Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.     

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.     

Intrinsic pigment cell stimulating activity in the skin of the leopard frog, Rana pipiens.

Consistent with the concept that specific pigment patterns of amphibians might result from the highly localized distribution of stimulators and inhibitors of pigment cell expression in the skin, the spot pattern of the leopard frog, Rana pipiens, was examined through the use of the Xenopus neural tube explant assay system (Fukuzawa and Ide, 1988). Media conditioned with pieces of skin from dorsal black spotted areas promoted melanization of neural crest cells at a significantly higher level than did media conditioned with dorsal interspot skin in the absence of extra tyrosine. All conditioned media contained exceedingly low concentrations of tyrosine. With the addition of supplemental tyrosine, the melanization capacity of conditioned media from the interspot areas was elevated to that of the spotted skin. Control media conditioned with ventral frog skin inhibited melanization, as usual, because of the presumed presence of melanization inhibiting factor (MIF). It is considered that dorsal skin contains a melanization stimulating factor (MSF) which is present in significantly higher levels in spotted skin than in interspot areas and that expression of the particular pigmentary pattern of this leopard frog is regulated by the relative distribution of MIF, MSF, and possibly other intrinsic substances present in the skin.     

Melanization Stimulating Activity in the Skin of the Gilthead Porgy,Sparus auratus

The presence of a melanization-stimulating factor (MSF) was discovered in dorsal and/or ventral skin of Sparus auratus. Skin from this marine species was used to condition Steinberg's balanced salt solution (BSS), which was subsequently tested with the neural tube assay. BBS conditioned by dorsal and/or ventral skin of S. auratus at 25% and 50% concentrations had a profound stimulatory effect on the percentage of melanization of neural crest cells throughout the 3day assay period. In some cases 90% melanization occurred within the first 24 hr. Such stimulated cells showed a doubling of the number of dendrites per cell. assess the effects of MSF on other indices of melanization, dorsal and/or ventral skin was used to condition MEM used in the culture of B16-F10 murine melanoma cells. During the first 24 hr, B16-F10 murine melanoma cells responded to conditioned media by demonstrating a considerable increase in activities of tyrosine hydroxylase, dopa oxidase, and dopachrome tautomerase, but no effect was observed on melanin content. In contrast, melanin content increased after 48 hr of incubation, whereas the enzymatic activities were inhibited during this period. It seems that MSF activity, expressed in several ways, may be present generally among marine species.     

Hyperpigmented patches in the skin of the newt Notophthalmus viridescens

In the integument of the red-spotted newt there occasionally appear patches of skin which are at the same time melanistic and iridescent. Such hyperpigmented patches have been found on the back, on the tail and on the dorsal surface of both fore and hind limbs. Cytological examination of several such areas revealed the presence of large numbers of chromatophores distributed throughout the dermis. The majority of the chromatophores consisted of atypically large and dendritic melanophores, which contained typical pigment granules. The iridescence resulted from a high incidence of iridophores. Xanthophores also were found in considerable abundance. This extensive and apparently random intermingling of melanophores, iridophores and xanthophores in limited areas constitutes a striking exception to the usual distributional patterns of pigment cells in this animal.     

Localization of pigment cells in cultured frog skin

The pigmentation pattern of ventral skin of the frog Rana esculenta consists mainly of melanophores and iridophores, rather than the three pigment cells (xanthophores, iridophores, and melanophores) which form typical dermal chromatophore units in dorsal skin. The present study deals with the precise localization and identification of the types of pigment cells in relation to their position in the dermal tracts of uncultured or cultured frog skins. Iridophores were observed by dark-field microscopy; both melanophores and iridophores were observed by transmission electron microscopy. In uncultured skins, three levels were distinguished in the dermal tracts connecting the subcutaneous tissue to the upper dermis. Melanophores and iridophores were localized in the upper openings of the tracts directed towards the superficial dermis (level 1). The tracts themselves formed level 2 and contained melanophores and a few iridophores. The inner openings of the tracts made up level 3 in which mainly iridophores were present. These latter openings faced the subcutaneous tissue In cultured skins, such pigment-cell distribution remained unchanged, except at level 2 of the tracts, where pigment cells were statistically more numerous; among these, mosaic pigment cells were sometimes observed.     

Control of Melanoblast Differentiation in Amphibia by α-Melanocyte Stimulating Hormone,a Serum Melanization Factor,and a Melanization Inhibiting Factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide: Dev. Biol., 129:25–36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of α-melanocyte stimulating hormone (α-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the “outgrowth” and “melanization” of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW <30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493–1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW > 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system. We suggest that ventrally localized MIF plays an important role in amphibian pigment pattern formation and that the interacting effects of MIF and melanogenic factors influence melanoblast differentiation, migration, and/or proliferation of neural crest cells to effect the expression of pigmentary patterns.     

Ultrastructure of the dermal chromatophores in a lizard (Scincidae: Plestiodon latiscutatus) with conspicuous body and tail coloration

Microscopic observation of the skin of Plestiodon lizards, which have body stripes and blue tail coloration, identified epidermal melanophores and three types of dermal chromatophores: xanthophores, iridophores, and melanophores. There was a vertical combination of these pigment cells, with xanthophores in the uppermost layer, iridophores in the intermediate layer, and melanophores in the basal layer, which varied according to the skin coloration. Skin with yellowish-white or brown coloration had an identical vertical order of xanthophores, iridophores, and melanophores, but yellowish-white skin had a thicker layer of iridophores and a thinner layer of melanophores than did brown skin. The thickness of the iridophore layer was proportional to the number of reflecting platelets within each iridophore. Skin showing green coloration also had three layers of dermal chromatophores, but the vertical order of xanthophores and iridophores was frequently reversed. Skin showing blue color had iridophores above the melanophores. In addition, the thickness of reflecting platelets in the blue tail was less than in yellowish-white or brown areas of the body. Skin with black coloration had only melanophores.     

Control of melanoblast differentiation in amphibia by alpha-melanocyte stimulating hormone, a serum melanization factor, and a melanization inhibiting factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide:Dev. Biol., 129:25-36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of alpha-melanocyte stimulating hormone (alpha-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the "outgrowth" and "melanization" of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW less than 30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493-1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW greater than 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Divisionistic generation of skin hue and the change of shade in the scalycheek damselfish, Pomacentrus lepidogenys

In addition to melanophores and xanthophores, there existed two types of iridophore in the dermis of the scalycheek damselfish, Pomacentrus lepidogenys. There are dendritic iridophores which reflect white light-rays by Tyndall scattering, and the round or somewhat ellipsoidal iridophores which reflect rays with a relatively narrow spectral peak from blue to green through the non-ideal thin-film interference. Most of the dendritic iridophores were covered with xanthophores and were situated over melanophores, thus constituting a kind of chromatophore unit which produces a yellow or yellowish-green color. The characteristic yellowish-green hue of the integument results from a compound effect of small contributions by more elementary colors. During color changes of the skin, the position of the spectral peak does not shift. Unlike the iridophores of the blue damselfish, both types of iridophore of the scalycheek damselfish were found to be inactive. It appears, therefore, that the aggregation and dispersion of pigment within the melanophores is the primary mechanism responsible for the changes in color of this species.     

Melanization stimulating factors in the integument of the Mugil cephalus and Dicertranchus labrax

The pigment pattern expression resides in the chromatoblasts of the embryonic skin. The differentiation of these chromatoblasts is influenced by specific local factors such a melanization inhibiting factor (MIF) and a melanization-stimulating factor (MSF). We reveal the presence of these factors by means of a series of experiments on the skin of the marine species of fish Dicertranchus labrax and Mugil cephalus, each with different pigment pattern, the former having a light skin and the latter a darker one. Media conditioned by exposure to dorsal and/or ventral skin, stimulates the melanization of Xenopus laevis neural crest cells throughout a 3 day assay period. Similarly conditioned culture media tested on B16-F10 murine malignant melanocytes, revealed a considerable influence in enzymatic activities: dopachrome tautomerase (DCT), tyrosine hydroxylase and dopa oxidase. The use of media in a dose response basis suggests that the conditioned media may contain both melanophore stimulating and inhibiting factors. The results obtained may actually reflect the resultant activity of the two factors present.     

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.     

Stimulation of Cultured Iridophores by Amphibian Ventral Conditioned Medium

That the ventral integument of adult frogs (Rana pipiens) contains factor(s) that stimulate iridophore expression (adhesion, morphologic appearance, proliferation) was demonstrated on iridophores derived from tadpoles of R. pipiens and Pachymedusa dacnicolor, and maintained in primary culture in a growth medium based upon Leibovitz's L-15. Experimental growth medium (VCM) conditioned by a one-hour exposure to pieces of ventral skin of adult R. pipiens induced iridophores to assume a broad and stellate appearance, to form confluent sheets, and to proliferate over a nine-day period. Iridophores in control medium assumed long thin profiles, detached easily, and exhibited no signs of proliferation. Unknown cells containing reflecting platelets and unusual other organelles appeared uniquely in chromatophore cultures of P. dacnicolor in VCM. The intense stimulation of iridophore expression in VCM is consistent with the known inhibitory effect of this medium on melanization and with its purported role in the determination of dorsal/ventral pigment patterns of amphibians. The results are discussed in terms of a prevailing theory about pigment cell origins and development.     

Transgenic Medaka Fish Bearing the Mouse Tyrosinase Gene: Expression and Transmission of the Transgene Following Electroporation of the Orange-Colored Variant

Transgenic fish bearing the mouse tyrosinase gene (mg-Tyrs-J) were produced by transfection into fertilized eggs of the homozygous normal orange-colored variant of medaka fish, Oryzias latipes, by means of electroporation. Of 589 eggs transfected, 38 fish (6%) exhibited brownish wild-type skin pigmentation, which was discernible from control siblings. Light microscopy of the skin from the founders thus generated disclosed that 1) melanization occurred and was restricted to melanophores formed presumably from preexisting amelanotic melanophores, 2) there was a wide variation in the degree of melanization observed among melanophores, and 3) no melanin deposition was recognized in xanthophores or leucophores. Immunofluorescence using an antibody raised against mouse tyrosinase disclosed that melanophores at varying stages of maturation were reactive. Thus, it was shown that the transgene in medaka fish expressed its action in a cell type-specific manner. Crossing of transgenic founders with homozygous orange-colored variant fish yielded two groups of offspring expressing either the wild-type or the orange-colored skin pigmentation at an approximate ratio of 1:1. Crossing between founders exhibiting wild-type pigmentation yielded only offspring with melanized skin. Skin melanophores in these offspring formed vertical stripes, which are rare in this species. The hereditary basis of melanized skin was demonstrated in matings of Fl progenies, which resulted in similar degrees of melanization over whole skin melanophores. The sum of these findings implied that the transgene is expressed as a dominant character gene and is transmitted through germ cell lines according to the Mendelian law. PCR analysis combined with nested PCR technique strongly suggested that the transgene was integrated into the medaka genome, even though the copy number deduced from gel banding was largely diminished, possibly as a result of fragmentation or instability within the medaka genome.     

Formation of the Dermal Chromatophore Unit (DCU) in the Tree Frog Hyla Arborea

In the tadpole of the tree frog Hyla arborea, the color of the dorsal skin was dark brown. Dermal melanophores, xanthophores, and iridophores were scattered randomly under the subepidermal collagen layer (SCL). After metamorphosis, the dorsal color of the animal changed to green and the animal acquired the ability of dramatic color change, demonstrating that the dermal chromatophore unit (DCU) was formed at metamorphosis. Fibroblasts invaded the SCL and divided it into two parts: the stratum spongiosum (SS) and the stratum compactum (SC). The activity of collagenase increased at metamorphosis. The fibroblasts appeared to dissolve the collagen matrix as they invaded the SCL. Then, three types of chromatophores migrated through the SCL and the DCU was formed in the SS. The mechanism how the three types of chromatophores were organized into a DCU is uncertain, but different migration rates of the three chromatophore types may be a factor that determines the position of the chromatophores in the DCU. Almost an equal number of each chromatophore type is necessary to form the DCUs. However, the number of dermal melanophores in the tadpoles was less than the number of xanthophores and iridophores. It was suggested that epidermal melanophores migrated to the dermis at metamorphosis and developed into dermal melanophores. This change may account for smaller number of dermal melanophores available to form the DCU_s.     

Melanophore characteristics in a fresh-water teleost, Puntius conchonius (Ham.)

The present study is an attempt to determine the factors responsible for the melanophore pattern of the freshwater teleost, Puntius conchonius (Ham.) under normal background conditions (i.e., the existence of a large round black spot on the middle of the side above the posterior part of the anal fin and the dull shade of the rest of the body which is brownish on the dorsal part, referred here in the present study as the general body surface and silvery along the lateral and ventral part of the body). On the basis of nature of their branching pattern various morphological types of melanophores are classified in a scale from the dark spot area as well as the general body surface. There are as many as 7 types of melanophores termed as A, B, C, AB/AC (all system I deep melanophores) and a, b and c (all system I superficial melanophores). The integument of the fish, however, possesses 3 kinds of chromatophores namely--melanophores, xanthophores and iridophores. The identity of the dark spot as clearly maintained entity independant from the general body surface may be attributed to greater number of system I melanophores, greater melanin content in the system I and system II melanophores, smaller interspaces between system I and system II deep melanophores, greater anastomosing in the superficial melanophores, larger size of system I deep as well as superficial melanophores. The reverse order of the above mentioned factors is responsible for the dull-brownish shade of the general body surface of the fish. The dark spot toward the tail end in the fish, possibly may serve to intimidate or misdirect attack and thus facilitate escape.     

The influence of long-term chromatic adaptation on pigment cells and striped pigment patterns in the skin of the zebrafish, Danio rerio

The striped pigment patterns in the flanks of zebrafish result from chromatophores deep within the dermis or hypodermis, while superficial melanophores associated with dermal scales add a dark tint to the dorsal coloration. The responses of these chromatophores were compared during the long-term adaptation of zebrafish to a white or a black background. In superficial skin, melanophores, xanthophores, and two types of iridophores are distributed in a gradient along the dorso-ventral axis independent of the hypodermal pigment patterns. Within one week the superficial melanophores and iridophores changed their density and/or areas of distribution, which adopted the dorsal skin color and the hue of the flank to the background, but did not affect the striped pattern. The increases or decreases in superficial melanophores are thought to be caused by apoptosis or by differentiation, respectively. When the adaptation period was prolonged for more than several months, the striped color pattern was also affected by changes in the width of the black stripes. Some black stripes disappeared and interstripe areas were emphasized with a yellow color within one year on a white background. Such long-term alteration in the pigment pattern was caused by a decrease in the distribution of melanophores and a concomitant increase in xanthophores in the hypodermis. These results indicate that morphological responses of superficial chromatophores contribute to the effective and rapid background adaptation of dorsal skin and while prolonged adaptation also affects hypodermal chromatophores in the flank to alter the striped pigment patterns.     

Observations on the development of unusual melanization of leopard frog ventral skin

The ontogeny of ventral pigmentation of two species of leopard frog, Rana pipiens and R. chiricahuensis, was examined by light microscopy and transmission electron microscopy to reveal how the unusual melanistic ventral pigmentation of R. chiricahuensis is achieved at the cellular level. Ventral skin of R. pipiens is always white. Ventral skin of adult R. chiricahuensis is white when frogs are background-adapted to a white substrate, but ventral skin becomes nearly as dark colored as the dorsal skin when frogs darken in response to a black background. Skin samples from tadpoles of both species, newly metamorphosed frogs, and adult frogs were analyzed for chromatophore composition and distribution. Ventral skin of R. pipiens larvae, newly metamorphosed frogs, and adults and of R. chiricahuensis larvae was white due to abundant iridophores and no melanophores. Melanophore density in the ventral integument of R. chiricahuensis was 9.1 ± 2.8/mm² in newly metamorphosed frogs and 87.0 ± 4.8/mm² in adult frogs. Pigment within ventral melanophores migrated during physiological color change during background adaptation. © 1993 Wiley-Liss, Inc.     

Dermal and epidermal chromatophores of the Antarctic teleost Trematomus bernacchii

The physiological response and ultrastructure of the pigment cells of Trematomus bernacchii, an Antarctic teleost that lives under the sea ice north of the Ross Ice Shelf, were studied. In the integument, two types of epidermal chromatophores, melanophores and xanthophores, were found; in the dermis, typically three types of chromatophores--melanophores, xanthophores, and iridophores--were observed. The occurrence of epidermal xanthophore is reported for the first time in fish. Dermal melanophores and xanthophores have well-developed arrays of cytoplasmic microtubules. They responded rapidly to epinephrine and teleost melanin-concentrating hormone (MCH) with pigment aggregation and to theophylline with pigment dispersion. Total darkness elicited pigment aggregation in the majority of dermal xanthophores of isolated scales, whereas melanophores remained dispersed under both light and dark conditions. Pigment organelles of epidermal and dermal xanthophores that translocate during the pigmentary responses are carotenoid droplets of relatively large size. Dermal iridophores containing large reflecting platelets appeared to be immobile.     

A ventrally localized inhibitor of melanization in Xenopus laevis skin

Melanophores normally differentiate in dorsal but not in ventral skin of Xenopus laevis. We have sought factors which might regulate this differentiation pattern, and we have obtained a putative melanization inhibiting factor (MIF) from ventral but not from dorsal skin. Preliminary studies reveal that MIF is destroyed by heat or trypsin treatment, indicating its protein composition, and has a molecular weight in the range of 300 kDa. The effects of MIF on the differentiation of neural crest derivatives to melanophores were examined in vitro in the presence of tyrosine and fetal calf serum (FCS). Tyrosine enhances melanophore differentiation in vitro at concentrations equivalent to those estimated in adult Xenopus blood plasma (20 microM). FCS also stimulates melanization, by way of materials other than the tyrosine contained in FCS. MIF strongly inhibits outgrowth and melanization of neural crest cells from neural tube explants. MIF also inhibits the differentiation of melanoblasts contained in cultured explants of ventral skin. Inhibition of melanization or melanophore differentiation by MIF occurs even in the presence of L-tyrosine and/or FCS. We suggest that MIF plays an important role in the establishment of dorso-ventral pigment patterns in amphibia.