Xanthophore Differentiation Research Articles

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

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

Genetic basis of orange spot formation in the guppy (Poecilia reticulata)

BackgroundTo understand the evolutionary significance of female mate choice for colorful male ornamentation, the underlying regulatory mechanisms of such ornamentation must be understood for examining how the ornaments are associated with “male qualities” that increase the fitness or sexual attractiveness of offspring. In the guppy (Poecilia reticulata), an established model system for research on sexual selection, females prefer males possessing larger and more highly saturated orange spots as potential mates. Although previous studies have identified some chromosome regions and genes associated with orange spot formation, the regulation and involvement of these genetic elements in orange spot formation have not been elucidated. In this study, the expression patterns of genes specific to orange spots and certain color developmental stages were investigated using RNA-seq to reveal the genetic basis of orange spot formation.ResultsComparing the gene expression levels of male guppy skin with orange spots (orange skin) with those without any color spots (dull skin) from the same individuals identified 1102 differentially expressed genes (DEGs), including 630 upregulated genes and 472 downregulated genes in the orange skin. Additionally, the gene expression levels of the whole trunk skin were compared among the three developmental stages and 2247 genes were identified as DEGs according to color development. These analyses indicated that secondary differentiation of xanthophores may affect orange spot formation.ConclusionsThe results suggested that orange spots might be formed by secondary differentiation, rather than de novo generation, of xanthophores, which is induced by Csf1 and thyroid hormone signaling pathways. Furthermore, we suggested candidate genes associated with the areas and saturation levels of orange spots, which are both believed to be important for female mate choice and independently regulated. This study provides insights into the genetic and cellular regulatory mechanisms underlying orange spot formation, which would help to elucidate how these processes are evolutionarily maintained as ornamental traits relevant to sexual selection.

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

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). 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. 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.

Global tissue transcriptomic analysis to improve genome annotation and unravel skin pigmentation in goldfish

Goldfish is an ornamental fish with diverse phenotypes. However, the limited genomic resources of goldfish hamper our understanding of the genetic basis for its phenotypic diversity. To provide enriched genomic resources and infer possible mechanisms underlying skin pigmentation, we performed a large-scale transcriptomic sequencing on 13 adult goldfish tissues, larvae at one- and three-days post hatch, and skin tissues with four different color pigmentation. A total of 25.52 Gb and 149.80 Gb clean data were obtained using the PacBio and Illumina platforms, respectively. Onto the goldfish reference genome, we mapped 137,674 non-redundant transcripts, of which 5.54% was known isoforms and 78.53% was novel isoforms of the reference genes, and the remaining 21,926 isoforms are novel isoforms of additional new genes. Both skin-specific and color-specific transcriptomic analyses showed that several significantly enriched genes were known to be involved in melanogenesis, tyrosine metabolism, PPAR signaling pathway, folate biosynthesis metabolism and so on. Thirteen differentially expressed genes across different color skins were associated with melanogenesis and pteridine synthesis including mitf, ednrb, mc1r, tyr, mlph and gch1, and xanthophore differentiation such as pax7, slc2a11 and slc2a15. These transcriptomic data revealed pathways involved in goldfish pigmentation and improved the gene annotation of the reference genome.

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

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

Pigment cell interactions and differential xanthophore recruitment underlying zebrafish stripe reiteration and Danio pattern evolution.

Fishes have diverse pigment patterns, yet mechanisms of pattern evolution remain poorly understood. In zebrafish, Danio rerio, pigment-cell autonomous interactions generate dark stripes of melanophores that alternate with light interstripes of xanthophores and iridophores. Here, we identify mechanisms underlying the evolution of a uniform pattern in D. albolineatus in which all three pigment cell classes are intermingled. We show that in this species xanthophores differentiate precociously over a wider area, and that cis regulatory evolution has increased expression of xanthogenic Colony Stimulating Factor-1 (Csf1). Expressing Csf1 similarly in D. rerio has cascading effects, driving the intermingling of all three pigment cell classes and resulting in the loss of stripes, as in D. albolineatus. Our results identify novel mechanisms of pattern development and illustrate how pattern diversity can be generated when a core network of pigment-cell autonomous interactions is coupled with changes in pigment cell differentiation.

Cellular plasticity among axolotl neural crest-derived pigment cell lineages.

Many of the factors and mechanisms guiding the migration/differentiation of neural crest cells that give rise to a number of distinguishable cell types, including all dermal and epidermal pigment cells, remain unknown. The axolotl possesses three pigment cell types that differentiate according to specific developmentally programmed sequences and contribute to pigment pattern in the adult. A single lineage of the crest that becomes restricted to one of three pigment cell types gives us the opportunity to examine the existence of a neural crest stem cell population and the potential for trans-differentiation events. Interpretations of experiments involving drug-treated and mutant axolotls implicate cellular plasticity leading to observed phenotypes. We present results from recent in vitro studies designed to identify parameters influencing differentiation events of individual neural crest-derived pigment cell lineages. We demonstrate that the differentiation of xanthophores is enhanced, while that of the melanophores are inhibited in guanosine-supplemented neural crest cell cultures. Data suggest that the increase in one pigment cell population is at the expense of another, indicative of cellular plasticity. Videomicroscopy used in this study agrees with an abundance of correlative evidence supporting the hypothesis of transdifferentiation events among neural crest-derived pigment cell populations. The embryonic neural crest-derived pigment cell system is an ideal model to study differentiation of multipotential stem cells that play critical roles in patterning.

ELECTRON MICROSCOPIC DEMONSTRATION OF TYROSINASE IN PTERINOSOMES OF THE FROG XANTHOPHORE, AND THE ORIGIN OF PTERINOSOMES.

In the frog, Rana japonica, the successive appearance of types I, II and III pterinosomes, which were defined according to the degree of lamellar structure, is in keeping with the xanthophore differentiation at the larval stage, but these three types coexist in a single xanthophore in the adult. An intense tyrosinase reaction was found in type I-II intermediate form in the larval and adult xanthophores, but it was rarely observed in types I and III. A tyrosinase reaction was always found in the GERL (Golgi-associated Endoplasmic Reticulum) of larval and adult xanthophores, and it was similarly evident in small Golgi vesicles which were separated from the GERL and dispersed in the cytoplasm. The above findings suggest that tyrosinase and pterinosome originate from different parts of the cytoplasm. The hypothesis that small Golgi vesicles are transported to the tyrosinase-negative premelanosomes involved in the origin of the melanosome is also applicable to the origin of pterinosomes.

Electron microscopic study on the xanthophore differentiation in Xenopus laevis, with special reference to their pterinosomes

Structural changes in pterinosomes and behavior of cytoplasmic inclusions in the process of xanthophore differentiation were studied electron microscopically using Xenopus laevis . At the early stage of xanthophore development, type I pterinosomes with clear limiting membranes, inner amorphous materials, and fine-fibrous structures, well developed nucleolus, mitochondria, and Golgi bodies, and a large number of polysomes are seen. Characteristic at the transitional stage are type II pterinosomes with indistinct limiting membranes and a few lamellae, and the simultaneous appearance of lipid droplets and glycogen particles. At the late stage, type III pterinosomes have many concentric lamellae. Their limiting membranes are not distinguished from the outermost lamellae. On the basis of counts of pterinosomes according to type at various stages, it was concluded that there is successive transformation from type I to type III. Adult xanthophores contain all types of pterinosomes in single cells, in contrast to larval xanthophores. A different mechanism of pterinosome genesis in larval and in adult xanthophores is suggested.

Ultramicroscopic study of the developmental change of the xanthophore in the frog, Rana Japonica with special reference to pterinosomes.

Structural change in pterinosomes and the behavior of cytoplasmic inclusions in the process of xanthophore differentiation were studied electron microscopically using Rana japonica. At the early stage of xanthophore development, type I pterinosomes had clear limiting membranes and were empty or amorphous within. The nucleus and cytoplasm were characterized by a well-developed nucleolus, mitochondria and Golgi bodies, and a large number of polysomes. At the middle stage, type II pterinosomes had indistinct limiting membranes and a few lamellae. Lipid droplets appeared almost concurrently with glycogen particles in the cytoplasm. At the later stage, type III pterinosomes had concentrically arranged lamellae, lacking clear limiting membranes. Thus, the successive transformation from types I to III was concluded. Adult xanthophores contained types I to III pterinosomes in each cell. A different mechanism is suggested of the differentiation of pterinosomes between the larval and the adult xanthophores.

Structural change of pterinosome (pteridine pigment granule) during the xanthophore differentiation of Oryzias fish

Structural changes in pterinosomes found in xanthophores were studied electron microscopically using 5, 6, 7, 8, and 9 mm-long stages of larvae of Oryzias latipes . Herein designated types G-1 to G-5 pterinosomes appeared most abundantly, corresponding to the respective stage in the above sequence. They were round or ellipsoidal. Types G-1 and G-2 had clear limiting membranes and inner fibrous structures. Type G-2 had a few superficial lamellae. Type G-3 was characterized by multiple, concentric lamellae and a central fibrous aggregate. Type G-4 was discernible from type G-3 by the lack of a central aggregate. Type G-5 lacked or contained only fragments of lamellae. Each type of pterinosome at the respective stage was counted. Thus, it was concluded that type G-1 was successively transformed into types G-4 and/or G-5.

Studies on the Chromatophores of Oryzias latipes (Teleostean Fish): Behavior of the Pteridine, Fat and Carotenoid during Xanthophore Differentiation in the Color Varieties

Studies on the Chromatophores of Oryzias latipes (Teleostean Fish): Behavior of the Pteridine, Fat and Carotenoid during Xanthophore Differentiation in the Color Varieties