By T. Hama
About thirty varieties are known in this fish. The chromatophores responsible for the body color are melanophores, xanthophores, and leucophores. The chromatophores in the vertebrate have their origin in the neural crest. This fact was ascertained in this fish. The portion containing neural crest was cultured in a nutrient medium which consisted of 4.5 parts medium 199, 4 parts Hanks saline, 1 part horse serum and 1/2 part of chick embryo extract. Where the brownish-black (wild type, BR), orange-red (bR) and white (br) color varieties were used, the chromatophores manifested their respective genotypic character (Takata and Hama, unpublished). Although the chromatophores are the same in origin, the chemical constituent proper to the respective chromatophore is very different, that is, melanins in melanophores, carotenoids in xanthophores and purines in leucophores. It was difficult to find a chemical relation among the pathways to produce such substances. This fact has long made it impossible to obtain a general principle which covers the specific differentiation to the respective chromatophores. This difficulty was obviated by replacing the carotenoids with pteridines as a principal constituent of xanthophore.
The granules laden with the pteridines were designated as "pterino-somes" (Hama et / al., 1963). The recent advance of pteridine chemistry has clarified the intimate relation among the metabolisms of aromatic amino acids, pteridines and purines. The pteridines are derived from guanine triphosphate (GTP) by GTP cyclohydrolase, and tetrahydro-biopterin acts as a cofactor of both of phenylalanine- and tryosine hydroxylases which catalyse the transformation into tyrosine from phenylalanine and into dopa from tyrosine. The above facts permit the collective study of the chromatophore differentiation problem from the biochemical basis of tyrosine-melanin, pteridines and purines, and from the morphological viewpoint of their respective carriers, melanosomes, pterinosomes and guanine platelets. Some properties of each chromatophore and iridocyte are described separately in the following.
Melanophore
The melanophores appear first at the dorsal part of the head at about stage 26 (22 somite stage) three days after fertilization (21-23) and at the same time or a little later, red-colored chromatophores (larval type of leucophore) and xanthophores are observed. At the time of hatching (about ten days after fertilization, 4.5 to 5.0 mm long), these chromatophores are found scattered on the whole body singly or superimposing with other kinds of chromatophores. In the latter case, the xanthophores are located in the uppermost portion, the melanophores in the innermost and the leucophores in the intermediate.
a. Normal melanophore. The melanophores and xanthophores contract and the leucophores expand by light. The melanophores of the dark-colored varieties (wild and blue Br) are filled with melanosomes.
b. Amelanotic melanophore. The larval and adult melanophores of the light-colored varieties (orange-red and white) were melanized by the sulfhydryl reagents such as iodoacetamide or p-chloromercuribenzoic acid in the presence of tyrosine. This indicates that the tyrosinase exists in an inhibited state. Such a kind of melanophore has been called colorless or "amelanotic melanophore". The amelanotic melanophores are almost the same in shape, but greater in number and remarkably higher in tyrosinase activity compared with the normal ones (Hishida et al., 1961, Tomita and Hishida, 196la and b). The localization of tyrosinase was examined. The respective tyrosinase activity of melanosomes and amelanotic melanosomes which were isolated by the high-speed centrifugation of sucrose density gradient system was measured using iodoacetamide and labelled tyrosine. Almost no tyrosinase activity was found in the melanosomes, while a large amount of tyrosinase was detected in the amelanotic melanosomes (Ide and Hama, 1969). The embryo of the wild type reared in the phenylthiourea (melanin inhibitor) solution had no melanins and no tyrosinase activity. On return to the water, melanin formation was gradually recovered (Tomita and Hishida, 1961b). No pteridine derivatives were found in the melanosomes although the melanosomes of retinal pigment epithelium carried the pteridines (Hama, unpublished). In general, the melanins are either missing or very few in the amelanotic melanophores of the adult, but rather rich in those of hatching larvae. The melanins as such are released as "melanin complex" out of the cell in the process of differentiation. This phenomenon was electron microscopically confirmed. The electron microscopic study revealed that the amelanotic melanosomes are round, a little smaller than the melanosomes, and have a small amount of sparsed melanin depositions within. They were almost completely melanized by the treatment of sulfhydryl poisons (Hama et al., unpublished).
c. Melanophore of albino (ib). The melanization experiment was carried out using 108 hatching embryos. No melanization of the eyes was found. By careful examination, very faint melanization of melanophores was observed in 20 embryos. In this case, only one to five melanophores were detected in one individual. They were branched but abnormal in shape, and grayish (Hama, 1969). This fact indicates that the albino has no melanophores by nature or few degenerative melanophores. In other words, the neural crest of albino has lost the capacity to form melanophores. Contrary to this, the neural crest cells of light-colored varieties (+ib) have the capacity to form the melanophores, but lack the ability to activate the tyrosinase.
Xanthophore
According to Takeuchi (1960), the mode of xanthophore distribution with development is as follows. The xanthophores are recognized in the 5 mm long larvae at the time of hatching. A dorso-ventral gradient of xanthophore density is found along the trunk. In the head region, there are some places where no xanthophore appears and only a few are distributed on the lip. In the 8 mm long larvae at the time of scale formation, the xanthophores begin to appear on the spinal cord and on the dorsal and anal fins. They concentrate on the lip, while there is a sparse distribution on the head. The ventral fin formed at this stage has a few xanthophores. Generally speaking, the xanthophore distribution at this stage is similar to that of adult fish. In the adult, the xanthophore density is maximal at the upper and lower edges on the caudal fin. On the anal fin it varies somewhat, depending upon the sex of the fish. While the xanthophores are distributed almost uniformly in the female, they are distributed more densely in the more proximal part, displaying a dorso-ventral gradient in the male. In the distal part, they are distributed only scantily. The dorso-ventral gradient of xanthophore density is also seen on the dorsal and pectoral fins, and on the spinal cord. The above description is concerned with the carotenoid-containing xanthophores of the wild and orange-red color varieties. The xanthophores of the blue and white have no carotenoids.
The carotenoid-containing xanthophore has been called "carotenoid xanthophore" and the non-carotenoid containing type "acarotenoid xanthophore" (Hama and Hasegawa, 1967). As described in the section on melanophore, the xanthophores appear at about stage 26. The carotenoids appear at stage 34 of hatching only in the carotenoid xanthophores. At the onset of xanthophore differentiation, they are filled with yellow granules which were called "sepiapterinosomes" (Hama, 1966, Hama and Hasegawa, 1967). Sepiapterinosomes contain sepiapterins (sepiapterin and isosepiapterin) and colorless pteridines (tetrahydrobiopterin, biopterin, 2-amino-4-hydroxypteridine (AHP), AHP-6-CH20H, AHP-6-COOH, xanthopterin and isoxanthopterin). With the advance of development, sepiapterins decrease and the larval pattern of colorless pteridines is transformed into the adult pattern (Hama et al., 1965). This change is likely in parallel with the structural changes of pterinosomes. The structural change was studied electron microscopically using the 5 mm long larvae to adult. From the inner structure, pterinosomes were classified into the types of G-1 to G-5. They are round or ellipsoidal, 0.5-0.7micron in diameter and 0.8-0.9micron long, the largest compared with other kinds of pigment granules. Types G-1 and G-2 have distinct limiting membranes and inner fibrous structures. Type G-2 has a few superficial lamellae. Type G-3 is characterized by multiple, concentric lamellae and a central fibrous aggregate. Type G-4 is discernible from type G-3 by the lack of a central aggregate. Type G-5 Iacks fragments of lamellae or contains few of them. By statistic treatment of the types appearing at the respective developing stages, it was concluded that type G-1 was successively transformed into type G-5 (Kamei-Takeuchi and Hama, 1971).
As described before, the tyrosinase was found in the melanosomes of the melanophore. It is plausible that the sepiapterinosome, a homologous organelle of melanosome, contains tyrosinase. The sepiapterinosomes were isolated, being separated from the melanin granules by the method of low speed centrifugation of sucrose density gradient system using the adult skin of wild type. The sepiapterinosomes had inactivated tyrosinase, although in a smaller amount (Ide and Hama, 1969). And further, the melanization of sepiapterinosomes was electron microscopically demonstrated. In this case, the sepiapterinosomes at earlier stages were melanized to a greater degree than those of later ones (Hama et al., unpublished). The larvae reared in the 1/1,000 phenylthiourea solution became pronouncely yellowish. This depends on the remarkable increase of sepiapterins (Hama, 1963).
Concerning the carotenoids, many problems remain to be settled. The red eggs, which were spawn from the female fed with red pepper or receiving an injection of the same substance, developed into larvae having the reddish dorsal part and orange-red xanthophores. From this fact it was suggested that the carotenoids of larval xanthophores have their origin in the yolk carotenoids (Takeuchi, 1960 and 1967). Goodrich et al (1941) reported that the carotenoid in the orange-red medaka is lutein (xanthophyll). Our chromatographic and spectroscopic analyses of the skin carotenoids of adult orange-red variety showed that they consist of ten different constituents: beta-carotene, tunaxanthin, lutein and several unidentified carotenoids (M-I to M-VII) specific to this fish. Tunaxanthin occupied about 40 percent of the total, and an unidentified carotenoid M-V similar to tunaxanthin amounted to about the same. Lutein was determined to be only about 4 percent of the total. From these findings, this animal seems to be a fish of marine type since tunaxanthin is common to marine fish and has not been known in freshwater fish. Most xanthophylls were found to occur in esterified form except lutein and carotenoid M-VI, which were in both free and esterified forms (Hirao et al., 1969). A rapid disappearance of skin carotenoid of this fish which was transferred from outdoors to indoors and a very low concentration of carotenoids in the liver suggests a quick turn-over mechanism of carotenoid (Takeuchi, 1961).
Leucophore
The existence of leucophore-free (lf) (Tomita, 1973) and leucophore-abundant mutants (gray, BRci; light-blue, Brci; cream, bRci and milky, brci) (Takeuchi, 1969) serves for the experimental purposes on leucophore. In general, the leucophores and iridophores (iridocytes) have been called collectively guanophores, but these two chromatophores should be distinguished since the former contain the white granules laden with uric acid, while the latter the iridescent platelets. The processes of leucophore distribution and differentiation are as follows. In the wild type, the red pigment cells appear first at the part of the constriction separating the medulla from the midbrain beneath the brain at stage 26. These pigment cells were called "red pigment granules" by Matsui (1949). They are nothing else than the larval form of leucophore as described below. At the time of hatching, many larval leucophores are scattered underneath the whole brain, singly or superimposing with other kinds of chromatophores. They appear successively on the dorsal surface of head, on the middorsal part of the trunk-tail, and finally on the ventral line of the tail. Since the melanophores exist in an amelanotic state in the light-colored varieties, the presence and behavior of the larval leucophores are distinctly observable. With development, the larval leucophores increase in number. However, some of these begin to lose their red color. In the 10-12 mm long young, the reddish and the white-reddish leucophores are marked. Finally, these leucophores are transformed into the white and white-reflecting ones of the adult. Under careful examination, the leucophores are white on the dorsal portion and slightly reddish on the belly even in the adult. Thus, the color of leucophores change in the course of their differentiation (Hama, 1967). They were previously named "brown chromatophore" by Goodrich (1927), "lipophore" by Oka (1931) and "erythrophore" (1963) or "erythropterinophore" (1966) by Hama. All of these were identified as the larval or adult form of leucophore.
The red pigment granules of larval leucophore has been called "droso-pterinosomes" (Hama, 1966; Hama and Hasegawa, 1967) since they contain drosopterins (drosopterin, isodrosopterin and neodrosopterin) and colorless pteridines (the same as in the xanthophore). As described earlier in connection with sepiapterinosomes, the drosopterinosomes change in structure and constituent with development. The developmental change of structure was electron microscopically studied.
Three types were classified. Type-a, through type-b, is transformed into type-c with the pterinosome differentiation. Type-a was 0.5-0.6micron in diameter and of a single membrane. Type-b was limited with double membrane, the inner one of which was suggestive of a lamella which have resulted from the internal fiber-like structure. Type-c was 0.7-0.8micron in diameter and of densely stained double membrane with an intervening space, and some feature showed superficially located small empty spaces. It is a common experience that crystals or the like drop out when thin sections are being made for electron microscopy (Kamei-Takeuchi et al., 1968).
As the constituents of pterinosomes, AHP and isoxanthopterin was described. AHP is transformed into isoxanthopterin by xanthine dehydrogenase. If this enzyme exerts its action on both of purine and pteridine metabolisms, it is possible to find uric acid in the pterinosome. In fact, uric acid was identified by paper chromatographic and spectroscopic analyses. The presence of uric acid in the leucophore is the first finding of the kind, to this author's knowledge. The leucophores of "milky" mutant are large in number (3-4 times) and in size (Takeuchi, 1969). A large amount of uric acid roughly corresponding to them was found (Ide and Hama, unpublished). Uric acid was removed from the body by the treatment of melamine as described below. The animals treated as such totally lacked uric acid. When the eggs were immersed in the 1M/l00 melamine solution, they developed into larvae where only larval leucophore were missing. When larvae and adult were reared in the same solution, the leucophores of both became unrecognizable within 24 hours. Careful examination showed that the leucophores gradually became smaller until achieving the final stage as minimumsized, round and transparent cells. During this period of depigmentation, the pterinosomes were released as "pterinosome complex" (Takeuchi and Kajishima, 1973), that is, coalescent pterinosomes, being eliminated from the body through the renal organ and intestine. The above features were electron microscopically demonstrated. When the leucophore-free larvae or adults were transferred to the water, the leucophores appeared to almost regain the normal in one to two weeks (Hama, 1970; Hama and Takeuchi, unpublished). The melamine effect was recognized in the cultured leucophores, but the synthesis of pterinosomes was not observable by the removal of melamine in the culture medium. This problem remains to be solved. At any rate, from the above facts, it was concluded that the melamine inhibited the formation of drosopterinosomes and also exerted its effect on the degradation of drosopterinosomes and white pterinosomes in the respective larvae and adults.
As in the case of sepiapterins, when the hatching larvae were reared in the 1/1,000 phenylthiourea solution, the drosopterins were found to increase in inverse to a remarkable decrease of isoxanthopterin. Although it was difficult to count the xanthophores of the hatching larvae, it was rather easy to count the leucophores owing to their distinct red color. The difference in the number of leucophore between the normal and phenylthiourea-treated larvae was insignificant statistically. This fact suggests that the increase of drosopterins or sepiapterins is not dependent on that of leucophore or xanthophore number (Hama, 1970).
Iridocyte
The iridocytes are distributed singly or superimposing with other kinds of chromatophores. In the latter case, the xanthophore is found on the upper part of the iridocyte, and the melanophore on its under part. The iridocytes in this fish were electron microscopically noted in detail by Kawaguti and Takeuchi (1968). The following description is derived exclusively from their work. The iridocytes are different in form according to the body location, and their guanine platelets show a definite pattern in form and intracellular distribution. According to their properties, types fall into three classes: "silvery," "iridescent" and "blue".
a. Silvery iridocyte. These are mainly located in the skin with no relation to melanophores and xanthophores, and in the peritoneal wall. In the skin, they are found in the hypodermal loose connective tissue, being elongated cells with many small, rod-shaped guanine platelets about lmicron long. Some are grouped in a sort of parallel arrangement, but as a whole they are at various angles in relation to the body surface. The peritoneal wall consists of the iridocyte layer lined with the inner melanophore layer. Moreover, the iridocyte layer is composed of two kinds of silvery and iridescent iridocytes. Those of the former kind are very numerous and form a thick, compact layer of several cells, measuring about 40micron in thickness; the latter cover the outer surface of the silvery iridocyte layer.
b. Iridescent iridocyte. The iridescent iridocytes in the peritoneal wall just described are large in size and have long guanine platelets which exhibit a parallel arrangement. The silvery and iridescent iridocytes may act as a light shield to protect the fish from light harmful to the visceral organs with the assistance of the inner melanophore layer. The choroid membrane is composed of the capillary layer, two layers of melanophores and the iridocyte layer which consists of two kinds of iridocytes. One iridocyte type has large but thin cells with close-knit groups of parallel rods arranged so as to point in various directions while forming a comparatively thick layer. Another has some blue iridocytes containing long platelets. The iridocytes of the iris are very large and numerous, forming a thick layer. Each of the thick layers is separated from its neighboring ones with a wide interspace differed from the choroid iridocytes arranged in a compact layer. In the tail, a thin layer of iridescent iridocytes is found in the hypodermis on both sides.
c. Blue iridocyte. These are mainly distributed in the operculum and are similar to the iridocytes in blue spots of the neon tetra. A thick distribution of iridocytes is found under the main opercular bone. However, a few are also present under the secondary bone and under the tertiary bone. The iridocytes piling up under the main opercular bone are very large and assume great thickness. Moreover, they have long guanine platelets in parallel arrangements giving a lattice-like appearance.
In: "MEDAKA(killifish) : Biology and Strains"
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