Zebrafish Bioassay for Screening Therapeutic Candidates Based on Melanotrophic Activity.

In this study, we used the zebrafish animal model to establish a bioassay by which physiological efficacy differential of alpha-melanocyte-stimulating hormone (α-MSH) analogues could be measured by melanosome dispersion in zebrafish larvae. Brain-skin connection research has purported the interconnectedness between the nervous system and skin physiology. Accordingly, the neuropeptide α-MSH is a key regulator in several physiological processes, such as skin pigmentation in fish. In mammals, α-MSH has been found to regulate motivated behavior, appetite, and emotion, including stimulation of satiety and anxiety. Several clinical and animal model studies of autism spectrum disorder (ASD) have already demonstrated the effectiveness of α-MSH in restoring the social deficits of autism. Therefore, we sought to analyze the effect of synthetic and naturally-occurring α-MSH variants amongst different species. Our results showed that unique α-MSH derivatives from several fish species produced differential effects on the degree of melanophore dispersion. Using α-MSH human form as a standard, we could identify derivatives that induced greater physiological effects; particularly, the synthetic analogue melanotan-II (MT-II) exhibited a higher capacity for melanophore dispersion than human α-MSH. This was consistent with previous findings in an ASD mouse model demonstrating the effectiveness of MT-II in improving ASD behavioral symptoms. Thus, the melanophore assay may serve as a useful screening tool for therapeutic candidates for novel drug discovery.

P-Glycoprotein Inhibitor Tariquidar Plays an Important Regulatory Role in Pigmentation in Larval Zebrafish.

Zebrafish has emerged as a powerful model in studies dealing with pigment development and pathobiology of pigment diseases. Due to its conserved pigment pattern with established genetic background, the zebrafish is used for screening of active compounds influencing melanophore, iridophore, and xanthophore development and differentiation. In our study, zebrafish embryos and larvae were used to investigate the influence of third-generation noncompetitive P-glycoprotein inhibitor, tariquidar (TQR), on pigmentation, including phenotype effects and changes in gene expression of chosen chromatophore differentiation markers. Five-day exposure to increasing TQR concentrations (1 µM, 10 µM, and 50 µM) resulted in a dose-dependent augmentation of the area covered with melanophores but a reduction in the area covered by iridophores. The observations were performed in three distinct regions-the eye, dorsal head, and tail. Moreover, TQR enhanced melanophore renewal after depigmentation caused by 0.2 mM 1-phenyl-2-thiourea (PTU) treatment. qPCR analysis performed in 56-h post-fertilization (hpf) embryos demonstrated differential expression patterns of genes related to pigment development and differentiation. The most substantial findings include those indicating that TQR had no significant influence on leukocyte tyrosine kinase, GTP cyclohydrolase 2, tyrosinase-related protein 1, and forkhead box D3, however, markedly upregulated tyrosinase, dopachrome tautomerase and melanocyte inducing transcription factor, and downregulated purine nucleoside phosphorylase 4a. The present study suggests that TQR is an agent with multidirectional properties toward pigment cell formation and distribution in the zebrafish larvae and therefore points to the involvement of P-glycoprotein in this process.

Evaluation of hypopigmentation in embryonic zebrafish induced by emerging disinfection byproduct, 3, 5-di-I-tyrosylalanine.

Halogenated dipeptides, 3, 5-di-I-tyrosylalanine (DIYA), have been identified as novel disinfection byproducts (DBPs), following chloramination of authentic water. However, little is known about their toxicity. Zebrafish embryos were used to assess the toxicity of novel iodinated DBPs (I-DBPs). Although DIYA did not exhibit high acute toxicity to embryonic zebrafish (LC50 > 2 mM), it significantly inhibited pigmentation of melanophores and xanthophores on head, trunk and tail at 500 µM as determined by photographic analysis. Whereas N-phenylthiourea (PTU) as a pigment inhibitor did not inhibit development of yellow pigments. Colorimetric detection of melanin further confirmed these results. Quantitative real time polymerase chain reaction (qRT-PCR) measurements indicated that genes (dct, slc24a5, tyr, tyrp1a, tyrp1b, silva) associated with the melanogenesis pathway were dramatically down-regulated following exposure to 500 µM DIYA. In addition, enzymatic activity of tyrosinase (TYR) decreased, also demonstrating that the underlying mechanism of hypopigmentation was attributed to the disruption of melanogenesis pathway. Transcription levels of xanthophore genes (gch2, bnc2, csf1a, csf1b, pax7a and pax7b) were also monitored by qRT-PCR assay. DIYA exposure up-regulated expression of gch2 and bnc2, but not csf1 and pax7. Tested DIYA analogues, brominated tyrosine was unlikely to inhibit pigmentation, indicating that the iodine substitution and dipeptides structure are of important structural feature for the inhibition of pigmentation. In this study, we observed that DIYA inhibited melanogenesis related genes, which might contribute to pigmentation defects. Moreover, as an emerging I-DBPs, the developmental toxicity of aromatic dipeptides should be further studied.

Experimental manipulation of perceived predation risk and cortisol generates contrasting trait trajectories in plastic crucian carp.

Most animals constitute potential prey and must respond appropriately to predator-mediated stress in order to survive. Numerous prey also adaptively tailor their response to the prevailing level of risk and stress imposed by their natural enemies, i.e. they adopt an inducible defence strategy. Predator exposure may activate the stress axis, and drive the expression of anti-predator traits that facilitate survival in a high-risk environment (the predation-stress hypothesis). Here, we quantified two key morphological anti-predator traits, body morphology and coloration, in crucian carp reared in the presence or absence of a predator (pike) in addition to experimental manipulation of physiological stress via implants containing either cortisol or a cortisol inhibitor. We found that predator-exposed fish expressed a deeper-bodied phenotype and darker body coloration as compared with non-exposed individuals. Skin analyses revealed that an increase in the amount of melanophores caused the dramatic colour change in predator-exposed fish. Increased melanization is costly, and the darker body coloration may act as an inducible defence against predation, via a conspicuous signal of the morphological defence or by crypsis towards dark environments and a nocturnal lifestyle. By contrast, the phenotype of individuals carrying cortisol implants did not mirror the phenotype of predator-exposed fish but instead exhibited opposite trajectories of trait change: a shallow-bodied morphology with a lighter body coloration as compared with sham-treated fish. The cortisol inhibitor did not influence the phenotype of fish i.e. neither body depth nor body coloration differed between this group and predator-exposed fish with a sham implant. However, our results illuminate a potential link between stress physiology and morphological defence expression.

The Flavor Enhancer Maltol Increases Pigment Aggregation in Dermal and Neural Melanophores in Xenopus laevis Tadpoles.

Melanophores are pigmented cells that change the distribution of melanosomes, enabling animals to appear lighter or darker for camouflage, thermoregulation, and protection from ultraviolet radiation. A complex series of hormonal and neural mechanisms regulates melanophore pigment distribution, making these dynamic cells a valuable tool to screen toxicants as they rapidly respond to changes in the environment. We found that maltol, a naturally occurring flavor enhancer and fragrance agent, induces melanophore pigment aggregation in a dose-dependent manner in Xenopus laevis tadpoles. To determine if maltol affects camouflage adaptation, we placed tadpoles into maltol baths situated over either a white or a black background. Maltol induced pigment aggregation in a similar dose-dependent pattern regardless of background color. We also tested how maltol treatment compares to melatonin treatment and found that the degree of pigment aggregation induced by maltol is similar to treatment with melatonin but that maltol induces over a much longer time course. Last, maltol had no effect on mRNA expression in the brain of genes that regulate camouflage-related pigment aggregation. The present results suggest that maltol does not exert its effects via the camouflage adaptation mechanism or via melatonin-related mechanisms. These results are the first to identify a putative toxicological effect of maltol exposure in vivo and rule out several mechanisms by which maltol may exert its effects on pigment aggregation. Environ Toxicol Chem 2020;39:381-395. © 2019 SETAC.

Investigation of multiple-dynein transport of melanosomes by non-invasive force measurement using fluctuation unit χ.

Pigment organelles known as melanosomes disperse or aggregate in a melanophore in response to hormones. These movements are mediated by the microtubule motors kinesin-2 and cytoplasmic dynein. However, the force generation mechanism of dynein, unlike that of kinesin, is not well understood. In this study, to address this issue, we investigated the dynein-mediated aggregation of melanosomes in zebrafish melanophores. We applied the fluctuation theorem of non-equilibrium statistical mechanics to estimate forces acting on melanosomes during transport by dynein, given that the energy of a system is related to its fluctuation. Our results demonstrate that multiple force-producing units cooperatively transport a single melanosome. Since the force is generated by dynein, this suggests that multiple dyneins carry a single melanosome. Cooperative transport has been reported for other organelles; thus, multiple-motor transport may be a universal mechanism for moving organelles within the cell.

Whole-Cell Photoacoustic Sensor Based on Pigment Relocalization.

Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In a biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.

A novel anti-melanogenic agent, KDZ-001, inhibits tyrosinase enzymatic activity.

BACKGROUND: The demand for anti-melanogenic agents is increasing due to the unwanted side effects of current treatments. To find an effective anti-melanogenic agent, we used zebrafish as a whole animal model for phenotype-based drug and cosmetic discovery screening. OBJECTIVES: The aim of this study was to identify and explore a small molecule that could be used for skin-whitening cosmetics. METHODS: Using zebrafish embryos, we examined the effects of 1000 compounds on zebrafish development and pigmentation. Pigmentation production was assessed by tyrosinase (TYR) enzymatic activity and melanin contents. Pigmentation marker expression in the human melanoma cell line HMV-II was analyzed by western blot. We also tested reconstituted human skin tissue and analyzed KDZ-001 with computational molecular modeling. RESULTS: We identified three compounds that affected the pigmentation of developing melanophores in zebrafish. Among them, we identified KDZ-001, a novel anti-melanogenic agent, which strongly inhibits melanin synthesis in the developing melanophores of zebrafish, HMV-II cells, and reconstituted human skin with no toxicity. We found that KDZ-001 directly inhibits TYR enzymatic activity. Notably, computational molecular modeling of KDZ-001 suggested that its interaction with copper ions in the active site of TYR is essential for melanin synthesis, further demonstrating that KDZ-001 mainly acts as a TYR inhibitor to synthesize melanin. CONCLUSION: KDZ-001 inhibits melanin synthesis and has a potential for use in skin-whitening cosmetics.

Thyroid Hormones Regulate Zebrafish Melanogenesis in a Gender-Specific Manner.

Zebrafish embryos are treated with anti-thyroidal compounds, such as phenylthiourea, to inhibit melanogenesis. However, the mechanism whereby the thyroidal system controls melanin synthesis has not been assessed in detail. In this work, we tested the effect of the administration of diets supplemented with T3 (500µg/g food) on the pigment pattern of adult zebrafish. Oral T3 induced a pronounced skin paling in both adult female and male zebrafish that was reversible upon cessation of treatment. The number of visible melanophores was significantly reduced in treated fish. Accordingly, treatment down-regulated expression of tyrosinase-related protein 1 in both sexes. We also found sexually dimorphic regulation of some melanogenic genes, such as Dct/Tyrp2 that was dramatically up-regulated in females after T3 treatment. Thus, we demonstrated that melanogenesis is reversibly inhibited by thyroid hormones in adult zebrafish and make the discovery of gender-specific differences in the response of melanogenic gene expression. Thus, fish gender is now shown to be an important variable that should be controlled in future studies of fish melanogenesis.

Analogue of Melanotan II (MTII): A Novel Melanotropin with Superpotent Action on Frog Skin.

An α-MSH peptide analogue, named MTII (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]- NH2), is one of the most important ligands of melanotropic receptors but are relatively nonselective. In order to improve the melanotropic activities of the well-characterized MTII analogues, we report here a new analogue by modifying the core structure as well as the size of the cyclic region of MTII peptide. The analogue peptide, Ac-Nle-c[Asp-His-D-Phe-Lys-Trp-Gly-Lys]-OH (F Peptide), in which we replaced Arg at position 8 with Lys and added a Gly to position 10 of the MTII peptide sequence, was synthesized and used as a new melanotropic hormone in controlling rapid color changes in frogs by its actions on mobilizing pigment granule movements within chromatophores. The in vivo responses of chromatophores to MTII and the related analogue F Peptide were studied in frogs. The results show that the F Peptide was a superpotent agonist with similar melanotropic activity to the MTII peptide according to MTII peptide by in vivo studies. The analogue also exhibited ultraprolonged melanotropic activity. The F peptide can be useful in the study of numerous physiological processes, particularly when superpotent and prolonged melanotropic activity is desired.

Colour changes in Labeo rohita (Ham.) due to pigment translocation in melanophores, on exposure to municipal wastewater of Tung Dhab drain, Amritsar, India.

The present study was aimed to evaluate the effect of municipal wastewater of Tung Dhab drain on morphology of scale melanophores in freshwater fish Labeo rohita (Ham.). Chronic, non-renewal toxicity tests were performed with sub lethal concentrations (17.7, 26.6 and 35.4%) of wastewater for exposure durations of 15, 30 and 60 days. Recovery experiments were also performed for duration of 60 days. The scales were removed, processed and diameters of melanophores were measured using an ocular micrometer. The results showed concentration and duration dependent changes in melanophore morphology (size and dendricity) in experimental fish as compared to control fish. The values of mean melanophore size index (MMSI) varied from 5.37 ± 0.49 (17.7%) to 12.12 ± 0.81 (35.5%) in comparison to control values of 4.32 ± 0.32 and 4.55 ± 0.29 for 15 and 60 days respectively. The recovery experiments suggested that observed dark colouration due to pigment translocation is reversible, even after chronic exposure for 60 days.

Correlation of melanophore index with a battery of functional genomic stress indicators for measurement of environmental stress in aquatic ecosystem.

The correlation of primary stress indicator; melanophore index (MI) with set of genomic stress indicators is important for a better understanding of the cellular stress pathway induced by xenobiotics in aquatic species. This study presents a correlation between melanophore index (MI) and genomic stress indicators in Oreochromis mossambicus treated with lead nitrate, phenol and hexachlorocyclohexane (HCH). O. mossambicus was exposed to sub-lethal concentrations of the different LC50 values (96 h) of the tested chemicals at varying exposure periods and the response via genomic stress indicators and scale melanophores were assessed in accordance with standard protocols. Melanophore index decreased significantly (p<0.01) in a time dependent pattern to the tested chemicals. Gene expression showed significant time dependent increase in the expression of heat shock proteins (HSP70 and HSP60). Vitellogenin (Vtg) expression insignificantly altered. Significant increase in the expression of melanin concentrating hormone (MCH) was observed in response to hexachlorocyclohexane (HCH) in the treated fish. The findings demonstrated an inverse relationship between melanophore index and the set of genomic stress indicators.

Evidence for the presence of novel ß-melatonin receptors along with classical α-melatonin receptors in the fish Rasbora daniconius (Ham.).

The effects of melatonin (MT) were examined on the isolated scale melanophores from dorso-lateral (D-L) and band regions of a tropical fish Rasbora daniconius. Our study primarily aimed for further depiction of the signaling receptors involved in MT mediated pigment translocations in the fish. Melanophore Size Index (MSI) was employed as a recording parameter for the responses of melanophores to MT and various antagonists. MT has induced aggregation as well as dispersion in D-L region and aggregation in band region melanophores during summer season. During winter, MT-induced responses were only of aggregatory type in D-L region, while in the band region there was an increase in the sensitivity. The responses of the melanophores to MT were reversible. The aggregation of innervated melanophores induced by MT on the D-L and band regions was partially mediated through the neurotransmitters released under the influence of MT and partially by the specific MT receptors. Luzindole and K185 have completely blocked the aggregatory responses of D-L and band region melanophores. Aggregatory receptors may be of the conventional α-MT type. Dispersion of D-L and band region melanophores induced by MT in the presence of various antagonists and on denervated band region could be the result of activation of ß-MT receptors of dispersive nature. Presence of α and ß MT receptors is thus indicated in this fish melanophores.

Thyroid hormone-dependent adult pigment cell lineage and pattern in zebrafish.

Pigment patterns are useful for elucidating fundamental mechanisms of pattern formation and how these mechanisms evolve. In zebrafish, several pigment cell classes interact to generate stripes, yet the developmental requirements and origins of these cells remain poorly understood. Using zebrafish and a related species, we identified roles for thyroid hormone (TH) in pigment cell development and patterning, and in postembryonic development more generally. We show that adult pigment cells arise from distinct lineages having distinct requirements for TH and that differential TH dependence can evolve within lineages. Our findings demonstrate critical functions for TH in determining pigment pattern phenotype and highlight the potential for evolutionary diversification at the intersection of developmental and endocrine mechanisms.

Endothelin modulates the circadian expression of non-visual opsins.

The non-visual opsin, melanopsin, expressed in the mammalian retina, is considered a circadian photopigment because it is responsible to entrain the endogenous biological clock. This photopigment is also present in the melanophores of Xenopus laevis, where it was first described, but its role in these cells is not fully understood. X. laevis melanophores respond to light with melanin granule dispersion, the maximal response being achieved at the wavelength of melanopsin maximal excitation. Pigment dispersion can also be triggered by endothelin-3 (ET-3). Here we show that melanin translocation is greater when a blue light pulse was applied in the presence of ET-3. In addition, we demonstrated that mRNA levels of the melanopsins Opn4x and Opn4m exhibit temporal variation in melanophores under light/dark (LD) cycles or constant darkness, suggesting that this variation is clock-driven. Moreover, under LD cycles the oscillations of both melanopsins show a circadian profile suggesting a role for these opsins in the photoentrainment mechanism. Blue-light pulse decreased Opn4x expression, but had no effect on Opn4m. ET-3 abolishes the circadian rhythm of expression of both opsins; in addition the hormone increases Opn4x expression in a dose-, circadian time- and light-dependent way. ET-3 also increases the expression of its own receptor, in a dose-dependent manner. The variation of melanopsin levels may represent an adaptive mechanism to ensure greater melanophore sensitivity in response to environmental light conditions with ideal magnitude in terms of melanin granule dispersion, and consequently color change.

Berberine-induced pigment dispersion in Bufo melanostictus melanophores by stimulation of beta-2 adrenergic receptors.

Reduced production of melanin by decreased or the absence of melanocytes leads to various hypopigmentation disorders, and the development of melanogenetic agents for photoprotection and hypopigmentation disorders is one of the top priority areas of research. Hence, the present study was carried out to elucidate the ability of berberine, a principal active ingredient present in the roots of the herb Berberis vulgaris to stimulate pigment dispersion in the isolated skin melanophores of the toad Bufo melanostictus. In the present study, mean melanophore size index of the isolated skin melanophores of B. melanostictus was assayed after treating with various concentrations of berberine. A marked melanin dispersion response leading to skin darkening was observed in the isolated melanophores of toad in response to berberine, which was found to be mediated through beta-2 adrenergic receptors. The physiologically significant dose-related melanin dispersion effects of berberine per se were found to be completely abolished by propranolol, which is a specific beta-2 adrenergic receptor blocker. These per se melanin dispersal effects were also found to be markedly potentiated by isoprenaline, which is a specific beta-adrenoceptor agonist. The results indicate that berberine causes a tremendous, dose-dependent, physiologically significant pigment dispersing in the isolated skin melanophores of B. melanostictus.

Role of catecholamines and nitric oxide on pigment displacement of the chromatophores of freshwater snakehead teleost fish, Channa punctatus.

We are reporting for the first time that the catecholamines (adrenaline and noradrenaline) inhibit the effect of nitric oxide (NO) on melanosome dispersion in freshly isolated scales of the freshwater snakehead fish, Channa punctatus. We studied the effect of NO and catecholamines on the pigment displacement by observing the changes in the melanophore index. The scales when treated with solution containing NO donor sodium nitroprusside (SNP) showed dispersion of melanosomes, whereas NO synthase blocker N-omega-Nitro-L-arginine suppresses this action of SNP. Treatment with adrenaline and noradrenaline on the isolated scales caused aggregation of melanosomes. Scales treated with solution containing catecholamines and SNP resulted in aggregation of melanosomes suggesting that catecholamines mask the effect of SNP. These results suggest that the catecholamines are inhibiting the effect of NO and causing the aggregation of the melanosomes may be via surface receptors.

oca2 Regulation of chromatophore differentiation and number is cell type specific in zebrafish.

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410-kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2-deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar-type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore-specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post-fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.

Maintenance of melanophore morphology and survival is cathepsin and vps11 dependent in zebrafish.

Here, we characterize a Danio rerio zebrafish pigment cell mutant (melanophore integrity mutant), which displays a defect in maintenance of melanophore and iridophore number. Mapping and candidate gene analysis links the melanophore integrity mutant mutation to the vacuolar protein sorting 11 (vps11(w66)) gene. Quantification of vps11(w66) chromatophores during larval stages suggests a decrease in number as compared to wildtype siblings. TUNEL analysis and treatment with the caspase inhibitor, zVAD-fmk, indicate that vps11(w66) chromatophore death is caspase independent. Western blot analysis of PARP-1 cleavage patterns in mutant lysates suggests that increases in pH dependent cathepsin activity is involved in the premature chromatophore death observed in vps11(w66) mutants. Consistently, treatment with ALLM and Bafilomycin A1 (cathepsin/calpain and vacuolar-type H+-ATPase inhibitors, respectively), restore normal melanophore morphology and number in vps11(w66) mutants. Last, LC3B western blot analysis indicates an increase in autophagosome marker, LC3B II in vps11(w66) mutants as compared to wildtype control, but not in ALLM or Bafilomycin A1 treated mutants. Taken together, these data suggest that vps11 promotes normal melanophore morphology and survival by inhibiting cathepsin release and/or activity.

Establishment of HRAS(G12V) transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs.

Most targeted anticancer drugs have been identified by screening at the molecular or cellular level in vitro. However, many compounds selected by such costly and time-consuming screening do not prove effective against tumors in vivo. The development of anticancer drugs would thus be facilitated by the availability of an in vivo screening system based on a multicellular organism. We have now established a transgenic line of the freshwater fish medaka in which melanophores (melanocytes) proliferate in a manner dependent on heat shock-induced signaling by a human RAS oncoprotein. The human HRAS(G12V) oncogene was expressed under the control of a melanophore-specific gene promoter in order to allow visualization of tumor growth in live fish maintained in a water tank. The expression of HRAS(G12V) was induced as a result of Cre-mediated recombination by exposure of the fish to a temperature of 37°C for 30 min, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. The transgenic medaka established here thus represents a promising in vivo system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes.