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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.

This is a biographical sketch of my research and its related personal episodes with respect to brightly colored pigmentation in lower vertebrates. It includes a brief story of the studies on; (a) pterinosomes as a specific site of pteridine deposition in xanthophores or erythrophores of fish and amphibians, (b) a mosaic phenotype of chromatophores occurring in the reptiles and its implication for their developmental origin and differentiation mechanisms, (c) erythrophoroma as a tumor of erythrophores in goldfish, (d) the pluripotentials of erythrophoroma cells for expression of neural crest-derived characters in vitro, (e) pigment disorders occurring in hatchery-raised flounders and (f) recognition of pigment cell types by murine tyrosinase genes transfected into an orange-colored variant of medaka fish. Some of the personal affairs associated with the history of the Japanese community for pigment cell research were described to illustrate the background of these studies.

The rapid-freezing and freeze-substitution method was applied for the ultrastructural study of the dermal chromatophores of a teleost, Oryzias latipes. The method was found to be suitable for preserving fragile membranous structures within melanophores and xanthophores. In addition, relatively high electron density in overall profile indicates that the procedure is effective in reducing the extraction of cytoplasmic ground substances that inevitably occurs during the process of conventional chemical fixation and the following dehydration. The improved ultrastructural images clearly show that the pterinosomes, the characteristic pigmentary organelles of xanthophores, are formed through several distinct developmental stages starting from the loose congregations of vesicles derived from the Golgi complex. The earlier stages of development are similar to those found in melanosome formation. Whereas carotenoid pigments in xanthophores in conventional aldehyde-osmium-fixed materials are found to be electrondense membrane-free particles, they are identified as membrane-bounded organelles in the present study. The envelope of these carotenoid vesicles does not exhibit a typical trilaminar structure but appears to be an extremely thin membrane. Carotenoid vesicles are, in most cases, in direct contact with the outer surface of tubular endoplasmic reticulum.

1. 1. Pigment organelle (pterinosomes and carotenoid vesicles) movements within isolated swordtail ( Xiphophorus helleri ) erythrophores cultured in vitro were studied. 2. 2. Pterinosome and carotenoid vesicles migrate centripetally to a juxtanuclear position or centrifugally into the dendritic processes of erythrophores in response to catecholamines. 3. 3. Cateeholamine stimulation of alpha adrenoceptors results in centripetal pigment organelle migration whereas beta adrenoceptors mediate centrifugal movements. 4. 4. Melatonin also induces perinuclear aggregation of pigment organelles but by receptor mechanisms separate from adrenoceptors. 5. 5. MSH and ACTH stimulate pigment organelle dispersion. Centrifugal dispersion of erythrophore pigments is also stimulated by cyclic AMP and dibutyryl cyclic AMP.

A biochemical and transmission electron microscopic description of the wild-type pigment phenotype in developing Mexican axolotls (Ambystoma mexicanunm) is presented. There are three pigment cell types found in adult axolotl skin - melanophores, xanthophores and iridophores. Both pigments and pigment cells undergo specific developmental changes in axolotls. Melanophores are the predominant pigment cell type throughout development; xanthophores occur secondarily and in fewer numbers than melanophores; iridophores do not appear until well into the larval stage and remain thereafter as the least frequently encountered pigment cell type. Ultrastructural differences in xanthophore organelle (pterinosome) structure at different developmental stages correlate with changes in the pattern of pteridine biosynthesis. Sepiapterin, a yellow pteridine, is present in larval axolotl skin but not in adults. Riboflavin (also yellow) is present in minimal quantities in larval skin and large quantities in adult axolotl skin. Pterinosomes undergo a morphological “reversion” at some point prior to or shortly after axolotls attain sexual maturity. Correlated with the neotenic state of the axolotl, certain larval pigmentary features are retained throughout development. Notably, the pigment cells remain scattered in the dermis such that no two pigment cell bodies overlap, although cell processes may overlap. This study forms the basis for comparison of the wild type pigment phenotype to the three mutant phenotypes-melanoid, axanthic and albino-found in the axolotl.

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.

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.

Morphological and biochemical studies on bright-colored pigment cells of adult Rana japonica were carried out. It was revealed that two types of pigment cells, xanthophores and erythrophores, are similar in structure. The possibility of interconversion between these cells was discussed, Ultrastructural characteristics of pigment granules (pterinosomes) of these cells are very similar to those observed in xiphophorin fish, hut significantly different from those of goldfish. Frog pterinosomes, as those of swordtail, are limited by a triple-layered membrane containing a concentric arrangement of internal lamellae, but are different than goldfish pterinosomes. Biochemical analyses were carried out on cellular components of skin fractionated by density gradient centrifugation, and evidence is presented that the pterinosome is the only cytoplasmic organelle which contains pteridine derivatives in abundance. Carotenoids, which are present in only a small amount, seem to be associated with smooth membranous structure. In order to find out the possibility of transformation of pterinosomes into melanosomes which has been pointed out in goldfish, tyrosinase activity of the pterinosome fraction was measured. No appreciable degree of enzyme activity was found in this case.

Bright integumental pigmentation of amphibians is imparted by the action of reflecting pigment cells (iridophores), yellow pigment cells (xanthophores), or red pigment cells (erythrophores). Iridophores are sensitive to intermedin and respond by concentrating their pigment. Common features of the intermedin molcule are involved in the stimulation of both iridophores and melanophores. The purines, guanine, hypoxanthine, and adenine are the primary iridophore pigments and are found in discrete organelles, the reflecting platelets. Intermedin markedly affects the form of reflecting platelets as well as the amounts of purine they contain. Xanthophores and erythrophores contain pteridine and carotenoid pigments. Pteridines are found within an organelle, the pterinosome, a spheroid approximately 500 mμ in diameter, formed by a series of concentric lamellae. Carotenoids appear to be contained in droplets interspersed between the pterinosomes. Migration of pigment within xanthophores is not generally controlled by intermedin although such may be the case in adult Hyla arenicolor . The carotenoid content of skin is higher in normal larvae than in hypophysioprivic larvae. Pteridine content of xanthophores is much higher in normal larvae than in hypophysioprivic larvae. Pteridine content of xanthophores is much higher in normal larvae than in hypophysioprivic animals. In the latter, administration of intermedin elevates pteridine levels. Profound changes in pteridine content occur at metamorphosis under the direct control of thyroxine. Adult amphibians which can change their colors rapidly, do so by means of a group of dermal chromatophores constituting the “dermal chromatophore unit”. Cytological and endocrinological relationships of the unit are discussed in terms of color change mechanisms.

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

1. 1. The properties, subcellular distribution and status of particulate tyrosinase in xanthic goldfish skin were studied using fractionated materials. 2. 2. Tyrosinase occurred in two molecular forms that exhibited different electrophoretic mobilities in acrylamide-gel electrophoresis. 3. 3. Freshly prepared particulate or pterinosome suspension in an isotonic medium had no measurable tyrosinase activity. The enzyme activity was detected after mechanical or chemical breakage of the structural integrity of the particles. 4. 4. No endogenous tyrosinase inhibitor was found in the particulate fractions.

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.

Structural changes in drosopterinosomes (red pigment granules) of Rana japonica in the process of erythrophore differentiation were studied by light and electron microscopy. On the basis of the degree of pterinosome differentiation, three types can be recognized: Typ-I drosopterinosomes appear first during metamorphosis and have clear limiting membranes and amorphous materials within. Those of type-II are found in abundance shortly after metamorphosis and have inner structures, consisting of fibrillae and/or small lamellae in dense concentric arrangement. Type-III is found abundantly in adults and acquires an almost homogeneously electron-dense mature morphology, probably from the deposition of electron-dense materials. On the basis of counts of pterinosomes, a successive transformation from type I to III is suggested. The differences among red drosopterinosomes, yellow sepiapterinosomes in xanthophore and melanosomes are not always distinguishable electron microscopically. Discrimination is possible by careful examination of lamellar patterns characteristic of the respective granules and by a simultaneous application of light and electron microscopy. From this viewpoint, a re-evaluation of the identification of granules previously reported was effected.

We have identified two simple methods to analyse xanthophore and pterinosome biogenesis in zebrafish. The first uses methylene blue (methylthionium chloride), a redox dye which specifically labels xanthophores and pterinosomes, while the second uses autofluorescence to detect pteridine levels; these methods may be used to detect the number, location and shape of xanthophores and pterinosomes. These assays were applied to two zebrafish mutants--brie and yobo--and revealed that both mutants have pterinosome biogenesis and pteridine synthesis defects. Additionally, using capillary electrophoresis, we provide evidence that sepiapterin is responsible for the yellow colour and blue-light induced fluorescence in zebrafish embryos.