Generation of Electric Potential Difference by Chromatophores from Photosynthetic Bacteria in the Presence of Trehalose under Continuous Illumination.

Measurement of electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides associated with the surface of a nitrocellulose membrane filter (MF) impregnated with a phospholipid solution in decane or immersed into it in the presence of exogenous mediators and disaccharide trehalose demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The mediators were the ascorbate/N,N,N'N'-tetramethyl-p-phenylenediamine pair and ubiquinone-0 (electron donor and acceptor, respectively). Although stabilization of photoelectric responses upon long-term continuous illumination was observed for both variants of chromatophore immobilization, only the samples immersed into the MF retained the functional activity of reaction centers (RCs) for a month when stored in the dark at room temperature, which might be due to the preservation of integrity of chromatophore proteins inside the MF pores. The stabilizing effect of the bioprotector trehalose could be related to its effect on both the RC proteins and the phospholipid bilayer membrane. The results obtained will expand current ideas on the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into its electrochemical form.

DNA-binding and protein structure of nuclear factors likely acting in genetic information processing in the Paulinella chromatophore.

The chromatophores in Paulinella are evolutionary-early-stage photosynthetic organelles. Biological processes in chromatophores depend on a combination of chromatophore and nucleus-encoded proteins. Interestingly, besides proteins carrying chromatophore-targeting signals, a large arsenal of short chromatophore-targeted proteins (sCTPs; <90 amino acids) without recognizable targeting signals were found in chromatophores. This situation resembles endosymbionts in plants and insects that are manipulated by host-derived antimicrobial peptides. Previously, we identified an expanded family of sCTPs of unknown function, named here "DNA-binding (DB)-sCTPs". DB-sCTPs contain a ~45 amino acid motif that is conserved in some bacterial proteins with predicted functions in DNA processing. Here, we explored antimicrobial activity, DNA-binding capacity, and structures of three purified recombinant DB-sCTPs. All three proteins exhibited antimicrobial activity against bacteria involving membrane permeabilization, and bound to bacterial lipids in vitro. A combination of in vitro assays demonstrated binding of recombinant DB-sCTPs to chromatophore-derived genomic DNA sequences with an affinity in the low nM range. Additionally, we report the 1.2 Å crystal structure of one DB-sCTP. In silico docking studies suggest that helix α2 inserts into the DNA major grove and the exposed residues, that are highly variable between different DB-sCTPs, confer interaction with the DNA bases. Identification of photosystem II subunit CP43 as a potential interaction partner of one DB-sCTP, suggests DB-sCTPs to be involved in more complex regulatory mechanisms. We hypothesize that membrane binding of DB-sCTPs is related to their import into chromatophores. Once inside, they interact with the chromatophore genome potentially providing nuclear control over genetic information processing.

Mechanisms of alkali pH-shifted colour changes in squid (Uroteuthis edulis) subjected to frozen storage.

The skin discoloration of squid subjected to frozen storage negatively affects market price. In this study, various alkali treatments were investigated for effects on red granules and yellow pigments of squid skin and corresponding mechanisms were investigated at the tissue, cellular and molecular level. A significant colour improvement was observed when subjected to a pH 12 treatment, supported by decreased Δb* and increased Δa* values. Neither lower nor harsher alkali treatments than pH 12 can not obtain such results. HE staining and the UV-vis spectrum suggest that the improved red colour in skin was ascribed to the release of red pigment granules from damaged chromatophores by alkaline treatment and the release of red pigments in alkaline aqueous solutions from granules. However, based on TEM and particle size analysis, an excessive alkali treatment of pH 13 would degrade granules into smaller particles. The degradation of yellowness pigments indicated high sensitivity to alkali environments according to HPLC results. This study provides a valuable reference for improving the colour appearance of squid skin subjected to frozen storage.

Effect of Dipyridamole on Membrane Energization and Energy Transfer in Chromatophores of Rba. sphaeroides.

Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of photosynthetic electron transport chain in the bacterial chromatophores of Rba. sphaeroides was investigated. DIP was found to affect efficiency of energy transfer from the light-harvesting complex LH2 to the LH1-reaction center core complex and to produce the long-wavelength ("red") shift of the absorption band of light-harvesting bacteriochlorophyll molecules in the IR spectral region at 840-900 nm. This shift is associated with the membrane transition to the energized state. It was shown that DIP is able to reduce the photooxidized bacteriochlorophyll of the reaction center, which accelerated electron flow along the electron transport chain, thereby stimulating generation of the transmembrane potential on the chromatophore membrane. The results are important for clarifying possible mechanisms of DIP influence on the activity of membrane-bound functional proteins. In particular, they might be significant for interpreting numerous therapeutic effects of DIP.

Disproportionate effect of cationic antiseptics on the quantum yield and fluorescence lifetime of bacteriochlorophyll molecules in the LH1-RC complex of R. rubrum chromatophores.

Photosynthetic membrane complexes of purple bacteria are convenient and informative macromolecular systems for studying the mechanisms of action of various physicochemical factors on the functioning of catalytic proteins both in an isolated state and as part of functional membranes. In this work, we studied the effect of cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine) on the fluorescence intensity and the efficiency of energy transfer from the light-harvesting LH1 complex to the reaction center (RC) of Rhodospirillum rubrum chromatophores. The effect of antiseptics on the fluorescence intensity and the energy transfer increased in the following order: chlorhexidine, picloxydine, miramistin, octenidine. The most pronounced changes in the intensity and lifetime of fluorescence were observed with the addition of miramistin and octenidine. At the same concentration of antiseptics, the increase in fluorescence intensity was 2-3 times higher than the increase in its lifetime. It is concluded that the addition of antiseptics decreases the efficiency of the energy migration LH1 → RC and increases the fluorescence rate constant kfl. We associate the latter with a change in the polarization of the microenvironment of bacteriochlorophyll molecules upon the addition of charged antiseptic molecules. A possible mechanism of antiseptic action on R. rubrum chromatophores is considered. This work is a continuation of the study of the effect of antiseptics on the energy transfer and fluorescence intensity in chromatophores of purple bacteria published earlier in Photosynthesis Research (Strakhovskaya et al. in Photosyn Res 147:197-209, 2021).

A bipartite chromatophore transit peptide and N-terminal protein processing in the Paulinella chromatophore.

The amoeba Paulinella chromatophora contains photosynthetic organelles, termed chromatophores, which evolved independently from plastids in plants and algae. At least one-third of the chromatophore proteome consists of nucleus-encoded (NE) proteins that are imported across the chromatophore double envelope membranes. Chromatophore-targeted proteins exceeding 250 amino acids (aa) carry a conserved N-terminal extension presumably involved in protein targeting, termed the chromatophore transit peptide (crTP). Short imported proteins do not carry discernable targeting signals. To explore whether the import of proteins is accompanied by their N-terminal processing, here we identified N-termini of 208 chromatophore-localized proteins by a mass spectrometry-based approach. Our study revealed extensive N-terminal acetylation and proteolytic processing in both NE and chromatophore-encoded (CE) fractions of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Surprisingly, only the N-terminal ∼50 aa (part 1) become cleaved upon import. This part contains a conserved adaptor protein-1 complex-binding motif known to mediate protein sorting at the trans-Golgi network followed by a predicted transmembrane helix, implying that part 1 anchors the protein co-translationally in the endoplasmic reticulum and mediates trafficking to the chromatophore via the Golgi. The C-terminal part 2 contains conserved secondary structural elements, remains attached to the mature proteins, and might mediate translocation across the chromatophore inner membrane. Short imported proteins remain largely unprocessed. Finally, this work illuminates N-terminal processing of proteins encoded in an evolutionary-early-stage organelle and suggests host-derived posttranslationally acting factors involved in regulation of the CE chromatophore proteome.

Epigenetic dynamics shaping melanophore and iridophore cell fate in zebrafish.

BACKGROUND: Zebrafish pigment cell differentiation provides an attractive model for studying cell fate progression as a neural crest progenitor engenders diverse cell types, including two morphologically distinct pigment cells: black melanophores and reflective iridophores. Nontrivial classical genetic and transcriptomic approaches have revealed essential molecular mechanisms and gene regulatory circuits that drive neural crest-derived cell fate decisions. However, how the epigenetic landscape contributes to pigment cell differentiation, especially in the context of iridophore cell fate, is poorly understood. RESULTS: We chart the global changes in the epigenetic landscape, including DNA methylation and chromatin accessibility, during neural crest differentiation into melanophores and iridophores to identify epigenetic determinants shaping cell type-specific gene expression. Motif enrichment in the epigenetically dynamic regions reveals putative transcription factors that might be responsible for driving pigment cell identity. Through this effort, in the relatively uncharacterized iridophores, we validate alx4a as a necessary and sufficient transcription factor for iridophore differentiation and present evidence on alx4a's potential regulatory role in guanine synthesis pathway. CONCLUSIONS: Pigment cell fate is marked by substantial DNA demethylation events coupled with dynamic chromatin accessibility to potentiate gene regulation through cis-regulatory control. Here, we provide a multi-omic resource for neural crest differentiation into melanophores and iridophores. This work led to the discovery and validation of iridophore-specific alx4a transcription factor.

Transcriptomic Analysis of Skin Color in Anole Lizards.

Color and color pattern are critical for animal camouflage, reproduction, and defense. Few studies, however, have attempted to identify candidate genes for color and color pattern in squamate reptiles, a colorful group with over 10,000 species. We used comparative transcriptomic analyses between white, orange, and yellow skin in a color-polymorphic species of anole lizard to 1) identify candidate color and color-pattern genes in squamates and 2) assess if squamates share an underlying genetic basis for color and color pattern variation with other vertebrates. Squamates have three types of chromatophores that determine color pattern: guanine-filled iridophores, carotenoid- or pteridine-filled xanthophores/erythrophores, and melanin-filled melanophores. We identified 13 best candidate squamate color and color-pattern genes shared with other vertebrates: six genes linked to pigment synthesis pathways, and seven genes linked to chromatophore development and maintenance. In comparisons of expression profiles between pigment-rich and white skin, pigment-rich skin upregulated the pteridine pathway as well as xanthophore/erythrophore development and maintenance genes; in comparisons between orange and yellow skin, orange skin upregulated the pteridine and carotenoid pathways as well as melanophore maintenance genes. Our results corroborate the predictions that squamates can produce similar colors using distinct color-reflecting molecules, and that both color and color-pattern genes are likely conserved across vertebrates. Furthermore, this study provides a concise list of candidate genes for future functional verification, representing a first step in determining the genetic basis of color and color pattern in anoles.

Generation of no-yellow-pigment Xenopus tropicalis by slc2a7 gene knockout.

BACKGROUND: Amphibians possess three kinds of dermal chromatophore: melanophores, iridophores, and xanthophores. Knockout Xenopus tropicalis that lack the pigmentation of melanophores and iridophores have been reported. The identification of the causal genes for xanthophore pigmentation or differentiation could lead to the creation of a see-through frog without three chromatophores. The genes causing xanthophore differentiation mutants are slc2a11b and slc2a15b in Japanese medaka (Oryzias latipes). RESULTS: To obtain a heritable line of X tropicalis mutants without yellow pigment, we generated slc2a7 and slc2a15a knockout animals because they have the greatest similarity to the O latipes slc2a11b and slc2a15b genes. The slc2a7 knockout frog had a bluish skin and there were no visible yellow pigments in stereo microscope and skin section observations. Furthermore, no pterinosomes, which are characteristic of xanthophores, were observed via transmission electron microscopy in the skin of knockout animals. CONCLUSIONS: We report the successful generation of a heritable no-yellow-pigment X tropicalis mutant after knock out of the slc2a7 gene. This finding will enable the creation of a see-through frog with no chromatophores.

Molecular parallelisms between pigmentation in the avian iris and the integument of ectothermic vertebrates.

Birds exhibit striking variation in eye color that arises from interactions between specialized pigment cells named chromatophores. The types of chromatophores present in the avian iris are lacking from the integument of birds or mammals, but are remarkably similar to those found in the skin of ectothermic vertebrates. To investigate molecular mechanisms associated with eye coloration in birds, we took advantage of a Mendelian mutation found in domestic pigeons that alters the deposition of yellow pterin pigments in the iris. Using a combination of genome-wide association analysis and linkage information in pedigrees, we mapped variation in eye coloration in pigeons to a small genomic region of ~8.5kb. This interval contained a single gene, SLC2A11B, which has been previously implicated in skin pigmentation and chromatophore differentiation in fish. Loss of yellow pigmentation is likely caused by a point mutation that introduces a premature STOP codon and leads to lower expression of SLC2A11B through nonsense-mediated mRNA decay. There were no substantial changes in overall gene expression profiles between both iris types as well as in genes directly associated with pterin metabolism and/or chromatophore differentiation. Our findings demonstrate that SLC2A11B is required for the expression of pterin-based pigmentation in the avian iris. They further highlight common molecular mechanisms underlying the production of coloration in the iris of birds and skin of ectothermic vertebrates.

Evolution of the potassium channel gene Kcnj13 underlies colour pattern diversification in Danio fish.

The genetic basis of morphological variation provides a major topic in evolutionary developmental biology. Fish of the genus Danio display colour patterns ranging from horizontal stripes, to vertical bars or spots. Stripe formation in zebrafish, Danio rerio, is a self-organizing process based on cell-contact mediated interactions between three types of chromatophores with a leading role of iridophores. Here we investigate genes known to regulate chromatophore interactions in zebrafish that might have evolved to produce a pattern of vertical bars in its sibling species, Danio aesculapii. Mutant D. aesculapii indicate a lower complexity in chromatophore interactions and a minor role of iridophores in patterning. Reciprocal hemizygosity tests identify the potassium channel gene obelix/Kcnj13 as evolved between the two species. Complementation tests suggest evolutionary change through divergence in Kcnj13 function in two additional Danio species. Thus, our results point towards repeated and independent evolution of this gene during colour pattern diversification.

Genome mapping of a LYST mutation in corn snakes indicates that vertebrate chromatophore vesicles are lysosome-related organelles.

Reptiles exhibit a spectacular diversity of skin colors and patterns brought about by the interactions among three chromatophore types: black melanophores with melanin-packed melanosomes, red and yellow xanthophores with pteridine- and/or carotenoid-containing vesicles, and iridophores filled with light-reflecting platelets generating structural colors. Whereas the melanosome, the only color-producing endosome in mammals and birds, has been documented as a lysosome-related organelle, the maturation paths of xanthosomes and iridosomes are unknown. Here, we first use 10x Genomics linked-reads and optical mapping to assemble and annotate a nearly chromosome-quality genome of the corn snake Pantherophis guttatus The assembly is 1.71 Gb long, with an N50 of 16.8 Mb and L50 of 24. Second, we perform mapping-by-sequencing analyses and identify a 3.9-Mb genomic interval where the lavender variant resides. The lavender color morph in corn snakes is characterized by gray, rather than red, blotches on a pink, instead of orange, background. Third, our sequencing analyses reveal a single nucleotide polymorphism introducing a premature stop codon in the lysosomal trafficking regulator gene (LYST) that shortens the corresponding protein by 603 amino acids and removes evolutionary-conserved domains. Fourth, we use light and transmission electron microscopy comparative analyses of wild type versus lavender corn snakes and show that the color-producing endosomes of all chromatophores are substantially affected in the LYST mutant. Our work provides evidence characterizing xanthosomes in xanthophores and iridosomes in iridophores as lysosome-related organelles.

Neural innervation as a potential trigger of morphological color change and sexual dimorphism in cichlid fish.

Many species change their coloration during ontogeny or even as adults. Color change hereby often serves as sexual or status signal. The cellular and subcellular changes that drive color change and how they are orchestrated have been barely understood, but a deeper knowledge of the underlying processes is important to our understanding of how such plastic changes develop and evolve. Here we studied the color change of the Malawi golden cichlid (Melanchromis auratus). Females and subordinate males of this species are yellow and white with two prominent black stripes (yellow morph; female and non-breeding male coloration), while dominant males change their color and completely invert this pattern with the yellow and white regions becoming black, and the black stripes becoming white to iridescent blue (dark morph; male breeding coloration). A comparison of the two morphs reveals that substantial changes across multiple levels of biological organization underlie this polyphenism. These include changes in pigment cell (chromatophore) number, intracellular dispersal of pigments, and tilting of reflective platelets (iridosomes) within iridophores. At the transcriptional level, we find differences in pigmentation gene expression between these two color morphs but, surprisingly, 80% of the genes overexpressed in the dark morph relate to neuronal processes including synapse formation. Nerve fiber staining confirms that scales of the dark morph are indeed innervated by 1.3 to 2 times more axonal fibers. Our results might suggest an instructive role of nervous innervation orchestrating the complex cellular and ultrastructural changes that drive the morphological color change of this cichlid species.

Being red, blue and green: the genetic basis of coloration differences in the strawberry poison frog (Oophaga pumilio).

BACKGROUND: Animal coloration is usually an adaptive attribute, under strong local selection pressures and often diversified among species or populations. The strawberry poison frog (Oophaga pumilio) shows an impressive array of color morphs across its distribution in Central America. Here we quantify gene expression and genetic variation to identify candidate genes involved in generating divergence in coloration between populations of red, green and blue O. pumilio from the Bocas del Toro archipelago in Panama. RESULTS: We generated a high quality non-redundant reference transcriptome by mapping the products of genome-guided and de novo transcriptome assemblies onto a re-scaffolded draft genome of O. pumilio. We then measured gene expression in individuals of the three color phenotypes and identified color-associated candidate genes by comparing differential expression results against a list of a priori gene sets for five different functional categories of coloration - pteridine synthesis, carotenoid synthesis, melanin synthesis, iridophore pathways (structural coloration), and chromatophore development. We found 68 candidate coloration loci with significant expression differences among the color phenotypes. Notable upregulated examples include pteridine synthesis genes spr, xdh and pts (in red and green frogs); carotenoid metabolism genes bco2 (in blue frogs), scarb1 (in red frogs), and guanine metabolism gene psat1 (in blue frogs). We detected significantly higher expression of the pteridine synthesis gene set in red and green frogs versus blue frogs. In addition to gene expression differences, we identified 370 outlier SNPs on 162 annotated genes showing signatures of diversifying selection, including eight pigmentation-associated genes. CONCLUSIONS: Gene expression in the skin of the three populations of frogs with differing coloration is highly divergent. The strong signal of differential expression in pteridine genes is consistent with a major role of these genes in generating the coloration differences among the three morphs. However, the finding of differentially expressed genes across pathways and functional categories suggests that multiple mechanisms are responsible for the coloration differences, likely involving both pigmentary and structural coloration. In addition to regulatory differences, we found potential evidence of differential selection acting at the protein sequence level in several color-associated loci, which could contribute to the color polymorphism.

Characterization and Biosynthesis of Lipids in Paulinella micropora MYN1: Evidence for Efficient Integration of Chromatophores into Cellular Lipid Metabolism.

The chromatophores found in the cells of photosynthetic Paulinella species, once believed to be endosymbiotic cyanobacteria, are photosynthetic organelles that are distinct from chloroplasts. The chromatophore genome is similar to the genomes of α-cyanobacteria and encodes about 1,000 genes. Therefore, the chromatophore is an intriguing model of organelle formation. In this study, we analyzed the lipids of Paulinella micropora MYN1 to verify that this organism is a composite of cyanobacterial descendants and a heterotrophic protist. We detected glycolipids and phospholipids, as well as a betaine lipid diacylglyceryl-3-O-carboxyhydroxymethylcholine, previously detected in many marine algae. Cholesterol was the only sterol component detected, suggesting that the host cell is similar to animal cells. The glycolipids, presumably present in the chromatophores, contained mainly C16 fatty acids, whereas other classes of lipids, presumably present in the other compartments, were abundant in C20 and C22 polyunsaturated fatty acids. This suggests that chromatophores are metabolically distinct from the rest of the cell. Metabolic studies using isotopically labeled substrates showed that different fatty acids are synthesized in the chromatophore and the cytosol, which is consistent with the presence of both type I and type II fatty acid synthases, supposedly present in the cytosol and the chromatophore, respectively. Nevertheless, rapid labeling of the fatty acids in triacylglycerol and phosphatidylcholine by photosynthetically fixed carbon suggested that the chromatophores efficiently provide metabolites to the host. The metabolic and ultrastructural evidence suggests that chromatophores are tightly integrated into the whole cellular metabolism.

Generation of a white-albino phenotype from cobalt blue and yellow-albino rainbow trout (Oncorhynchus mykiss): Inheritance pattern and chromatophores analysis.

Albinism is the most common color variation described in fish and is characterized by a white or yellow phenotype according to the species. In rainbow trout Oncorhynchus mykiss, aside from yellow-albino phenotypes, cobalt blue variants with autosomal, recessive inheritance have also been reported. In this study, we investigated the inheritance pattern and chromatophores distribution/abundance of cobalt blue trouts obtained from a local fish farm. Based on crosses with wild-type and dominant yellow-albino lines, we could infer that cobalt blue are dominant over wild-type and co-dominant in relation to yellow-albino phenotype, resulting in a fourth phenotype: the white-albino. Analysis of chromatophores revealed that cobalt blue trouts present melanophores, as the wild-type, and a reduced number of xanthophores. As regards to the white-albino phenotype, they were not only devoid of melanophores but also presented a reduced number of xanthophores. Cobalt blue and white-albino trouts also presented reduced body weight and a smaller pituitary gland compared to wild-type and yellow-albino phenotypes. The transcription levels of tshb and trh were up regulated in cobalt blue compared to wild type, suggesting the involvement of thyroid hormone in the expression of blue color. These phenotypes represent useful models for research on body pigmentation in salmonids and on the mechanisms behind endocrine control of color patterning.

Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish.

Actinopterygian fishes harbor at least eight distinct pigment cell types, leading to a fascinating diversity of colors. Among this diversity, the cellular origin of the white color appears to be linked to several pigment cell types such as iridophores or leucophores. We used the clownfish Amphiprion ocellaris, which has a color pattern consisting of white bars over a darker body, to characterize the pigment cells that underlie the white hue. We observe by electron microscopy that cells in white bars are similar to iridophores. In addition, the transcriptomic signature of clownfish white bars exhibits similarities with that of zebrafish iridophores. We further show by pharmacological treatments that these cells are necessary for the white color. Among the top differentially expressed genes in white skin, we identified several genes (fhl2a, fhl2b, saiyan, gpnmb, and apoD1a) and show that three of them are expressed in iridophores. Finally, we show by CRISPR/Cas9 mutagenesis that these genes are critical for iridophore development in zebrafish. Our analyses provide clues to the genomic underpinning of color diversity and allow identification of new iridophore genes in fish.

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.