Heterogeneity of neural crest-derived melanocytes

The majority of melanocytes originate from the neural crest cells (NCC) that migrate, spread on the whole embryo’s body to form elements of the nervous system and skeleton, endocrinal glands, muscles and melanocytes. Human melanocytes differentiate mainly from the cranial and trunk NCC. Although melanocyte development has traditionally been associated with the dorsally migrating trunk NCC, there is evidence that a part of melanocytes arise from cells migrating ventrally. The ventral NCC differentiate into neurons and glia of the ganglia or Schwann cells. It has been suggested that the precursors for Schwann cells differentiate into melanocytes. As melanoblasts travel through the dermis, they multiply, follow the process of differentiation and invade the forming human fetal epidermis up to third month. After birth, melanocytes lose the ability to proliferate, except the hair melanocytes that renew during the hair cycle. The localization of neural crest-derived melanocytes in non-cutaneous places e.g. eye (the choroid and stroma of the iris and the ciliary body), ear (cells of the vestibular organ, cochlear stria vascularis), meninges of the brain, heart seems to indicate that repertoire of melanocyte functions is much wider than we expected e.g. the protection of tissues from potentially harmful factors (e.g. free radicals, binding toxins), storage ions, and anti-inflammatory action.     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Transplantation of Melanocytes Obtained from the Skin Ameliorates Apomorphine-Induced Abnormal Behavior in Rodent Hemi-Parkinsonian Models

Tyrosinase, which catalyzes both the hydroxylation of tyrosine and consequent oxidation of L-DOPA to form melanin in melanocytes, is also expressed in the brain, and oxidizes L-DOPA and dopamine. Replacement of dopamine synthesis by tyrosinase was reported in tyrosine hydroxylase null mice. To examine the potential benefits of autograft cell transplantation for patients with Parkinson’s disease, tyrosinase-producing cells including melanocytes, were transplanted into the striatum of hemi-parkinsonian model rats or mice lesioned with 6-hydroxydopamine. Marked improvement in apomorphine-induced rotation was noted at day 40 after intrastriatal melanoma cell transplantation. Transplantation of tyrosinase cDNA-transfected hepatoma cells, which constitutively produce L-DOPA, resulted in marked amelioration of the asymmetric apomorphine-induced rotation in hemi-parkinsonian mice and the effect was present up to 2 months. Moreover, parkinsonian mice transplanted with melanocytes from the back skin of black newborn mice, but not from albino mice, showed marked improvement in the apomorphine-induced rotation behavior up to 3 months after the transplantation. Dopamine-positive signals were seen around the surviving transplants in these experiments. Taken together with previous studies showing dopamine synthesis and metabolism by tyrosinase, these results highlight therapeutic potential of intrastriatal autograft cell transplantation of melanocytes in patients with Parkinson’s disease.     

In Situ and In Vitro Expression of Protein Kinase C Alpha in Human Melanocytes

Protein kinase C (PKC) is a multigene family of at least 12 isoforms involved in the transduction of extracellular signals. We investigated whether PKC-α, a major isoform known to be relatively abundant in brain tissue, is increased in human melanocytes relative to keratinocytes in vitro and in situ. Immunohistochemical staining for PKC-α in frozen neonatal human foreskin exhibited intermittent 2–3+ staining along the basal cell layer consistent with melanocytes, and 0–1+ staining of keratinocytes (on a scale of 0–3). Microscopic densitometry of the intermittent cellular staining was at least 3-fold greater than that of adjacent keratinocyte cell cytoplasm. Sequential frozen sections revealed similar intermittent cell staining with PKC-α and Mel-5 (tyrosinase related protein-1), known to specifically react with melanocytes. Northern blot analysis with a specific cDNA probe for PKC-α showed strong PKC-α mRNA expression in cultured melanocytes, whereas PKC-α mRNA in cultured non-stratifying keratinocytes was expressed at low levels. Western blot analysis revealed a prominent PKC-α band at approximately 80 kDa in melanocytes as opposed to a weak band in keratinocytes. Densitometry of the northern and western blots revealed that melanocytes had at least 10-fold more PKC-α mRNA and approximately 6-fold more PKC-α protein expression than keratinocytes. Total PKC activity measured in vitro revealed that melanocytes had 5-fold more activity than keratinocytes. The marked difference in melanocyte and keratinocyte expression of PKC-α provides further evidence for cell type specificity in the balance of PKC-α expression and may implicate differential PKC isoform signaling pathways in neuro-ectodermally derived cells.     

Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast/melanocyte-proliferation medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion). LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes. Moreover, anti-LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. These results suggest that LIF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.     

Inducible expression of V600EBraf using tyrosinase-driven Cre recombinase results in embryonic lethality

We recently demonstrated that expression of ^V600EBraf in mature mouse melanocytes induces melanoma. Here, we show that expression of ^V600EBraf using the tyrosinase promoter leads to an unexpected embryonic lethality, with the animals dying before, at, or shortly after birth. The mice suffer from a range of developmental defects in the skin, the brain, the eyes and the heart, tissues that are normally colonized by melanocytes. We show that the ^V600EBraf expressing cells are potential melanocytic precursors that are fully transformed, suggesting that ^V600EBraf stimulates proliferation and blocks differentiation of these cells. Our data suggests that the presence of these cells in the organs that are normally occupied by melanocytes leads to severe developmental disruption, resulting in catastrophic defects and leading to death of the individual.     

The slaty mutation affects the morphology and maturation of melanosomes in the mouse melanocytes

The slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase in melanocytes and to reduce the melanin content in skin, hairs and eyes. Although the melanosomes in slaty melanocytes are reported to be eumelanosome-like, detailed melanosome biogenesis is not well studied. To address this point, melanosomes in neonatal epidermal melanocytes from wild-type (Dct+/Dct+) mice at the slaty locus as well as its congenic mouse mutant (Dct(slt)/Dct(slt)) in serum-free primary culture were observed under the electron microscope. Wild-type melanocytes possessed exclusively elliptical melanosomes with internal longitudinal structures, whereas in mutant melanocytes, numerous spherical melanosomes with globular depositions of pigment and elliptical melanosomes as well as mixed type of the two melanosomes were observed. Mature stage IV melanosomes were greatly decreased in mutant melanocytes, whereas immature stage III melanosomes were more numerous than in wild-type melanocytes. These results suggest that the slaty mutation affects the morphology and maturation of melanosomes in mouse melanocytes.     

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Growth Competition between W Mutant and Wild-type Cells in Mouse Aggregation Chimeras

The dominant spotting (W) locus of the mouse has been demonstrated to be identical with the c-kit proto-oncogene. The c-kit is strongly expressed in hematopoietic organs and the brain of mice. In homozygotes and double heterozygotes of the W mutant alleles (hereafter W mutant), development of erythrocytes, mast cells, melanocytes and germ cells is deficient. The deficiency of erythrocytes, mast cells and melanocytes is attributed to a defect of precursor cells, but the cause of the germ cell deficiency is not clear. We investigated the effect of the W mutation on proliferative potential of cells composing various organs by examining aggregation chimeras between W mutant and wild-type (+/+) embryos. Proportions of +/+ components were significantly greater in the male germ cells and hematopoietic cells. In contrast, the average proportions of +/+ components were comparable to those of W mutant components in other organs including the brain. The present result suggests that the W (c-kit ) gene plays an important role in development of the male germ cells and hematopoietic cells and that it does not promote the proliferation of major cell population in the brain, in spite of the strong expression of the W (c-kit ) gene in the brain.     

Regulation of the catalytic activity of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures

The activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, is higher in Black skin melanocytes than in melanocytes derived from Caucasian skin. This variation in enzyme activity is not due to differences in tyrosinase abundance or tyrosinase gene activity, but, rather, is due to differences in the catalytic activity of preexisting tyrosinase. In melanocytes, tyrosinase is localized to the membrane of melanosomes and in Caucasian melanocytes the melanosome-bound enzyme is largely inactive. Conversely, in melanosomes of Black melanocytes, tyrosinase has high catalytic activity. Treatment of Caucasian melanocytes with the lysosomotropic compound ammonium chloride or with the ionophores nigericin and monensin results in a rapid and pronounced increase in tyrosinase activity. This increase occurs without any change in tyrosinase abundance, indicating that these compounds are increasing the catalytic activity of preexisting enzyme. Inhibition of the vacuolar proton pump V-ATPase by treatment of Caucasian melanocytes with bafilomycin also increases tyrosinase activity. In contrast to the 10-fold increase in tyrosinase observed in Caucasian melanocytes, neither ammonium chloride, monensin, nigericin, nor bafilomycin is able to increase the already high level of tyrosinase activity present in melanosomes of melanocytes derived from Black skin. Finally, staining of Caucasian melanocytes with the fluorescent weak base acridine orange shows that melanosomes of Caucasian, but not Black, melanocytes are acidic organelles. These data support a model for racial pigmentation that is based on differences in melanosome pH in Black and Caucasian skin types. The models suggests that melanosomes of Caucasian melanocytes are acidic, while those of Black individuals are more neutral. Since tyrosinase is inactive in an acid environment, the enzyme is largely inactive in Caucasian melanosomes but fully active in Black melanosomes.     

Effects of gamma-radiation on the differentiation of mouse melanocytes in the hair follicles

Pregnant mice were whole-body irradiated with a single acute dose of gamma-rays (60Co) to investigate the effect of gamma-radiation on embryonic melanoblasts. The effect was studied by scoring changes in the differentiation of melanocytes in the hair follicles of mice heterozygous for the recessive coat color mutation pink-eyed dilution (p). Abnormal round melanocytes were found in the hair matrix and the dermal papilla of F1 offspring 3.5 days after birth. However, these round melanocytes possessed a melanin deposition of similar intensity to normal hair follicular melanocytes. The frequency of the abnormal hair follicles increased in a dose-dependent manner. Moreover, higher frequencies were found in the animals irradiated at earlier stages of embryonic development. These results indicate that gamma-radiation affects dendritogenesis and the location of mouse melanocytes in the hair follicles, with greater effects seen at the earlier stages of development.     

The effects of single gene substitution on the mammalian melanocyte system—a qualitative and quantitative histological study in the C57BL and DBL mice

About 160 skin samples from the tail, sole, palm, ear and scrotum of DBL and C57BL/St mice were “split” with NaBr and treated with DOPA. A quantitative and qualitative microscopic analysis showed that: (a) the two strains did not differ consistently in the frequency of epidermal or dermal melanocytes; nor did the sexes differ from each other in this respect; (b) the melanocytes of the two strains differed morphologically. The DBL melanocytes were generally larger, with melanin-congested perikarya. They had fewer dendrites than the C57BL melanocytes and their dendrites were shorter; and (c) the melanocytes of the DBL and C57BL strains differed in activity, the DBL melanocytes donating less melanin to Malpighian cells than did the C57BL melanocytes. It was concluded that the morphology of DBL and C57BL melanocytes is largely autonomously determined, although regional differences in melanocyte morphology suggest that the cellular environment also plays some part in influencing melanoblast differentiation.     

Role of antioxidants in the survival of normal and vitiliginous avian melanocytes.

Mutant feather melanocytes from Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens are currently being used as avian models of vitiligo. Feather melanocytes in BPR and WL chickens die prematurely in vivo due to low (50-66%) antioxidant glutathione and superoxide dismutase levels when compared to the wild type Jungle Fowl (JF) melanocytes. Excess superoxide anions, generated by xanthine:xanthine oxidase (X:XO), caused a 15-20% increase in mortality after 1 and 2 hrs. in all three genotypes of in vitro melanocytes as compared to control values that received no X:XO. Overall, the JF wild type melanocytes had the lowest mortality rate, WL melanocytes had the highest mortality rate and the BPR melanocytes had an intermediate mortality rate. Superoxide anion and hydroxyl radical production in the WL feather were double the production in the JF wild type feather. The production of reactive oxygen species in BPR was intermediate to the other two genotypes. In an effort to mimic the low antioxidant levels of the BPR and WL feathers in the JF feather, JF in vitro feather melanocytes were treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. With BSO added to the medium, the JF mortality rates increased by 20-25%, reaching the mortality levels of the mutant BPR melanocytes. The addition of iron to the JF melanocyte X:XO medium increased their mortality rate by 20%, probably via the Fenton reaction. Thus, antioxidants play an extremely important role in both the viability of normal avian melanocytes and the premature death of the vitiliginous avian melanocytes. A working hypothesis, supported in part by the current results, is that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. This same mechanistic hypothesis could apply as "a" cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their antioxidant protection system and blood flow to an area may be restricted.     

Quantitative control of end products in the melanocyte lineage of the ascidian embryo.

Terminal amounts of tyrosinase (EC 1.10.3.1) activity and melanin pigment in the giant melanocytes of cleavage-arrestedCiona intestinalis (L.) embryos are regulated independently of cell size and number of nuclei in the cells. Embryos were cleavage-arrested in cytochalasin B at a time before the last two divisions of the melanocyte lineage took place. The resulting two giant melanocytes, one from each of the two bilateral melanocyte lineages, developed tyrosinase and melanin. The cells were about three times larger in volume than the normal larval melanocytes and each contained four nuclei instead of just one. Quantitative measurements of melanin synthesized and tyrosinase activity in embryos with the giant melanocytes revealed amounts identical to those found in normal embryos. This specification of exact quantities differs markedly from the situation in mammalian melanocytes where cell volume and gene dosage influence the extent of melanotic differentiation. Quantitative control of differentiation in ascidian melanocytes appears to be mediated by a cytoplasmic determinant segregated through the melanocyte lineage and inherited by one daughter at each division of the lineages.     

Melanocytes in the Stria Vascularis and Vestibular Labyrinth of the Mongolian Gerbil (Meriones unguiculatus)

Inner ear melanocytes are mainly present in the cochlea, vestibular organ, and endolymphatic sac, but their exact biological function has not been determined. In this investigation, we study the pigment cells in the membranous labyrinth of the gerbil. The inner ear melanocytes of M. unguiculatus show an irregular dendritic shape with cytoplasmic processes. These cells are disposed following the distribution of striai marginal and vestibular dark cells that have an important metabolic activity. Gerbil inner ear melanocytes are characterized by the presence of melanosomes, which are homogeneously dense organelles, of variable size and shape, that are surrounded by a membrane. In these cells, the Golgi apparatus plays a important role in melanin synthesis. When melanocytes were incubated in L-DOPA solution, the vesicles and cisterns of the Golgi apparatus exhibited a positive tyrosinase reaction. An interesting observation is the relation between melanocytes and inner ear capillaries. Sometimes, near to sensory vestibular areas, the melanocytes were in contact with Schwann cells and with myelinated fibres of vestibular nerve. The ultrastructural findings of this investigation are consistent with the hypothesis that melanocytes may have functional significance in the inner ear.     

Changes in the proliferation and differentiation of neonatal mouse pink-eyed dilution melanocytes in the presence of excess tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild-type at the pink-eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum-free primary culture, with or without the addition of L-Tyr. Incubation with added L-Tyr inhibited the proliferation of P/P melanocytes in a concentration-dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L-Tyr stimulated the proliferation of p/p melanoblasts-melanocytes derived from 0.5-day-old mice, but inhibited their proliferation when derived from 3.5- or 7.5-day-old mice. L-Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5-, 3.5- and 7.5-day-old p/p mice, but L-Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole-2,3,5-tricarboxylic acid as well as the pheomelanin marker, 4-amino-3-hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6-dihydroxyindole-2-carboxylic acid, as well as the contents of pheomelanin and its precursor, 5-S-cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L-Tyr increased the content of eumelanin and pheomelanin two- to threefold in cultured cells and media derived from 0.5-, 3.5- and 7.5-day-old mice. These results suggest that the proliferation of p/p melanoblasts-melanocytes is stimulated by L-Tyr, and that the differentiation of melanocytes is induced by L-Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

Hepatocyte growth factor controls the proliferation of cultured epidermal melanoblasts and melanocytes from newborn mice

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with hepatocyte growth factor (HGF) from 14 days (keratinocyte depletion). The HGF increased the number of melanoblasts and melanocytes, but not the percentage of differentiated melanocytes in the melanoblast-melanocyte population in the absence of keratinocytes. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and/or G2/M phases of the cell cycle were increased by the treatment with HGF. Moreover, an anti-HGF antibody supplemented to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes. These results suggest that HGF is a keratinocyte-derived factor involved in regulating the proliferation of epidermal melanoblasts and melanocytes from newborn mice in cooperation with cAMP elevators and/or bFGF.     

Interleukin-1alpha stimulates the differentiation of melanocytes but inhibits the proliferation of melanoblasts from neonatal mouse epidermis

Interleukin (IL)-1alpha is one of the important cytokines involved in regulating immunological reactions in the mouse skin. However, it is not known whether IL-1alpha regulates the proliferation and differentiation of mouse epidermal melanocytes. In this study, to investigate the role of IL-1alpha in the regulation of the proliferation and differentiation of mouse epidermal melanocytes, IL-1alpha was supplemented to serum-free primary cultures of epidermal cell suspensions from the initiation of the primary culture (keratinocytes and melanoblasts-melanocytes) as well as to pure cultures of melanoblasts-melanocytes (keratinocyte-depleted cultures, after 14 days), and its effect was tested. IL-1alpha inhibited the proliferation of undifferentiated melanoblasts irrespective of the presence or absence of keratinocytes, whereas the cytokine inhibited the proliferation of differentiated melanocytes only in the presence of keratinocytes. Moreover, IL-1alpha induced the differentiation of melanocytes and, in addition, stimulated tyrosinase activity, melanin synthesis, and dendritogenesis of melanocytes irrespective of the presence or absence of keratinocytes. These results suggest that IL-1alpha is involved in inhibiting the proliferation of neonatal murine epidermal melanoblasts and in stimulating the differentiation, melanogenesis, and dendritogenesis of melanocytes. The results also suggest that IL-1alpha inhibits the proliferation of differentiated melanocytes in cooperation with keratinocyte-derived factors.