Mutation of mpv17 results in loss of iridophores due to mitochondrial dysfunction in tilapia.

Mpv17 (mitochondrial inner membrane protein MPV17) deficiency causes severe mitochondrial DNA depletion syndrome in mammals and loss of pigmentation of iridophores and a significant decrease of melanophores in zebrafish. The reasons for this are still unclear. In this study, we established an mpv17 homozygous mutant line in Nile tilapia. The developing mutants are transparent due to loss of iridophores and aggregation of pigment granules in the melanophores and disappearance of the vertical pigment bars on the side of the fish. Transcriptome analysis using skin of fish at 30 dpf (days post fertilization) revealed that the genes related to purine (especially pnp4a) and melanin synthesis were significantly downregulated. However, administration of guanine diets failed to rescue the phenotype of the mutants. In addition, no obvious apoptosis signals were observed in the iris of the mutants by TUNEL staining. Significant downregulation of genes related to iridophore differentiation was detected by qPCR. Insufficient ATP, as revealed by ATP assay, α-MSH treatment and adcy5 mutational analysis, might account for the defects of melanophores in mpv17 mutants. Several tissues displayed less mtDNA and decreased ATP levels. Taken together, these results indicated that mutation of mpv17 led to mitochondrial dTMP deficiency, followed by impaired mtDNA content and mitochondrial function, which in turn, led to loss of iridophores and a transparent body color in tilapia.

It's not easy being green: Comparing typical skin colouration among amphibians with colour abnormalities associated with chromatophore deficits.

Amphibians can obtain their colour from a combination of several different pigment and light reflecting cell types called chromatophores, with defects in one or several of the cells leading to colour abnormalities. There is a need for better recording of colour abnormalities within wild amphibian populations, as this may provide baseline data that can be used to determine changes in environmental conditions and population dynamics, such as inbreeding. In this study, we provide records of several types of chromatophore deficiencies, including those involving iridophores, xanthophores and melanophores, among two Australian tree frog species; the green and golden bell frog, Litoria aurea, and the eastern dwarf tree frog, L. fallax. We explore these colour abnormalities in terms of the chromatophores that have likely been affected and associated with their expression, in combination with typical colour phenotypes, colour variations and colour changes for these species. We intend for our photographs to be used as a visual guide that addresses the need for more accessible information regarding the physical manifestation of different chromatophore defects among amphibians.

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

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

A Soft, Adhesive Self-Healing Naked-Eye Strain/Stress Visualization Patch.

Learning about the strain/stress distribution in a material is essential to achieve its mechanical stability and proper functionality. Conventional techniques such as universal testing machines only apply to static samples with standardized geometry in laboratory environment. Soft mechanical sensors based on stretchable conductors, carbon-filled composites, or conductive gels possess better adaptability, but still face challenges from complicated fabrication process, dependence on extra readout device, and limited strain/stress mapping ability. Inspired by the camouflage mechanism of cuttlefish and chameleons, here an innovative responsive hydrogel containing light-scattering "mechano-iridophores" is developed. Force induced reversible phase separation manipulates the dynamic generation of mechano-iridophores, serving as optical indicators of local deformation. Patch-shaped mechanical sensors made from the responsive hydrogel feature fast response time (<0.4 s), high spatial resolution (≈100 µm), and wide dynamic ranges (e.g., 10-150% strain). The intrinsic adhesiveness and self-healing material capability of sensing patches also ensure their excellent applicability and robustness. This combination of chemical and optical properties allows strain/stress distributions in target samples to be directly identified by naked eyes or smartphone apps, which is not yet achieved. The great advantages above are ideal for developing the next-generation mechanical sensors toward material studies, damage diagnosis, risk prediction, and smart devices.

Myron Gordon Award Lecture 2023: Painting the neural crest: How studying pigment cells illuminates neural crest cell biology.

It has been 30 (!!) years since I began working on zebrafish pigment cells, as a postdoc in the laboratory of Prof. Christiane Nüsslein-Volhard. There, I participated in the first large-scale mutagenesis screen in zebrafish, focusing on pigment cell mutant phenotypes. The isolation of colourless, shady, parade and choker mutants allowed us (as a postdoc in Prof. Judith Eisen's laboratory, and then in my own laboratory at the University of Bath since 1997) to pursue my ambition to address long-standing problems in the neural crest field. Thus, we have studied how neural crest cells choose individual fates, resulting in our recent proposal of a new, and potentially unifying, model which we call Cyclical Fate Restriction, as well as addressing how pigment cell patterns are generated. A key feature of our work in the last 10 years has been the use of mathematical modelling approaches to clarify our biological models and to refine our interpretations. None of this would have been possible without a hugely talented group of laboratory members and other collaborators from around the world-it has been, and I am sure will continue to be, a pleasure and privilege to work with you all!

African jewel fish (Hemichromis bimaculatus) distinguish individual faces based on their unique iridophore patterns.

Previous research has shown that African jewel fish (Hemichromis bimaculatus) recognize pair-bonded mates during their exchanges of egg-guarding duties. The current research examined the perceptual cues for face recognition by comparing two face models displaying anatomically realistic arrangements of blue iridophores derived from discriminant function analysis of distinct sibling groups. Four groups each consisting of 9 subadults were examined using a narrow compartment restraining lateral movement where face models were presented at eye level for eight trials. Because respiratory movement of the operculum can mechanically displace the eye thereby shifting the retinal image, jewel fish reduce their respiration rate during increased attention. When two experimental groups were presented with the same face models on four trials following initial model presentations, both groups exhibited stable respiration rates indicative of model habituation. When the habituated face models were switched to novel face models on the fifth trial, the rates of respiration decreased as measured by reliable increases in the elapsed times of opercular beats. Switching the models back to the habituated models on the sixth trial caused reliable decreases in the elapsed times of opercular beats, resembling the earlier trials for the habituated models. Switching the face models again to the formerly novel models on the seventh trial produced respiration rates that resembled those of the habituated models. The two control groups viewing the same models for all eight trials exhibited no substantial change in respiration rates. Together, these findings indicate that jewel fish can learn to recognize novel faces displaying unique arrangements of iridorphores after one trial of exposure.

Atlantic mackerel (Scomber scombrus) change skin colour in response to crowding stress.

Wild capture can be stressful for fish. Stress has the potential to induce mortality in released unwanted catches or negative flesh quality consequences in retained ones. Such effects compromise sustainable natural resource management and industry profitability. Mitigating stress during capture is therefore desirable. Biological indicators of stress can objectively inform fishers as to the functional welfare status of catches during fishing operations. If they are to be of practical use in mitigating stress during wild capture events, such indicators must be quantifiable, respond rapidly, reflect the level of induced stress and be easily observable. Atlantic mackerel (Scomber scombrus) are extensively targeted by purse seine fisheries in European waters but are particularly vulnerable to stress. Excessive crowding in the net is thought to be the principal stress mechanism. There is therefore a need to develop indicators of crowding stress for this species so that catch welfare can be improved. Here, we demonstrate that S. scombrus exhibit a skin colour change from predominately green to predominately blue when exposed to crowding stress. In sea cage trials, we induced various degrees of stress in groups of wild-caught S. scombrus by manipulating crowding density and its duration. Skin colour was quantified in air using digital photography. The colour change occurred rapidly (within the typical duration of crowding events in the fishery), and its magnitude was correlated to the severity and duration of crowding. Bluer fish were also associated with higher levels of plasma lactate. No appreciable colour change was observed in uncrowded (control) groups during the treatment period. Nonetheless, unstressed S. scombrus did turn blue <1 h after death. Together, these results indicate that skin colour change has the potential to be a useful real-time indicator of crowding stress for S. scombrus and could therefore be used to improve welfare during wild capture fishing.

Contribution of sox9b to pigment cell formation in medaka fish.

SoxE-type transcription factors, Sox10 and Sox9, are key regulators of the development of neural crest cells. Sox10 specifies pigment cell, glial, and neuronal lineages, whereas Sox9 is reportedly closely associated with skeletogenic lineages in the head, but its involvement in pigment cell formation has not been investigated genetically. Thus, it is not fully understood whether or how distinctly these genes as well as their paralogs in teleosts are subfunctionalized. We have previously shown using the medaka fish Oryzias latipes that pigment cell formation is severely affected by the loss of sox10a, yet unaffected by the loss of sox10b. Here we aimed to determine whether Sox9 is involved in the specification of pigment cell lineage. The sox9b homozygous mutation did not affect pigment cell formation, despite lethality at the early larval stages. By using sox10a, sox10b, and sox9b mutations, compound mutants were established for the sox9b and sox10 genes and pigment cell phenotypes were analyzed. Simultaneous loss of sox9b and sox10a resulted in the complete absence of melanophores and xanthophores from hatchlings and severely defective iridophore formation, as has been previously shown for sox10a-/- ; sox10b-/- double mutants, indicating that Sox9b as well as Sox10b functions redundantly with Sox10a in pigment cell development. Notably, leucophores were present in sox9b-/- ; sox10a-/- and sox10a-/- ; sox10b-/- double mutants, but their numbers were significantly reduced in the sox9b-/- ; sox10a-/- mutants. These findings highlight that Sox9b is involved in pigment cell formation, and plays a more critical role in leucophore development than Sox10b.

Pigment pattern morphospace of Danio fishes: evolutionary diversification and mutational effects.

Molecular and cellular mechanisms underlying variation in adult form remain largely unknown. Adult pigment patterns of fishes in the genus Danio, which includes zebrafish, Danio rerio, consist of horizontal stripes, vertical bars, spots and uniform patterns, and provide an outstanding opportunity to identify causes of species level variation in a neural crest derived trait. Understanding pigment pattern variation requires quantitative approaches to assess phenotypes, yet such methods have been mostly lacking for pigment patterns. We introduce metrics derived from information theory that describe patterns and pattern variation in Danio fishes. We find that these metrics used singly and in multivariate combinations are suitable for distinguishing general pattern types, and can reveal even subtle phenotypic differences attributable to mutations. Our study provides new tools for analyzing pigment pattern in Danio and potentially other groups, and sets the stage for future analyses of pattern morphospace and its mechanistic underpinnings.

Immunoglobulin superfamily receptor Junctional adhesion molecule 3 (Jam3) requirement for melanophore survival and patterning during formation of zebrafish stripes.

Adhesive interactions are essential for tissue patterning and morphogenesis yet difficult to study owing to functional redundancies across genes and gene families. A useful system in which to dissect roles for cell adhesion and adhesion-dependent signaling is the pattern formed by pigment cells in skin of adult zebrafish, in which stripes represent the arrangement of neural crest derived melanophores, cells homologous to melanocytes. In a forward genetic screen for adult pattern defects, we isolated the pissarro (psr) mutant, having a variegated phenotype of spots, as well as defects in adult fin and lens. We show that psr corresponds to junctional adhesion protein 3b (jam3b) encoding a zebrafish orthologue of the two immunoglobulin-like domain receptor JAM3 (JAM-C), known for roles in adhesion and signaling in other developing tissues, and for promoting metastatic behavior of human and murine melanoma cells. We found that zebrafish jam3b is expressed post-embryonically in a variety of cells including melanophores, and that jam3b mutants have defects in melanophore survival. Jam3b supported aggregation of cells in vitro and was required autonomously by melanophores for an adherent phenotype in vivo. Genetic analyses further indicated both overlapping and non-overlapping functions with the related receptor, Immunoglobulin superfamily 11 (Igsf11) and Kit receptor tyrosine kinase. These findings suggest a model for Jam3b function in zebrafish melanophores and hint at the complexity of adhesive interactions underlying pattern formation.

Fast, easy and early (larval) identification of transparent mutant zebrafish using standard fluorescence microscopy.

The availability of transparent zebrafish mutants (either TraNac: tra b6/b6; nac w2/w2 or casper: roy a9/a9; nac w2/w2 ) for live imaging studies together with the ease of generating transgenic lines are two of the strengths of the zebrafish model organism. The fact that transparent casper ( roy a9/a9;nac w2/w2) and silver nacre ( nac w2/w2) mutants are indistinguishable by eye at early stages (1-5 days post-fertilization; dpf) means many fish must be raised and later culled if they are not transparent. To identify translucent mutants early and easily at the early larval stage (≤5 dpf) before they are classified as protected animals, we developed a simple screening method using standard fluorescence microscopy. We estimate that this procedure could annually save 60,000 animals worldwide.

Genome and Transcriptome Sequencing of casper and roy Zebrafish Mutants Provides Novel Genetic Clues for Iridophore Loss.

casper has been a widely used transparent mutant of zebrafish. It possesses a combined loss of reflective iridophores and light-absorbing melanophores, which gives rise to its almost transparent trunk throughout larval and adult stages. Nevertheless, genomic causal mutations of this transparent phenotype are poorly defined. To identify the potential genetic basis of this fascinating morphological phenotype, we constructed genome maps by performing genome sequencing of 28 zebrafish individuals including wild-type AB strain, roy orbison (roy), and casper mutants. A total of 4.3 million high-quality and high-confidence homozygous single nucleotide polymorphisms (SNPs) were detected in the present study. We also identified a 6.0-Mb linkage disequilibrium block specifically in both roy and casper that was composed of 39 functional genes, of which the mpv17 gene was potentially involved in the regulation of iridophore formation and maintenance. This is the first report of high-confidence genomic mutations in the mpv17 gene of roy and casper that potentially leads to defective splicing as one major molecular clue for the iridophore loss. Additionally, comparative transcriptomic analyses of skin tissues from the AB, roy and casper groups revealed detailed transcriptional changes of several core genes that may be involved in melanophore and iridophore degeneration. In summary, our updated genome and transcriptome sequencing of the casper and roy mutants provides novel genetic clues for the iridophore loss. These new genomic variation maps will offer a solid genetic basis for expanding the zebrafish mutant database and in-depth investigation into pigmentation of animals.

Reflector of the body photophore in lanternfish is mechanistically tuned to project the biochemical emission in photocytes for counterillumination.

Lanternfish, a family Myctophidae, use ventro-lateral body photophores for camouflage of the ventral silhouette, a strategy called counterillumination. While other deep-sea fishes possess pigmented filters and silver reflectors to match sunlight filtering down through the depths, myctophids developed a blue-green reflector for this purpose. In this study, we showed in a lanternfish Diaphus watasei that the reflector comprised monolayered iridophores containing multilayered guanine crystals which enable high reflection with light interference colouration. Platelets shape in body photophores is an unique near-regular hexagonal, probably to allow the homogeneity of reflection angle of the luminescence from photocytes. Focus point of the parabola-like reflector is positioned on the photocytes that ensures the light produced from the photocytes is redirected to the ventral direction. In vitro luminescence reaction using purified luciferase and the substrate coelenterazine showed the light emission at λmax 454 nm, while reflection spectra of the iridophores exhibit peaks at longer wavelength, which accomplish to alter the luminescence emitted from photocytes to longer wavelength to fit the mesopelagic light environment. Taken together, we revealed multiple mechanistic elaborations in myctophid body photophores to achieve effective control of biochemical luminescence for counterillumination.

Genetic Characteristic and RNA-Seq Analysis in Transparent Mutant of Carp-Goldfish Nucleocytoplasmic Hybrid.

In teleost, pigment in the skin and scales played important roles in various biological processes. Iridophores, one of the main pigment cells in teleost, could produce silver pigments to reflect light. However, the specific mechanism of the formation of silver pigments is still unclear. In our previous study, some transparent mutant individuals were found in the carp-goldfish nucleocytoplasmic hybrid (CyCa hybrid) population. In the present study, using transparent mutants (TM) and wild type (WT) of the CyCa hybrid as a model, firstly, microscopic observations showed that the silver pigments and melanin were both lost in the scales of transparent mutants compared to that in wild types. Secondly, genetic study demonstrated that the transparent trait in the CyCa hybrid was recessively inherent, and controlled by an allele in line with Mendelism. Thirdly, RNA-Seq analysis showed that differential expression genes (DEGs) between wild type and transparent mutants were mainly enriched in the metabolism of guanine, such as hydrolase, guanyl nucleotide binding, guanyl ribonucleotide binding, and GTPase activity. Among the DEGs, purine nucleoside phosphorylase 4a (pnp4a) and endothelin receptor B (ednrb) were more highly expressed in the wild type compared to the transparent mutant (p < 0.05). Finally, miRNA-Seq analysis showed that miRNA-146a and miR-153b were both more highly expressed in the transparent mutant compared to that in wild type (p < 0.05). Interaction analysis between miRNAs and mRNAs indicated that miRNA-146a was associated with six DEGs (MGAT5B, MFAP4, GP2, htt, Sema6b, Obscn) that might be involved in silver pigmentation.

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

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

Bar, stripe and spot development in sand-dwelling cichlids from Lake Malawi.

BACKGROUND: Melanic patterns such as horizontal stripes, vertical bars and spots are common among teleost fishes and often serve roles in camouflage or mimicry. Extensive research in the zebrafish model has shown that the development of horizontal stripes depends on complex cellular interactions between melanophores, xanthophores and iridophores. Little is known about the development of horizontal stripes in other teleosts, and even less is known about bar or spot development. Here, we compare chromatophore composition and development of stripes, bars and spots in two cichlid species of sand-dwellers from Lake Malawi-Copadichromis azureus and Dimidiochromis compressiceps. RESULTS: (1) In D. compressiceps, stripes are made of dense melanophores underlaid by xanthophores and overlaid by iridophores. Melanophores and xanthophores are either loose or absent in interstripes, and iridophores are dense. In C. azureus, spots and bars are composed of a chromatophore arrangement similar to that of stripes but are separated by interbars where density of melanophores and xanthophores is only slightly lower than in stripes and iridophore density appears slightly greater. (2) Stripe, bar and spot chromatophores appear in the skin at metamorphosis. Stripe melanophores directly differentiate along horizontal myosepta into the adult pattern. In contrast, bar number and position are dynamic throughout development. As body length increases, new bars appear between old ones or by splitting of old ones through new melanophore appearance, not migration. Xanthophore and iridophore distributions follow melanophore patterns. (3) Metamorphic pigmentation arises in cichlids in a fashion similar to that described in zebrafish: melanophore progenitors derived from the medial route of neural crest migration migrate from the vicinity of the neural tube to the skin during metamorphosis. CONCLUSION: The three pigment cell types forming stripes, bars and spots arise in the skin at metamorphosis. Stripes develop by differentiation of melanophores along horizontal myosepta, while bars do not develop along patent anatomical boundaries and increase in number in relation with body size. We propose that metamorphic melanophore differentiation and migratory arrest upon arrival to the skin lead to stripe formation, while bar formation must be supported by extensive migration of undifferentiated melanophores in the skin.

Live Imaging of Neural Crest and Pigment Cells and Transient Transgenic Manipulation of Gene Activity.

Neural crest cells are a highly multipotent and migratory cell type that are important for adult pigment pattern formation, cellular homeostasis, and regeneration. The optical transparency and accessibility of fish embryos makes them particularly well-suited to high-resolution analysis of neural crest development. However, the dispersive nature of these cells adds to the challenge of their study. We describe key protocols for the analysis of neural crest development in zebrafish and medaka, including live imaging of neural crest cells and differentiating pigment cells and transient transgenesis assays that can be used to manipulate neural crest development.

The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria.

Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in nuclear genes involved in mitochondrial DNA (mtDNA) maintenance, and characterized by both a strong reduction in mtDNA content and severe mitochondrial defects in affected tissues. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been associated with hepatocerebral forms of MDS. The zebrafish mpv17 null mutant lacks the guanine-based reflective skin cells named iridophores and represents a promising model to clarify the role of Mpv17. In this study, we characterized the mitochondrial phenotype of mpv17-/- larvae and found early and severe ultrastructural alterations in liver mitochondria, as well as significant impairment of the respiratory chain, leading to activation of the mitochondrial quality control. Our results provide evidence for zebrafish Mpv17 being essential for maintaining mitochondrial structure and functionality, while its effects on mtDNA copy number seem to be subordinate. Considering that a role in nucleotide availability had already been postulated for MPV17, that embryos blocked in pyrimidine synthesis do phenocopy mpv17-/- knockouts (KOs) and that mpv17-/- KOs have impaired Dihydroorotate dehydrogenase activity, we provided mpv17 mutants with the pyrimidine precursor orotic acid (OA). Treatment with OA, an easily available food supplement, significantly increased both iridophore number and mtDNA content in mpv17-/- mutants, thus linking the loss of Mpv17 to pyrimidine de novo synthesis and opening a new simple therapeutic approach for MPV17-related MDS.

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.

Unusual light-reflecting pigment cells appear in the Xenopus neural tube culture system in the presence of guanosine.

Isolation and culture of Xenopus laevis neural tubes resulted in differentiation of melanophores and iridophores from neural crest cells; the differentiated melanophores and iridophores were then maintained in culture for more than 6 months. Guanosine has been reported to promote reflecting platelet formation in melanin-producing pigment cells; however, the process of pigment organellogenesis is still unclear. In the present study, unusual light-reflecting pigment cells were observed upon addition of guanosine to the neural tube culture system, which contained melanosomes specific to melanophores, and reflecting platelets specific to iridophores. Ultrastructural studies suggested that irregularly shaped reflecting platelets were formed from stage II melanosomes (the early stage of melanosome formation) in these unusual pigment cells.