Insights from zebrafish on human pigment cell disease and treatment.

Black pigment cells, melanocytes, arise early during development from multipotent neural crest cells. Melanocytes protect human skin from DNA damaging sunrays and provide color for hair, eyes, and skin. Several disorders and diseases originate from these cells, including the deadliest skin cell cancer, melanoma. Thus, melanocytes are critical for a healthy life and for protecting humans from disease. Due to the ease of visualizing pigment cells through transparent larvae skin and conserved roles for zebrafish melanophore genes to mammalian melanocyte genes, zebrafish larvae offer a biologically relevant model for understanding pigment cell development and disease in humans. This review discusses our current knowledge of melanophore biology and how zebrafish are contributing to improving how diseases of melanocytes are understood and treated in humans. Developmental Dynamics 246:889-896, 2017. © 2017 Wiley Periodicals, Inc.

Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

Zebrafish neurofibromatosis type 1 genes have redundant functions in tumorigenesis and embryonic development.

Neurofibromatosis type 1 (NF1) is a common, dominantly inherited genetic disorder that results from mutations in the neurofibromin 1 (NF1) gene. Affected individuals demonstrate abnormalities in neural-crest-derived tissues that include hyperpigmented skin lesions and benign peripheral nerve sheath tumors. NF1 patients also have a predisposition to malignancies including juvenile myelomonocytic leukemia (JMML), optic glioma, glioblastoma, schwannoma and malignant peripheral nerve sheath tumors (MPNSTs). In an effort to better define the molecular and cellular determinants of NF1 disease pathogenesis in vivo, we employed targeted mutagenesis strategies to generate zebrafish harboring stable germline mutations in nf1a and nf1b, orthologues of NF1. Animals homozygous for loss-of-function alleles of nf1a or nf1b alone are phenotypically normal and viable. Homozygous loss of both alleles in combination generates larval phenotypes that resemble aspects of the human disease and results in larval lethality between 7 and 10 days post fertilization. nf1-null larvae demonstrate significant central and peripheral nervous system defects. These include aberrant proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), dysmorphic myelin sheaths and hyperplasia of Schwann cells. Loss of nf1 contributes to tumorigenesis as demonstrated by an accelerated onset and increased penetrance of high-grade gliomas and MPNSTs in adult nf1a(+/-); nf1b(-/-); p53(e7/e7) animals. nf1-null larvae also demonstrate significant motor and learning defects. Importantly, we identify and quantitatively analyze a novel melanophore phenotype in nf1-null larvae, providing the first animal model of the pathognomonic pigmentation lesions of NF1. Together, these findings support a role for nf1a and nf1b as potent tumor suppressor genes that also function in the development of both central and peripheral glial cells as well as melanophores in zebrafish.

Expression regulation triggers oncogenicity of xmrk alleles in the Xiphophorus melanoma system.

The Xiphophorus melanoma model has gained attention in biomedical research as a genetic model for tumor formation. Melanoma development in interspecific hybrids of Xiphophorus is connected to pigment cell specific overexpression of the mutationally activated receptor tyrosine kinase Xmrk. In purebred fish the oncogenic function of xmrk is suppressed by a so far unknown regulator locus R. To test the hypothesis that R is involved in transcriptional regulation of xmrk and consequently acts upstream of the xmrk signal, we performed a quantitative analysis of xmrk transcript levels in normal and melanoma tissues of different Xiphophorus genotypes carrying either a highly tumorigenic or a non-tumorigenic xmrk allele. Our results demonstrate that expression of the tumorigenic xmrk allele is highly increased in malignant melanomas compared to benign lesions, macromelanophore spots, and healthy skin. Transcription of the non-tumorigenic xmrk allele in pigment cells, in contrast, is not influenced by the presence or absence of R. These findings strongly indicate that differential transcriptional regulation of the xmrk promoter determines the tumorigenic potential of xmrk alleles in the Xiphophorus melanoma system, thereby supporting the hypothesis that R suppresses the oncogenic function of xmrk on the level of transcriptional control.

A sub-acute study of metronidazole toxicity assessed in Egyptian Tilapia zillii.

Metronidazole (MTZ), an antiparasitic and antibacterial compound, is one of the world's most widely used drugs. Despite being considered as a rodent mutagen and a carcinogen, it is still widely used in humans for the treatment of infections with anaerobic organisms. Therefore, the main objective of the current study was to evaluate the in vivo toxicity of MTZ using the micronucleus (MN) assay and random amplified polymorphism DNA (RAPD-PCR) analysis as well as histopathological examination in Tilapia zillii. Moreover, the protective effect of vitamin C (VitC) against toxicity of MTZ was investigated in the present study. Fish were treated with three doses of MTZ (5, 10 and 20 mg l(-1)) alone or in combination with VitC (200 mg kg(-1) food) at several time intervals (2 days, 7 days and 14 days). The results of the present study showed a significant effect of MTZ on micronucleus formation and changes in polymorphic band patterns as well as induction of different histopathological alterations in Tilapia zillii. The effects of the drug were reduced when fish were exposed to a combination of MTZ and VitC.

Pigmentation development, defects, and patterning in summer flounder (Paralichthys dentatus).

Flounders offer unique opportunities to study the cytological basis of vertebrate pigmentation. Individual skin pigment cells are clearly visible at hatching, and flounder ontogeny includes a dramatic shift in overall pigmentation (from symmetrical to asymmetrical) during metamorphosis. Moreover, several types of malpigmentation occur in hatchery populations; although much effort has gone into reducing the frequency of such defects, their etiology remains poorly understood, and they have rarely been described at the cellular level. In this paper, we use light and fluorescence microscopy to describe the cytological basis of normal developmental changes and of common types of malpigmentation. We then discuss the implications of these observations for underlying patterning mechanisms.

Melanoma development and pigment cell transformation in xiphophorus.

As early as 1927, it was recognised that hybridisation of platyfish (Xiphophorus maculatus) and swordtails (Xiphophorus helleri) results in offspring that develop tumours according to Mendelian laws. Most obviously, the primary event, namely the cell lineage-specific overexpression of a structurally altered receptor tyrosine kinase, finds its parallel in many tumours of birds and mammals. Once expressed at high levels, this receptor, the Xiphophorus melanoma inducing receptor kinase Xmrk, shows constitutive activation. By using different pathways, Xmrk induces both proliferative as well as anti-apoptotic signalling in pigment cells finally leading to cell transformation, tumour induction, and progression. Analyses of the different signalling cascades induced by the Xmrk-receptor led to the identification of the src-kinase Fyn, the MAP kinases ERK1 and ERK2, the "Signal Transducer and Activator of Transcription" STAT5, and the PI3-kinase as its major downstream substrates. This review describes some of the genetic findings, as well as the results from the recent molecular analyses of the factors involved in the initiation and manifestation of pigment cell transformation and melanoma development in Xiphophorus.

A comparison of melanin bleaching and azure blue counterstaining in the immunohistochemical diagnosis of malignant melanoma.

Distinguishing heavily pigmented melanocytes from melanophages on routine hematoxylin and eosin slides can be difficult. Melanin bleaching with potassium permanganate solution is a traditional means of removing melanin from tissues and can be used before immunohistochemical staining to remove any pigment that might be confused with the brown chromogen diaminobenzidine. Azure B stains melanin granules green-blue, easily contrasts with diaminobenzidine, and may be used as a counterstain on unbleached sections after immunohistochemical staining. To our knowledge, studies comparing melanin bleaching with azure B counterstaining in the immunohistochemical evaluation of malignant melanomas have not been performed. Paraffin sections from 33 heavily pigmented malignant melanomas were bleached with a 3.0-g/L potassium permanganate solution, immunohistochemically stained for S-100 and HMB-45, and counterstained with hematoxylin. Unbleached sections were similarly stained for S-100 and HMB-45 and counterstained with azure B. To establish optimal permanganate concentrations, a variable number of sections were bleached with lower permanganate concentrations ranging from 0.125 to 2.5 g/L. S-100 antigenicity was preserved at all permanganate concentrations, whereas HMB-45 antigenicity was abolished at concentrations of 0.5 g/L and greater. At permanganate concentrations from 0.125 to 0.5 g/L, both antigenicities were preserved; however, melanin was incompletely removed. Complications of bleaching included tissue damage and loss of cytologic detail. Positive immunohistochemical staining was observed in azure B counterstained sections. Azure B stained melanin greenblue and was easily distinguished from the brown diaminobenzidine chromogen, regardless of the antibody tested. Neither tissue damage nor loss of cytologic detail was observed. We conclude that the use of azure B counterstaining is superior to permanganate bleaching in the histologic evaluation of heavily pigmented cutaneous malignant melanomas.

Zebrafish pigmentation mutations and the processes of neural crest development.

Neural crest development involves cell-fate specification, proliferation, patterned cell migration, survival and differentiation. Zebrafish neural crest derivatives include three distinct chromatophores, which are well-suited to genetic analysis of their development. As part of a large-scale mutagenesis screen for embryonic/early larval mutations, we have isolated 285 mutations affecting all aspects of zebrafish larval pigmentation. By complementation analysis, we define 94 genes. We show here that comparison of their phenotypes permits classification of these mutations according to the types of defects they cause, and these suggest which process of neural crest development is probably affected. Mutations in eight genes affect the number of chromatophores: these include strong candidates for genes necessary for the processes of pigment cell specification and proliferation. Mutations in five genes remove part of the wild-type pigment pattern, and suggest a role in larval pigment pattern formation. Mutations in five genes show ectopic chromatophores in distinct sites, and may have implications for chromatophore patterning and proliferation. 76 genes affect pigment or morphology of one or more chromatophore types: these mutations include strong candidates for genes important in various aspects of chromatophore differentiation and survival. In combination with the embryological advantages of zebrafish, these mutations should permit cellular and molecular dissection of many aspects of neural crest development.

Increasing melanoma incidence: putatively explainable by retrotransposons. Experimental contributions of the xiphophorine Gordon-Kosswig melanoma system.

The worldwide accelerating increase of neoplasia in humans is difficult to explain. We use the Xiphophorus tumor model to approach this problem by melanoma provocation with X-rays. Melanoma develops following inappropriate expression of x-erb B-conducted developmental genes and their controllers. These oncodeterminants are inherited according to Mendelian rules. We detected a new type of oncodeterminants that, following a single treatment of embryos with X-rays, generates a self-generating non-Mendelian melanoma transmission and accelerating increase of its incidence in succeeding generations (e.g., 0-->18-->33-->52%). To localize these oncodeterminants, we crossed nonirradiated fish having half of their chromosomes irradiated with nonirradiated fish having none of, half of, or all of their chromosomes irradiated. Because tumor rate and expression in the following generations correspond to the rates of treated chromosomes, we conclude that the new oncodeterminants are distributed over the chromosomes of the fish, where they may increase in the changing generations. By means of xiphophorine-specific retroviral DNA, we isolated two retrotransposons that behave hereditarily like the new transgenerational oncodeterminants. Sequence analysis revealed three ORFs flanked by LTRs containing motives of regulatory sequences typical for known retroviral and retrotransposal LTRs. Pol- and env-resembling sequences are lacking. Southern and in situ hybridization showed their multiple and repetitive nature distributed throughout the chromosomes and indications for their capability to increase in number without further treatment. Their transcripts are expressed in concert with those of most of the other known xiphophorine tumor determinants. Their expression is extremely high in cell cultures from tumorous embryos derived from ancestors treated as embryos with X-rays.

A case of extramammary Paget's disease with depigmented macules as the sole manifestation.

We report a 76-year-old man who had four depigmented macules in the genital area as the sole manifestation of extramammary Paget's disease (EMP). Histologically, many scattered, dissociated, plump Paget cells, and small intraepidermal nests of these cells were seen in all four lesions. The distribution of Paget cells extended beyond the margin of the depigmented areas into adjacent normally pigmented skin. Fontana-Masson staining revealed a reduction in, or absence of, melanin deposition along the basal layer of the depigmented lesions, in contrast with an abundance of melanin along the basal layer of the adjacent normal skin. Pigment-blockade melanocytes and melanophages were seen within or below the affected epidermis. The depigmentation in this case could have been caused by a symbiotic disorder between melanocytes and keratinocytes (including melanocyte destruction), and by a disorder in melanosome transmission to the keratinocytes. This case illustrates that a depigmented macule may be a diagnostic feature of EMP. Moreover, depigmentation is probably one of the earliest clinical features of EMP, and not a neighbouring secondary change such as occurs in the Sutton's halo naevus phenomenon.

Chromatophoromas and chromatophore hyperplasia in Pacific rockfish (Sebastes spp.).

Pacific rockfish from Cordell Bank, off central California (United States), were collected and histologically examined from 1985 to 1990. Hyperplastic and neoplastic cutaneous lesions, involving dermal chromatophores, were observed in five species; yellowtail rockfish (Sebastes flavidus), bocaccio (S. paucispinis), olive rockfish (S. serranoides), widow rockfish (S. entomelas), and chilipepper rockfish (S. goodei). Yearly prevalences were highest in S. paucispinis (29-38%). Prevalence was initially low in S. flavidus, but increased more than 3-fold from 1985 (7.5%) to 1990 (25%). The majority of lesions were black, but white, yellow, orange, red, and mixed-color variants were also seen. Lesions were found in skin, fins, lips, gingiva, tongue, urogenital papilla, conjunctiva, and cornea of the eye. Flat lesions were consistent with melanophore (black), xanthophore (yellow or orange), and erythrophore (red) hyperplasia. Neoplastic lesions included melanophoromas, amelanotic melanophoromas, xanthophoromas, erythrophoromas, and mixed chromatophoromas. Although etiology has not been determined, interest is currently focused on potential exposure to chemical and radioactive carcinogens from the Farallon Island Radioactive Waste Dump, 30 km to the south.

Biosynthetic pathways of pteridines and their association with phenotypic expression in vitro in normal and neoplastic pigment cells from goldfish.

The distribution of GTP-cyclohydrolase I, pyruvoyl tetrahydropterin (dysopropterin) synthase, and pyruvoyl tetrahydropterin reductase in goldfish erythrophores, melanophores, and erythrophoroma cells in vitro has been revealed by specific biochemical assays. The activity of pyruvoyl tetrahydropterin synthase in the erythrophores is nearly the same as that in rat kidney and pineal gland. Results of the simultaneous quantification of unconjugated pteridines (biopterin, sepiapterin, neopterin, and pterin) by HPLC indicate that the total amounts of these derivatives present in these cells and in the respective culture media are closely correlated with the activities of these enzymes. These findings imply that these cells are capable of the autonomous synthesis of pteridines, which most likely proceeds from GTP to 6-lactoyl-5,6,7,8-tetrahydropterin (reduced sepiapterin), via dihydroneopterin triphosphate and pyruvoyl tetrahydropterin, through reactions catalyzed by these enzymes. A comparison of pteridine metabolism between clones of the stem cell type and the yellow-pigmented clones induced from erythrophoroma cells suggests that brightly colored pigmentation involves two separate phases: the biosynthesis of pteridines and their deposition in the pigment organelles. The presence of the highly active pteridine-synthesizing enzymes in melanophores and melanogenic erythrophoroma cells strongly suggests a loose commitment to the expression of pigment phenotypes in this species.

Dermatosis cenicienta: presentación de cinco observaciones / Ashy dermatosis: a report of five patients

Se presentan 5 observaciones de dermatosis cenicienta en tres hombres y dos mujeres, con edades que oscilan entre 30 y 54 años, evolución entre tres meses y 17 años y crecimiento progresivo. En ninguno hubo antecedentes de consumo de drogas antiparasitarias, vitaminas o antecedentes de infecciones , inflamaciones, inmunizaciones ni exposición al sol por largas horas. La histopatología fue típica en todos ellos, tanto en las porciones centrales como en los bordes activos. El tratamiento con crioterapia parece tener algún efecto beneficioso, pero necesitamos ensayarlo en más casos.

Dermatosis cenicienta: presentación de cinco observaciones / Ashy dermatosis: a report of five patients

Se presentan 5 observaciones de dermatosis cenicienta en tres hombres y dos mujeres, con edades que oscilan entre 30 y 54 años, evolución entre tres meses y 17 años y crecimiento progresivo. En ninguno hubo antecedentes de consumo de drogas antiparasitarias, vitaminas o antecedentes de infecciones , inflamaciones, inmunizaciones ni exposición al sol por largas horas. La histopatología fue típica en todos ellos, tanto en las porciones centrales como en los bordes activos. El tratamiento con crioterapia parece tener algún efecto beneficioso, pero necesitamos ensayarlo en más casos. (AU)

An association of melanophores appearing at metamorphosis as vehicles of asymmetric skin color formation with pigment anomalies developed under hatchery conditions in the Japanese flounder, Paralichthys olivaceus.

The mechanisms for asymmetric skin color formation in the Japanese flounder are studied with particular concerns to causes for pigment disorder (hypomelanosis) occurring under hatchery conditions. For an analysis of normal pigmentation, fish were raised with wild zooplanktons in an indoor hatchery, whilst for hypomelanosis, they were raised with Brazilian Artemia nauplii, a diet used in the hatcheries. Morphological observations, counting of melanophores, histochemical assay of DOPA-positive immature cells (melanoblasts), and radiometric estimation of tyrosinase activities in skins of developing larvae and juveniles indicate that 1) the structural plan for pigmentation in this species is bilaterally symmetric until metamorphosis, utilizing large-sized melanophores (hence larval melanophores) as main vehicles, and 2) an asymmetric coloration characteristic to metamorphosed juveniles is formed by an intensive development of smaller-sized melanophores (hence adult-type melanophores) appearing selectively in the ocular side at the later stages of metamorphosis and by an absence of it in the blind. These findings apparently indicate that 1) two types of melanophores occur in this species which differ with respect to morphological properties and developmental fate, and 2) selective differentiation of adult type melanophores in the ocular side of the body at or after metamorphosis is primarily responsible for an asymmetric coloration of its adult form. The similar assays on the fish fed with Artemia nauplii indicate that defective development of adult-type melanophores results in hypomelanosis in their ocular-sided skins, yielding a pigmentary pattern seen in the blind side of the metamorphosed juveniles with normal pigmentation.(ABSTRACT TRUNCATED AT 250 WORDS)

The pigmentary system of developing axolotls. III. An analysis of the albino phenotype.

The albino mutant in the Mexican axolotl (Ambystoma mexicanum) is analysed with respect to the differentiation of pigment cells. Pigment cells were observed with the transmission electron microscope in order to determine any unusual structural characteristics and to determine what happens to each of the cell types as development proceeds. Chemical analyses of pteridine pigments were also carried out, and the pattern of pteridines in albino animals was found to be more complex than, and quantitatively enhanced (at all developmental stages examined) over, the pattern observed in comparable wild-type axolotls. The golden colour of albino axolotls is due primarily to sepiapterin (a yellow pteridine) and secondarily to riboflavin (and other flavins). Coincident with enhanced levels of yellow pigments, xanthophore pigment organelles (pterinosomes) in albino skin reach a mature state earlier than they do in wild-type axolotl skin. This morphology is conserved throughout development in albino animals whereas it is gradually lost in the wild type. Unpigmented melanophores from albino axolotls are illustrated for the first time, and in larval albino axolotls the morphology of these cells is shown to be very similar to xanthophore morphology. In older animals xanthophores are easily distinguished from unpigmented melanophores. Iridophores seem to appear in albino skin at an earlier stage than they have been observed in wild-type skin. Morphologically, wild-type and albino iridophores are identical.