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.

The dynamic behavior of chromatophores marks the transition from bands to spots in leopard geckos.

Reptilian skin coloration is spectacular and diverse, yet little is known about the ontogenetic processes that govern its establishment and the molecular signaling pathways that determine it. Here, we focus on the development of the banded pattern of leopard gecko hatchlings and the transition to black spots in the adult. With our histological analyses, we show that iridophores are present in the white and yellow bands of the hatchling and they gradually perish in the adult skin. Furthermore, we demonstrate that melanophores can autonomously form spots in the absence of the other chromatophores both on the regenerated skin of the tail and on the dorsal skin of the Mack Super Snow (MSS) leopard geckos. This color morph is characterized by uniform black coloration in hatchlings and black spots in adulthood; we establish that their skin is devoid of xanthophores and iridophores at both stages. Our genetic analyses identified a 13-nucleotide deletion in the PAX7 transcription factor of MSS geckos, affecting its protein coding sequence. With our single-cell transcriptomics analysis of embryonic skin, we confirm that PAX7 is expressed in iridophores and xanthophores, suggesting that it plays a key role in the differentiation of both chromatophores. Our in situ hybridizations on whole-mount embryos document the dynamics of the skin pattern formation and how it is impacted in the PAX7 mutants. We hypothesize that the melanophores-iridophores interactions give rise to the banded pattern of the hatchlings and black spot formation is an intrinsic capacity of melanophores in the postembryonic skin.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

Gain-of-function mutations in Aqp3a influence zebrafish pigment pattern formation through the tissue environment.

The development of the pigmentation pattern in zebrafish is a tightly regulated process that depends on both the self-organizing properties of pigment cells and extrinsic cues from other tissues. Many of the known mutations that alter the pattern act cell-autonomously in pigment cells, and our knowledge about external regulators is limited. Here, we describe novel zebrafish mau mutants, which encompass several dominant missense mutations in Aquaporin 3a (Aqp3a) that lead to broken stripes and short fins. A loss-of-function aqp3a allele, generated by CRISPR-Cas9, has no phenotypic consequences, demonstrating that Aqp3a is dispensable for normal development. Strikingly, the pigment cells from dominant mau mutants are capable of forming a wild-type pattern when developing in a wild-type environment, but the surrounding tissues in the mutants influence pigment cell behaviour and interfere with the patterning process. The mutated amino acid residues in the dominant alleles line the pore surface of Aqp3a and influence pore permeability. These results demonstrate an important effect of the tissue environment on pigment cell behaviour and, thereby, on pattern formation.

Impact of light intensity and quality on chromatophore and nuclear gene expression in Paulinella chromatophora, an amoeba with nascent photosynthetic organelles.

Plastid evolution has been attributed to a single primary endosymbiotic event that occurred about 1.6 billion years ago (BYA) in which a cyanobacterium was engulfed and retained by a eukaryotic cell, although early steps in plastid integration are poorly understood. The photosynthetic amoeba Paulinella chromatophora represents a unique model for the study of plastid evolution because it contains cyanobacterium-derived photosynthetic organelles termed 'chromatophores' that originated relatively recently (0.09-0.14 BYA). The chromatophore genome is about a third the size of the genome of closely related cyanobacteria, but 10-fold larger than most plastid genomes. Several genes have been transferred from the chromatophore genome to the host nuclear genome through endosymbiotic gene transfer (EGT). Some EGT-derived proteins could be imported into chromatophores for function. Two photosynthesis-related genes (psaI and csos4A) are encoded by both the nuclear and chromatophore genomes, suggesting that EGT in Paulinella chromatophora is ongoing. Many EGT-derived genes encode proteins that function in photosynthesis and photoprotection, including an expanded family of high-light-inducible (ncHLI) proteins. Cyanobacterial hli genes are high-light induced and required for cell viability under excess light. We examined the impact of light on Paulinella chromatophora and found that this organism is light sensitive and lacks light-induced transcriptional regulation of chromatophore genes and most EGT-derived nuclear genes. However, several ncHLI genes have reestablished light-dependent regulation, which appears analogous to what is observed in cyanobacteria. We postulate that expansion of the ncHLI gene family and its regulation may reflect the light/oxidative stress experienced by Paulinella chromatophora as a consequence of the as yet incomplete integration of host and chromatophore metabolisms.

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.

Regulation of eye and jaw colouration in three-spined stickleback Gasterosteus aculeatus.

Fish can change their skin and eye colour for background matching and signalling. Males of Gasterosteus aculeatus develop ornamental blue eyes and a red jaw during the reproductive season, colours that are further enhanced during courtship. Here, the effects of different hormones on physiological colour changes in the eyes and jaws of male and female G. aculeatus were investigated in vitro. In an in vivo experiment, G. aculeatus were injected with a receptor blocker of a pivotal hormone (noradrenaline) that controls colour change. In males, noradrenaline had aggregating effects on melanophore and erythrophore pigments resulting in blue eyes and a pale jaw, whereas melanocyte-concentrating hormone (MCH) and melatonin resulted in a pale jaw only. When noradrenalin was combined with melanocyte stimulating hormone (MSH) or prolactin, the jaw became red, while the eyes remained blue. In vivo injection of yohimbine, an alpha-2 adrenoreceptor blocker, resulted in dispersion of melanophore pigment in the eyes and inhibited the blue colouration. Altogether, the data suggest that noradrenalin has a pivotal role in the short-term enhancement of the ornamental colouration of male G. aculeatus, potentially together with MSH or prolactin. This study also found a sex difference in the response to MCH, prolactin and melatonin, which may result from different appearance strategies in males, versus the more cryptic females.

Myogenic activity and serotonergic inhibition in the chromatophore network of the squid Dosidicus gigas (family Ommastrephidae) and Doryteuthis opalescens (family Loliginidae).

Seemingly chaotic waves of spontaneous chromatophore activity occur in the ommastrephid squid Dosidicus gigas in the living state and immediately after surgical disruption of all known inputs from the central nervous system. Similar activity is apparent in the loliginid Doryteuthis opalescens, but only after chronic denervation of chromatophores for 5-7 days. Electrically stimulated, neurally driven activity in intact individuals of both species is blocked by tetrodotoxin (TTX), but TTX has no effect on spontaneous wave activity in either D. gigas or denervated D. opalescens Spontaneous TTX-resistant activity of this sort is therefore likely myogenic, and such activity is eliminated in both preparations by serotonin (5-HT), a known inhibitor of chromatophore activity. Immunohistochemical techniques reveal that individual axons containing L-glutamate or 5-HT (and possibly both in a minority of processes) are associated with radial muscle fibers of chromatophores in intact individuals of both species, although the area of contact between both types of axons and muscle fibers is much smaller in D. gigas Glutamatergic and serotonergic axons degenerate completely following denervation in D. opalescens Spontaneous waves of chromatophore activity in both species are thus associated with reduced (or no) serotonergic input in comparison to the situation in intact D. opalescens Such differences in the level of serotonergic inhibition are consistent with natural chromogenic behaviors in these species. Our findings also suggest that such activity might propagate via the branching distal ends of radial muscle fibers.

Leucophores are similar to xanthophores in their specification and differentiation processes in medaka.

Animal body color is generated primarily by neural crest-derived pigment cells in the skin. Mammals and birds have only melanocytes on the surface of their bodies; however, fish have a variety of pigment cell types or chromatophores, including melanophores, xanthophores, and iridophores. The medaka has a unique chromatophore type called the leucophore. The genetic basis of chromatophore diversity remains poorly understood. Here, we report that three loci in medaka, namely, leucophore free (lf), lf-2, and white leucophore (wl), which affect leucophore and xanthophore differentiation, encode solute carrier family 2, member 15b (slc2a15b), paired box gene 7a (pax7a), and solute carrier family 2 facilitated glucose transporter, member 11b (slc2a11b), respectively. Because lf-2, a loss-of-function mutant for pax7a, causes defects in the formation of xanthophore and leucophore precursor cells, pax7a is critical for the development of the chromatophores. This genetic evidence implies that leucophores are similar to xanthophores, although it was previously thought that leucophores were related to iridophores, as these chromatophores have purine-dependent light reflection. Our identification of slc2a15b and slc2a11b as genes critical for the differentiation of leucophores and xanthophores in medaka led to a further finding that the existence of these two genes in the genome coincides with the presence of xanthophores in nonmammalian vertebrates: birds have yellow-pigmented irises with xanthophore-like intracellular organelles. Our findings provide clues for revealing diverse evolutionary mechanisms of pigment cell formation in animals.