, 7-hydroxybiopterin, isoxanthopterin, and 6-carboxyisoxanthopterin. Density-gradient fractions indicate that carotenoids are mostly associated with the endoplasmic reticulum. Both tyrosinase and possibly a tyrosinase inhibitor containing sulfhydryl groups are present in the pterinosome. The possible existence of a tyrosinase inhibitor is suggested by the marked increase of tyrosinase activity upon the addition of iodoacetamide or p-chloromercuribenzoic acid. In the light of their fine structure, pigmentary composition, and enzymatic properties, the erythrophores and pterinosomes are discussed with respect to their probable functions and their relationship to melanophores.

Vertebrate regeneration system: culture in vitro.

With standard tissue-culture techniques and media, various components of the lizard tail regenerate have been maintained in culture for 8 months. Differentiation of two cell types, melanophores and striated muscle, has been obtained. Myoblast proliferation and fusion can be selectively controlled by altering the culture medium.

Pax7 identifies neural crest, chromatophore lineages and pigment stem cells during zebrafish development.

Using immunostaining during early zebrafish embryogenesis, we report that the cranial and trunk neural crest expresses the paired box protein Pax7, thus revealing a novel neural crest marker in zebrafish. In the head, we show that Pax7 is broadly expressed in the cranial crest cells, which indicates that duplication of the paralogous group Pax3/7 at the origin of vertebrates included the conserved expression of Pax7 in the head neural crest of all of the vertebrates species studied so far. In the trunk, Pax7 recognizes both premigratory and migratory neural crest cells. Notably, we observed the expression of Pax7 during the development of melanophore, xanthophore and iridophore precursor cells. In contrast to the case of melanocyte precursors in birds, Pax7 showed overlapping expression with early melanin pigment. Finally, during the larva to adult transition, we show that pigment stem cells recapitulate the expression of Pax7.

Signal transduction pathway modeling using sequences of chromatophore images.

Extensive research is being done in order to use chromatophore cells as biosensors for various substances. In this paper, a link between the biological aspect of chromatophores and digital image/video processing techniques used for chromatophore characterization is established for this purpose. A model of the Gs--AC--PKA--granule motion-image feature signal transduction pathway is proposed, starting from the concentration of the input ligand and ending in the pigment area extracted from a microscope image. The model extends an existing system biology differential equation based model of the Gs--AC--PKA transduction pathway obtained from the Database of Quantitative Cellular Signaling (DQCS). Examples are presented to demonstrate the effectiveness of the proposed system model.

Artificial UV-B induced changes in pigmentation of marine diatom Coscinodiscus gigas.

In vitro studies in marine diatom Coscinodiscus gigas revealed that artificial UV-B radiation (313 nm) at a dose level of 0.4W m(-2) for a continuous period of 3 hours in a UV treatment chamber caused disbursement of chromatophores from their normal loci and resulted in clumping / aggregation of chromatophores exhibiting a phenomenon called UV-B induced syntrophism. It is also understood that such clumping could cause only insignificant reduction in photosynthetic oxygen release.

In vivo imaging of coral tissue and skeleton with optical coherence tomography.

Application of optical coherence tomography (OCT) for in vivo imaging of tissue and skeleton structure of intact living corals enabled the non-invasive visualization of coral tissue layers (endoderm versus ectoderm), skeletal cavities and special structures such as mesenterial filaments and mucus release from intact living corals. Coral host chromatophores containing green fluorescent protein-like pigment granules appeared hyper-reflective to near-infrared radiation allowing for excellent optical contrast in OCT and a rapid characterization of chromatophore size, distribution and abundance. In vivo tissue plasticity could be quantified by the linear contraction velocity of coral tissues upon illumination resulting in dynamic changes in the live coral tissue surface area, which varied by a factor of 2 between the contracted and expanded state of a coral. Our study provides a novel view on the in vivo organization of coral tissue and skeleton and highlights the importance of microstructural dynamics for coral ecophysiology.

Homotypic cell competition regulates proliferation and tiling of zebrafish pigment cells during colour pattern formation.

The adult striped pattern of zebrafish is composed of melanophores, iridophores and xanthophores arranged in superimposed layers in the skin. Previous studies have revealed that the assembly of pigment cells into stripes involves heterotypic interactions between all three chromatophore types. Here we investigate the role of homotypic interactions between cells of the same chromatophore type. Introduction of labelled progenitors into mutants lacking the corresponding cell type allowed us to define the impact of competitive interactions via long-term in vivo imaging. In the absence of endogenous cells, transplanted iridophores and xanthophores show an increased rate of proliferation and spread as a coherent net into vacant space. By contrast, melanophores have a limited capacity to spread in the skin even in the absence of competing endogenous cells. Our study reveals a key role for homotypic competitive interactions in determining number, direction of migration and individual spacing of cells within chromatophore populations.

Paulinella longichromatophora sp. nov., a New Marine Photosynthetic Testate Amoeba Containing a Chromatophore.

The freshwater testate filose amoeba Paulinella chromatophora is the sole species in the genus to have plastids, usually termed "chromatophores", of a Synechococcus/Prochlorococcus-like cyanobacterial origin. Here, we report a new marine phototrophic species, Paulinella longichromatophora sp. nov., using light and electron microscopy and molecular data. This new species contains two blue-green U-shaped chromatophores reaching up to 40 µm in total length. Further, the new Paulinella species is characterized by having five oral scales surrounding the pseudostomal aperture. All trees generated using three nuclear rDNA datasets (18S rDNA, 28S rDNA, and the concatenated 18S + 28S rDNA) demonstrated that three photosynthetic Paulinella species (two freshwater species, P. chromatophora and Paulinella strain FK01, and one marine species, P. longichromatophora) congruently formed a monophyletic group with strong support (≥ 90% of ML and ≥ 0.90 of PP), but their relationship to each other within the clade remained unresolved in all trees. P. longichromatophora, nevertheless, clustered consistently together with Paulinella strain FK01 with very low support, but the clade received strong support in plastid phylogenies. Phylogenetic analyses inferred from plastid-encoded 16S rDNA and a concatenated dataset of plastid 16S+23S rDNA demonstrated that chromatophores of all photosynthetic Paulinella species were monophyletic. The monophyletic group fell within a cyanobacteria clade having a close relationship to an α-cyanobacterial clade containing Prochlorococcus and Synechococcus species with very robust support (100% of ML and 1.0 of PP). Additionally, phylogenetic analyses of nuclear 18S rDNA and plastid 16S rDNA suggested divergent evolution within the photosynthetic Paulinella population after a single acquisition of the chromatophore. After the single acquisition of the chromatophore, ancestral photosynthetic Paulinella appears to have diverged into at least two distinct clades, one containing the marine P. longichromatophora and freshwater Paulinella strain FK01, the other P. chromatophora CCAC 0185.

Zebrafish Leucocyte tyrosine kinase controls iridophore establishment, proliferation and survival.

The zebrafish striped pattern results from the interplay among three pigment cell types; black melanophores, yellow xanthophores and silvery iridophores, making it a valuable model to study pattern formation in vivo. It has been suggested that iridophore proliferation, dispersal and cell shape transitions play an important role during stripe formation; however, the underlying molecular mechanisms remain poorly understood. Using gain- and loss-of-function alleles of leucocyte tyrosine kinase (ltk) and a pharmacological inhibitor approach, we show that Ltk specifically regulates iridophore establishment, proliferation and survival. Mutants in shady/ltk lack iridophores and display an abnormal body stripe pattern. Moonstone mutants, ltk(mne) , display ectopic iridophores, suggesting hyperactivity of the mutant Ltk. The dominant ltk(mne) allele carries a missense mutation in a conserved position of the kinase domain that highly correlates with neuroblastomas in mammals. Chimeric analysis suggests a novel physiological role of Ltk in the regulation of iridophore proliferation by homotypic competition.