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.     

MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF GOLDFISH ERYTHROPHORES AND THEIR PTERINOSOMES

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.     

The pteridine pathway in zebrafish: regulation and specification during the determination of neural crest cell-fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72-h postfertilization, producing H4biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6-lactoyl-7,8-dihydropterin) accumulates as yellow pigment in xanthophores, together with 7-oxobiopterin, isoxanthopterin and 2,4,7-trioxopteridine. Sepiapterin is the key intermediate in the formation of 7-oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42-h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP-H4biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest-derived cells and the regulation of pteridine synthesis in mammals.     

The Pteridine Pathway in Zebrafish: Regulation and Specification during the Determination of Neural Crest Cell‐Fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72‐h postfertilization, producing H₄biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6‐lactoyl‐7,8‐dihydropterin) accumulates as yellow pigment in xanthophores, together with 7‐oxobiopterin, isoxanthopterin and 2,4,7‐trioxopteridine. Sepiapterin is the key intermediate in the formation of 7‐oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42‐h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP‐H₄biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest‐derived cells and the regulation of pteridine synthesis in mammals.     

The Erythrophore in the Larval and Adult Dorsal Skin of the Brown Frog,Rana ornativentris: Its Differentiation,Migration, and Pigmentary Organelle Formation

To determine whether or not the erythrophore originates from xanthophores in the dorsal skin of the brown frog, Rana ornativentris, we morphologically examined the differentiation and migration of the two chromatophore types and their pigmentary organelle formation. At an early tadpole stage, three kinds of chromatophores, xanthophores, iridophores, and melanophores, appeared in the subdermis, whereas the erythrophore did so just before the foreleg protrusion stage. By the middle of metamorphosis, most chromatophores other than erythrophores had migrated to the subepidermal space. Erythrophores, which appeared late in the subdermis, proliferated actively there during metamorphosis and finished moving into the subepidermal space by the completion of metamorphosis. Carotenoid vesicles and pterinosomes within the erythrophores and xanthophores showed several significant differences in structure. In xanthophores, carotenoid vesicles were abundant throughout life, whereas those in erythrophores decreased in number with the growth of the frogs. The fibrous materials contained in the pterinosomes were initially scattered but soon formed a concentric lamellar structure. In erythrophores, the lamellar structure began to form at the periphery of the organelles but at the center in xanthophores. In addition, the pterinosomes of erythrophores were uniform in size throughout development, while those of xanthophores showed a tendency to become smaller after metamorphosis. The pterinosomes of xanthophores were significantly larger than those of erythrophores. These findings suggest that an erythrophore is not a transformed xanthophore, although they resemble each other closely in many respects.     

STUDIES ON FINE STRUCTURE AND CYTOCHEMICAL PROPERTIES OF ERYTHROPHORES IN SWORDTAIL, XIPHOPHORUS HELLERI, WITH SPECIAL REFERENCE TO THEIR PIGMENT GRANULES (PTERINOSOMES)

The fine structure and the composition of pteridine pigments of erythrophores in adults of the swordtail fish, Xiphophorus helleri, were studied by means of cytochemistry, paper chromatography, ionophoresis, centrifugal fractionation, and electron microscopy. It was found that water-soluble pigments of erythrophores consisted exclusively of pteridine derivatives including large amounts of drosopterin, isodrosopterin, neodrosopterin, and moderate amounts of sepiapterin. While these substances were responsible for red pigmentation, moderate quantities of colorless pteridines, biopterin, Rana-chrome 3, xanthopterin, isoxanthopterin, and others, were also detectable. The ultrastructure of the erythrophore is characterized by numerous pigment granules and a well developed tubular endoplasmic reticulum. The former consist of a three-layered limiting membrane and inner lamellae which appear to be whorl-like due to a concentric arrangement of parallel membranes. All of the mentioned pteridines are primarily contained in this organelle which is designated, accordingly, "pterinosome." The possible functions of erythrophores and pterinosomes are discussed in the light of their structure and pigmentary constitution.     

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.     

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.     

Brightly colored pigmentation in lower vertebrates: wonder searching its mechanisms and significance in the context of phylogeny

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.     

Pteridines as Reflecting Pigments and Components of Reflecting Organelles in Vertebrates

This paper reviews evidence for the presence of pteridines in iridophores, leucophores, and xanthophores in a wide variety of vertebrate chromatophores, and argues that the chemical and functional distinction between pterinosomes and reflecting platelets is not as clear-cut as previously believed. Observations indicate that: (1) Pteridines may, either alone or in conjunction with purines, form pigment granules that reflect light, (2) these pigment granules are highly variable ranging from fibrous pterinosomes to typical reflecting platelets and may be colored, reflect white light, or be iridescent, and (3) many “leucophores” probably contain typical pterinosomes and presumed associated colorless pteridines and are therefore more closely related to erythrophores and xanthophores than to iridophores with which they are usually classified. We propose that the classification of pigment cells should be modified to reflect these facts.     

Protonic and cationic changes during cyclical cation transport driven by electron transfer

A method is described for the subcellular fractionation of goldfish xanthophores. The procedure produces relatively pure fractions of caroteniod droplets, pterinosomes, cytosol and what appears to be plasma membrane. The presence of a distinct pattern of proteins is shown to be associated with the carotenoid droplets. Treatment of the xanthophores with ACTH affects the buoyant density of some carotenoid droplets and stimulates the phosphorylation of a polypeptide associated with the carotenoid droplets.     

Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores

The cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.     

Ultrastructural and biochemical analysis of epidermal xanthophores and dermal chromatophores of the teleost Sparus aurata

We have studied the pigmentary system of the teleost Sparus aurata skin by electron microscopy and chromatographic analysis. Under electron microscopy, we found the dermis to contain the three major types of recognized chromatophores: melanophores, xanthophores and iridophores. Melanophores were more abundant in the dorsal region, whereas the iridophores were more abundant in the ventral region. The most important discovery was that of epidermal xanthophores. Epidermal xanthophores were the only chromatophores in the epidermis, something only found in S aurata and in a teleost species living in the Antartic sea. In contrast, the biochemical analysis did not establish any special characteristics: we found pteridine and flavin pigments located mostly in the pigmented dorsal region. Riboflavin and pterin were two of the most abundant coloured pigment types, but other colourless pigments such as xanthopterin and isoxanthopterin were also detected.     

Different distribution of melanophores and xanthophores in early tailbud and larval stages inTriturus alpestris

Summary The subepidermal distribution of xanthophores and melanophores is investigated in embryos ofTriturus alpestris with a uniform (stage 28+) and a banded melanophore pattern (stage 35/36). In ultrathin head and trunk sections from stage 35/36 embryos which externally show longitudinal dorsal and lateral melanophore bands in the trunk and less compact continuations of the dorsal bands in the head, xanthophores were discovered in addition to melanophores. Melanophores contain melanosomes while xanthophores which are not externally visible, are recognized by their pterinosomes. Both chromatophore cell types are mutually exclusively distributed on the epidermal basement membrane (bm). Mesenchymal cells seemed not to be able to replace them, except on the bm of the corneal epithelium where there were only mesenchymal cells. In head and trunk sections from stage 28+ embryos which externally show a distribution of uniformly scattered melanophores on the dorsolateral halves, melanophores were found on the dorsolateral neural crest migration route. No epidermal bm was present and xanthophores were undetectable. In ventrolateral and ventral portions of embryos of both stages no chromatophores occurred. This investigation defines the histological localization of melanophores and xanthophores in embryos with a typical uniform and banded melanophore arrangement; a subsequent study analyzes when xanthophores appear and how they arrange with melanophores in alternating zones.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

Pigments and ultrastructures of pigment cells in xanthic sailfin mollies (Poecilia latipinna).

Electron micrographs of skin from xanthic (gold) sailfin mollies revealed numerous xanthophores, as well as scattered melanophores. The melanophores were seen to contain premelanosomes in various stages of development. This is consistent with the fact that xanthic mollies have been shown to be tyrosinase positive. Melanosomes in xanthic mollies appear to develop by one of two pathways: 1) from an endoplasmic reticulum-derived vesicle which develops an internal lamellar framework, and 2) by fusion of multiple Golgi-derived vesicles which lack an internal lamellar framework. Analysis of the pigments in the skin of the xanthic mollies identified four colorless pteridine pigments (xanthopterin, isoxanthopterin, neopterin, and pterin) and a carotenoid with an absorbance spectrum similar to beta-carotene. It appears that, unlike some other poeciliid fishes, sailfin mollies do not use pteridine pigments for orange coloration. Rather, they appear to rely primarily on carotenoids.     

pyewacket, a new zebrafish fin pigment pattern mutant

Many mutants that disrupt zebrafish embryonic pigment pattern have been isolated, and subsequent cloning of the mutated genes causing these phenotypes has contributed to our understanding of pigment cell development. However, few mutants have been identified that specifically affect development of the adult pigment pattern. Through a mutant screen for adult pigment pattern phenotypes, we identified pyewacket (pye), a novel zebrafish mutant in which development of the adult caudal fin pigment pattern is aberrant. Specifically, pye mutants have fin melanocyte pigment pattern defects and fewer xanthophores than wild-type fins. We mapped pye to an interval where a single gene, the zebrafish ortholog of the human gene DHRSX, is present. pye will be an informative mutant for understanding how xanthophores and melanocytes interact to form the pigment pattern of the adult zebrafish fin.     

Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes

Skin pigment pattern formation in zebrafish requires pigment‐cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N‐glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries.     

Pigment cell differentiation in the fire-bellied toad,Bombina orientalis. I. Structural,chemical, and physical aspects of the adult pigment pattern

Wild-collected adults of Bombina orientalis are bright green dorsally and red to red-orange ventrally. As a prelude to an analysis of the differentiation of pigment cells in developing B. orientalis, we describe structural and chemical aspects of the fully differentiated pigment pattern of the “normal” adult. Structurally, differences between dorsal green and ventral red skin are summarized as follows: (1) Dorsal green skin contains a “typical” dermal chromatophore unit comprised of melanophores, iridophores, and xanthophores. Red skin contains predominantly carotenoid-containing xanthophores (erythrophores), and skin from black spot areas contains only melanophores. (2) In ventral red skin, there is also a thin layer of deep-lying iridophores that presumably are not involved in the observed color pattern. (3) Xanthophores of red and green skin are morphologically distinguishable from each other. Dorsal skin xanthophores contain both pterinosomes and carotenoid vesicles; ventral skin xanthophores contain only carotenoid vesicles. Carotenoid vesicles in dorsal xanthophores are much larger but less electron dense than comparable structures in ventral xanthophores. The presence of carotenes in ventral skin accounts for the bright red-orange color of the belly of this frog. Similar pigments are also present in green skin, but in smaller quantities and in conjunction with both colored (yellow) and colorless pteridines. From spectral data obtained for xanthophore pigments and structural data obtained from the size and arrangement of reflecting platelets in the iridophore layer, we attempt to explain the phenomenon of observed green color in B. orientalis.