Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis

The murine recessive yellow (Mc1r(e)) is a loss-of-function mutation in the receptor for alpha-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r) and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes. In this study, the proliferation and differentiation of recessive yellow epidermal melanocytes cultured in dibutyryl cyclic AMP-supplemented serum-free medium were investigated in detail. The melanocytes produced mainly eumelanin in this culture system. The proliferation of recessive yellow melanocytes was decreased compared with that of wild-type at the e-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (TYR) activity and TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess l-tyrosine (l-Tyr) added to the culture media rescued the reduced activity of proliferation of melanocytes. l-Tyr also stimulated TYR activity and TRP1 and TRP2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes and l-Tyr rescues the reduced proliferative and differentiative activities by stimulating TYR activity and TRP1 and TRP2 expressions as well as melanosome maturation.     

Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture

The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/Pmelanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/Pmelanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

The mouse ruby‐eye 2d (ru2d/Hps5ru2‐d) allele inhibits eumelanin but not pheomelanin synthesis

The novel mutation named ru2^d/Hps5^ru2‐d, characterized by light‐colored coats and ruby‐eyes, prohibits differentiation of melanocytes by inhibiting tyrosinase (Tyr) activity, expression of Tyr, Tyr‐related protein 1 (Tyrp1), Tyrp2, and Kit. However, it is not known whether the ru2^d allele affects pheomelanin synthesis in recessive yellow (e/Mc1r^e) or in pheomelanic stage in agouti (A) mice. In this study, effects of the ru2^d allele on pheomelanin synthesis were investigated by chemical analysis of melanin present in dorsal hairs of 5‐week‐old mice from F₂ generation between C57BL/10JHir (B10)‐co‐isogenic ruby‐eye 2^d and B10‐congenic recessive yellow or agouti. Eumelanin content was decreased in ruby‐eye 2^d and ruby‐eye 2^d agouti mice, whereas pheomelanin content in ruby‐eye 2^d recessive yellow and ruby‐eye 2^d agouti mice did not differ from the corresponding Ru2^d/‐ mice, suggesting that the ru2^d allele inhibits eumelanin but not pheomelanin synthesis.     

The slaty mutation affects eumelanin and pheomelanin synthesis in mouse melanocytes

The slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase (DCT) in melanocytes. However, it is unknown whether the reduced DCT activity leads to a defect in the proliferation and differentiation of mouse melanocytes. To address this point, the proliferation and differentiation of neonatal melanocytes from Dct(slt)/Dct(slt) congenic mice in serum-free primary culture were investigated in detail. The proliferation of slaty epidermal melanoblasts/melanocytes in culture did not differ from that of wild-type mice. However, the differentiation was greatly inhibited. Tyrosinase (TYR) activity detected by dopa reaction as well as staining of DCT in slaty melanocytes was greatly reduced. The content of eumelanin in cultured slaty melanocytes was reduced, whereas the content of pheomelanin in media derived from cultured 7.5-day-old slaty melanocytes was greatly increased. The contents of eumelanin and pheomelanin in the neonatal slaty epidermis and dermis were reduced, except that the pheomelanin content in 3.5-day-old dermis was increased. These results suggest that the slaty mutation affects both eumelanin and pheomelanin synthesis in developmental stage-specific and skin site-specific manners, and, in addition, the gene controls the differentiation of melanocytes via the regulation of activity of TYR in addition to its own DCT.     

Effects of genic substitution at the pink-eyed dilution locus on the proliferation and differentiation of mouse epidermal melanocytes in vivo and in vitro

Cells positive to the dopa reaction (melanocytes) as well as to the combined dopa-premelanin reaction (melanoblasts and melanocytes) in the epidermis of C57BL/10JHir-p/p (pink-eyed dilution) mice were fewer and less reactive than in C57BL/10JHir (black, P/P) mice, suggesting that the proliferation and differentiation of p/p melanocytes are inhibited. To confirm the inhibitory effects of p gene on the proliferation and differentiation of epidermal melanocytes, we cultured epidermal cell suspensions of neonatal skins from P/P and p/p in a serum-free medium. The proliferation and differentiation of p/p melanoblasts/melanocytes in primary culture were greatly inhibited as compared to P/P melanoblasts/melanocytes. The morphology of p/p melanoblasts/melanocytes cultured in melanocyte growth medium, though non-pigmented, was similar to P/P melanocytes; namely, dendritic, polygonal, or epithelioid. About 8% of p/p cells cultured in melanocyte growth medium were positive to the dopa reaction, and about 25% were reactive to the combined dopa-premelanin reaction. Eumelanin content in p/p was extremely reduced compared to P/P. The immunocytochemical staining of p/p melanoblasts/melanocytes revealed that they are negative to tyrosinase, but reactive to tyrosinase-related protein (TRP)-1, TRP-2, and c-kit. However, the reactivities in p/p were lower than in P/P. Although the differentiation of p/p melanoblasts was not induced by endothelin (ET)-1, ET-2, and ET-3, the proliferation of p/p melanoblasts was stimulated by them. These results suggest for the first time that p gene exerts its influence on the proliferative activities of mouse epidermal melanoblasts by affecting the regulatory mechanisms dependent on the function of ETs.     

Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte-macrophage colony-stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte-derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.     

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.     

Origin of melanosome structures and cytochemical localizations of tyrosinase activity in differentiating epidermal melanocytes of newborn mouse skin

The mode of differentiation of epidermal melanocytes was studied by ultrastructural cytochemistry in the skin of newborn mice of strain C57BL/10J. From observations of epidermal melanoblasts and melanocytes, stage I melanosomes, including both unit membranes and inner matrices, appear to be formed from Golgi vacuoles or rough endoplasmic reticulum (RER). Stage I melanosomes were positive to ammoniacal silver-nitrate reaction in the melanoblasts of 1-day-old mice. All stages of melanosomes were similarly positive in the differentiating melanocytes of 2-day-old mice. However, Golgi apparatus, RER, and vesicles were negative. Therefore, it is conceivable that structural proteins, originated from Golgi vacuoles or RER, are developed into specialized proteins and are detected by this reaction in stage I melanosomes. Stage I melanosomes were dopa-negative in the melanoblasts. Stage I and II melanosomes were similarly negative in the differentiating melanocytes. Thus, the melanoblasts are thought to begin production of stage I melanosomes prior to the onset of tyrosinase activity. In the differentiating melanocytes, dopa-melanin depositions were observed in stage III and IV melanosomes, trans Golgi saccules, and small vesicles derived from these saccules, but not in RER. These vesicles were in contact with, or fused to, melanosomes. These findings suggest that tyrosinase may be transferred by Golgi vesicles into stage I and II melanosomes originating from Golgi vacuoles or RER.     

How are proliferation and differentiation of melanocytes regulated?

Coat colors are determined by melanin (eumelanin and pheomelanin). Melanin is synthesized in melanocytes and accumulates in special organelles, melanosomes, which upon maturation are transferred to keratinocytes. Melanocytes differentiate from undifferentiated precursors, called melanoblasts, which are derived from neural crest cells. Melanoblast/melanocyte proliferation and differentiation are regulated by the tissue environment, especially by keratinocytes, which synthesize endothelins, steel factor, hepatocyte growth factor, leukemia inhibitory factor and granulocyte-macrophage colony-stimulating factor. Melanocyte differentiation is also stimulated by alpha-melanocyte stimulating hormone; in the mouse, however, this hormone is likely carried through the bloodstream and not produced locally in the skin. Melanoblast migration, proliferation and differentiation are also regulated by many coat color genes otherwise known for their ability to regulate melanosome formation and maturation, pigment type switching and melanosome distribution and transfer. Thus, melanocyte proliferation and differentiation are not only regulated by genes encoding typical growth factors and their receptors but also by genes classically known for their role in pigment formation.     

Spatiotemporal gene control by the Cre-ERT2 system in melanocytes

The organ-specific and temporal control of gene activation/inactivation is a key issue in the understanding of protein function during normal and pathological development and during oncogenesis. We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase (Tyr::Cre-ERT2) gene expressed under the control of a 6.1 kb murine tyrosinase promoter in order to facilitate targeted spatiotemporally controlled somatic recombination in melanoblasts/melanocytes. Cre-ERT2 production was detected in tissues containing melanocytes. After tamoxifen induction at various times during embryogenesis and adulthood in a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts and melanocytes of the skin. Thus, the Tyr::Cre-ERT2 transgenic mice provides a valuable tool for following this cell lineage and for investigating gene function in melanocyte development and transformation.     

Ultrastructural change of melanosomes associated with agouti pattern formation in mouse hair.

We have studied the structural alteration of melanosomes in the melanocytes of agouti mice whose genetic characteristic is to produce eumelanin and phaeomelanin alternately in a single hair bulb. Melanocytes of hair bulbs from 1 to 2 day old mice of the black phase were observed to contain rod-shaped melanosomes of the eumelanin type (eumelanosome). In the melanocytes of the hair bulbs from 4 to 6-day old skin, which exclusively contain phaeomelanin, spherical melanosomes (phaeomelanosomes) were seen. On the other hand, the mice of the transitional phase from black to yellow possessed melanocytes that contained both eumelanosomes and phaeomelanosomes within a single cell. This result indicates that the shift from the eumelanin formation to the phaeomelanin formation or vice versa in agouti hair occurs within a single melanocyte.We observed multivesicular bodies in both the agouti melanocytes of the yellow phase and the genotypically yellow melanocytes. These bodies are considered to be the precursor of the phaeomelanin-containing melanosome. They are sometimes observed to have continuity with E. R. suggesting that the melanosomes are derived from E. R. in the phaeomelanin-forming melanocytes.     

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.     

Plumage pigmentation and expression of its regulatory genes during quail development--histochemical analysis using Bh (black at hatch) mutants

The plumage on the dorsal trunk of normal quail embryos exhibits longitudinal black and brown stripes of pigments produced by melanocytes. However, this pigmentation pattern disappeared in Bh (black at hatch) heterozygous and homozygous embryos because of overall black and brown pigmentation of plumages, respectively. To investigate the mechanisms of the pigment pattern formation of plumage and clarify the roles of the Bh locus in the pattern formation, we examined the expression pattern of genes relating to melanocyte development (Mitf, MelEM antigen, Kitl, Kit and EdnrB2) and melanin pigment production (Dct, Tyrp1, Tyr and Mmp115) in Bh mutant and wild-type embryos throughout development. As a result, we found that MelEM antigen was expressed in melanoblasts committed to produce black pigment before apparent melanogenic gene expression, and that Bh heterozygotes and homozygotes showed abnormal expression patterns of the MelEM antigen. These results indicate that MelEM antigen is a good marker for melanoblasts committed to produce black pigment, and suggests that the Bh locus directs melanocytes to produce eumelanin in proper positions.     

Melanocyte stimulating hormone induces the differentiation of mouse epidermal melanocytes in serum-free culture.

In serum-free primary culture of dissociated mouse epidermal cells, alpha-melanocyte stimulating hormone (alpha-MSH) and dibutyryl cyclic AMP (DBcAMP) induced the differentiation of melanocytes. Moreover, the proliferation of melanocytes was also induced in the dishes cultured with DBcAMP, but not with alpha-MSH. In order to clarify the role of keratinocytes in melanocyte proliferation and differentiation, pure cultures of keratinocytes were established in serum-free medium. Subconfluent primary keratinocytes were trypsinized and seeded into pure primary melanoblasts cultured with serum-free medium that did not contain alpha-MSH or DBcAMP. Melanoblasts were cultured with alpha-MSH or DBcAMP in the presence or absence of keratinocytes. alpha-MSH failed to induce melanocyte differentiation in the absence of keratinocytes. DBcAMP failed to induce melanocyte proliferation in the absence of keratinocytes, although it induced melanocyte differentiation even in the absence of keratinocytes. These results suggest that keratinocyte-derived factors are required not only for the induction of melanocyte differentiation by alpha-MSH but also for the induction of melanocyte proliferation by DBcAMP.     

Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

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 a 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 granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.     

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.