The patchwork mouse phenotype: implication for melanocyte replacement in the hair follicle.

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

Effects of low-dose γ-rays on the embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs

The effects of low-dose γ-rays on the embryonic development of animal cells are not well studied. The mouse melanocyte is a good model to study the effects of low-dose γ-rays on the development of animal cells, as it possesses visible pigment (melanin) as a differentiation marker. The aim of this study is to investigate in detail the effects of low-dose γ-rays on embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs at cellular level. Pregnant females of C57BL/10J mice at nine days of gestation were whole-body irradiated with a single acute dose of γrays (0.1, 0.25, 0.5, and 0.75 Gy), and the effects of γ-rays were studied by scoring changes in the development of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes at 18 days in gestation. The number of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes in the dorsal and ventral skins was markedly decreased even at 0.1 Gy-treated embryos (P < 0.001), and gradually decreased as dose increased. The effects on the ventral skin were greater than those on the dorsal skin. The dramatic reduction in the number of melanocytes compared to melanoblasts was observed in the ventral skin, but not in the dorsal skin. These results suggest that low-dose γ-rays provoke the death of melanoblasts and melanocytes, or inhibit the proliferation and differentiation of melanoblasts and melanocytes, even at the low dose.     

Different Populations of Melanocytes Are Present in Hair Follicles and Epidermis

Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath of anagen hair follicles. Comparative study of melanocytes in these different locations has been difficult as hair follicle melanocytes could not be cultured. In this study we used a recently described method of growing hair follicle melanocytes to characterize and compare hair follicle and epidermal melanocytes in the scalp of the same individual. Three morphologically and antigenically distinct types of melanocytes were observed in primary culture. These included (1) moderately pigmented and polydendritic melanocytes derived from epidermis; (2) small, bipolar, amelanotic melanocytes; and (3) large, intensely pigmented melanocytes; the latter two were derived from hair follicles. The three sub-populations of cells all reacted with melanocyte-specific monoclonal antibody. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the intensely pigmented hair follicle melanocytes did not. Amelanotic hair follicle melanocytes differed from epidermal melanocytes in being less differentiated, and they expressed less mature melanosome antigens. In addition, hair follicle melanocytes expressed some antigens associated with alopecia areata, but not antigens associated with vitiligo, whereas the reverse was true for epidermal melanocytes. Thus, antigenically different populations of melanocytes are present in epidermis and hair follicle. This could account for the preferential destruction of hair follicle melanocytes in alopecia areata and of epidermal melanocytes in vitiligo.     

Premature Graying as a Consequence of Compromised Antioxidant Activity in Hair Bulb Melanocytes and Their Precursors

Intricate coordinated mechanisms that govern the synchrony of hair growth and melanin synthesis remain largely unclear. These two events can be uncoupled in prematurely gray hair, probably due to oxidative insults that lead to the death of oxidative stress-sensitive melanocytes. In this study, we examined the gene expression profiles of middle (bulge) and lower (hair bulb) segments that had been micro-dissected from unpigmented and from normally pigmented hair follicles from the same donors using quantitative real-time RT-PCR (qPCR) arrays. We found a significant down-regulation of melanogenesis-related genes (TYR, TYRP1, MITF, PAX3, POMC) in unpigmented hair bulbs and of marker genes typical for melanocyte precursor cells (PAX3, SOX10, DCT) in unpigmented mid-segments compared with their pigmented analogues. qPCR, western blotting and spin trapping assays revealed that catalase protein expression and hydroxyl radical scavenging activities are strongly repressed in unpigmented hair follicles. These data provide the first clear evidence that compromised antioxidant activity in gray hair follicles simultaneously affects mature hair bulb melanocytes and their immature precursor cells in the bulge region.     

Melanocytes Fail to Survive in Hair Bulbs of the Shiba Goat (Capra hircus) With the Dominant Black-Eyed White Phenotype

Dominant black-eyed white phenotypes are one of the most commonly observed traits in domestic animals. Their genetic control mechanisms, however, have not been elucidated. As the first step to approach the problem, we examined histologically the patterns of the distribution of pigment cells in Shiba goats (two each of day-73-postcoitum and day-112-postcoitum fetuses, and a 15-week-old kid) with the dominant black-eyed white phenotype. Melanocytes were present and fully pigmented in the choroid and the sclera of eyes, as well as in dorsal skin epidermis of the fetuses and of the kid. Melanocytes were also found in approximately 6% of the hair bulbs in the fetal dorsal skin, while the rest (94%) lacked them. Hair follicles of the kid did not harbor melanocytes except for some in the early anagen stage. The results suggest that the survival of melanocytes was inhibited specifically in the hair follicles of the Shiba goat with the dominant black-eyed white phenotype and that the ostensibly similar phenotypes in the Shiba goat and in the SI or W mutants of the mouse, where melanocytes die en route to the hair bulbs, are regulated by different mechanisms.     

Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice

Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non‐cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non‐cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte‐related diseases and melanomas.     

Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit expression.

Disruption of the c-Kit/stem cell factor (SCF) signaling pathway interferes with the survival, migration, and differentiation of melanocytes during generation of the hair follicle pigmentary unit. We examined c-Kit, SCF, and S100 (a marker for precursor melanocytic cells) expression, as well as melanoblast/melanocyte ultrastructure, in perinatal C57BL/6 mouse skin. Before the onset of hair bulb melanogenesis (i.e., stages 0-4 of hair follicle morphogenesis), strong c-Kit immunoreactivity (IR) was seen in selected non-melanogenic cells in the developing hair placode and hair plug. Many of these cells were S100-IR and were ultrastructurally identified as melanoblasts with migratory appearance. During the subsequent stages (5 and 6), increasingly dendritic c-Kit-IR cells successively invaded the hair bulb, while S100-IR gradually disappeared from these cells. Towards the completion of hair follicle morphogenesis (stages 7 and 8), several distinct follicular melanocytic cell populations could be defined and consisted broadly of (a) undifferentiated, non-pigmented c-Kit-negative melanoblasts in the outer root sheath and bulge and (b) highly differentiated melanocytes adjacent to the hair follicle dermal papilla above Auber's line. Widespread epithelial SCF-IR was seen throughout hair follicle morphogenesis. These findings suggest that melanoblasts express c-Kit as a prerequisite for migration into the SCF-supplying hair follicle epithelium. In addition, differentiated c-Kit-IR melanocytes target the bulb, while non-c-Kit-IR melanoblasts invade the outer root sheath and bulge in fully developed hair follicles.     

Epigenetic and pharmacological control of pigmentation via Bromodomain Protein 9 (BRD9)

Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.     

The cell biology of human hair follicle pigmentation

Although we have made significant progress in understanding the regulation of the UVR‐exposed epidermal‐melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR‐shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis‐follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub‐populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species‐specific issues involved which the general reader of the pigmentation literature (with its substantial mouse‐based data) may not fully appreciate.     

MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles

Melanoblasts, the precursors of the pigment-producing cells of the skin and hair, are derived from the neural crest and migrate to the skin around 12 days of gestation in the mouse. In adult mice almost all the melanoblasts are confined to the hair follicles except for the epidermal layers of nonhairy skin. The receptor tyrosine kinase, KIT, is necessary for the survival, proliferation, and migration of melanoblasts. We have utilised an organ culture for embryonic skin taken from Dct-lacZ transgenic mice. The early patterning of the follicles and developing skin layers is retained within the cultures and the lacZ reporter allows visualisation of the melanoblasts within their native tissue environment. Soon after initiation of hair follicle development, melanoblasts localise in the follicles. Inhibition of follicle formation demonstrates that this localisation is an active process; in the absence of follicles, the melanoblasts proliferate but remain associated with the basement membrane. Implantation of beads releasing MGF, the ligand of KIT, does not result in melanoblast migration towards the bead, rather their localisation to the follicles is accelerated. Addition of soluble MGF induces the same effect; KIT therefore promotes melanocyte movement and acts as a chemokinetic, or motogenic, receptor. The melanoblasts must be guided to their correct location by other chemotactic signals or move at random and locate by ceasing movement when the follicle is engaged.     

Mutant gene expression in mouse aggregation chimeras. 8. The effect of the white gene on coat pigmentation

Aggregation of mouse embryos produced 11 chimaeras Miwh/+C/C----+/+c/c and 8 chimaeras +/+C/C----+/+c/c (control). Chimaerism was detected by mosaicism of coat retinal pigment epithelium and by electrophoretic pattern of glucose phosphate isomerase. All chimaeras showed a common pattern of pigmented and unpigmented hair regions that alternated as stripes of different length and width and extended from spine in lateral-ventral direction. However, white coat color predominated in Miwh/+C/C----+/+c/c chimaeras due to a higher proportion of unpigmented zones as well as to weakening of hair color in pigmented areas. Besides, distal regions of limbs were always unpigmented in Miwh/+C/C----+/+c/c chimaeras and completely or partially pigmented in +/+C/C----+/+c/c chimaeras. Pigmented hair regions are often located on the ventral trunk surface where the Miwh/+ heterozygotes usually had an unpigmented spot. The examination of hairs, taken from the same regions of gray coloration, revealed the presence of pigmented, unpigmented and mosaic hairs. The proportion of unpigmented hairs was much higher in Miwh/+C/C----+/+c/c chimaeras than in +/+C/C----+/+c/c chimaeras. The data obtained indicate that a single Miwh gene dose reduced proliferative activity of melanoblasts which resulted in weakening of coat pigmentation.     

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.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

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 adultmelanocytes 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.     

Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.     

The production of chimeric rats and their use in the analysis of the hooded pigmentation pattern

New, improved media and procedures for making rat chimeric embryos and culturing them in vitro have been developed. We have produced 27 rat chimeras: 20 males and 7 females. This ratio of males to females is consistent with that seen in mouse chimeras, suggesting that rat sex chimeras develop as phenotypic males. By aggregating embryos containing appropriate genetic markers for pigment cell differentiation, it is possible to produce chimeras that elucidate the site of action of the hooded gene. The coat color patterns of black ? black hooded chimeras display a white belly spot. In black ? albino hooded chimeras, small patches of white hair appear on the head and a large white spot occurs on the belly. Black ? agouti hooded chimeras display both agouti and nonagouti pigmentation over the entire surface of the chimera. These animals are fully pigmented with no white spots. In black ? albino non-hooded chimeras, rather small irregular patches of black and white hairs are distributed throughout the pelage. Histological examination of sections of hair follicles obtained from the white areas in the head of black ? albino hooded chimeras revealed amelanotic melanocytes. On the other hand, hair bulbs from the white belly spots do not contain any such melanocytes. Thus the white hairs of the head are due to the presence of albino melanocytes, but the white hairs of the belly are due to the total absence of melanocytes. All these observations are consistent with the conclusion that the hooded gene acts within melanoblasts, probably to retard their migration from the neural crest and/or to prevent their entrance into the hair follicles of the white areas of hooded rats.     

Appearance of hair follicle-inducible mesenchymal cells in the rat embryo

Rat vibrissa follicle morphogenesis starts around 13 days of gestation. By day 14 mesenchymal cells have already aggregated as 'condensations' beneath the initial hair bud. Some of the mesenchymal cells will form a dermal papilla, having profound effects on hair follicle formation. The appearance of follicle-inducing mesenchymal cells in the process of vibrissa follicle development was examined. Mesenchymal cells were isolated from the developing site of vibrissa follicles at 13 days or at later stages and amplified in mass culture, harvested and transplanted in association with the epithelium. It was demonstrated that 13-day mesenchymal cells did not induce any hair bulbs but those from 14 days or later stages could induce hair-producing new bulbs or new follicles depending on the association with the follicle epithelium or with the glabrous sole epidermis of the adult rats, respectively. Further, clones having hair bulb-inducing ability were obtained from 14- and 15-day mass-cultured mesenchymal cells. Based on these and other results, it was concluded that mesenchymal cells having follicle-inducing ability are present at least by 14 days in the future whisker pad region. This suggests that the differentiation of the dermal papilla cells must start before the initial hair bud stage.     

Extensive Hair-Shaft Elongation by Isolated Mouse Whisker Follicles in Very Long-Term Gelfoam? Histoculture

We have previously studied mouse whisker follicles in Gelfoam® histoculture to determine the role of nestin-expressing plutipotent stem cells, located within the follicle, in the growth of the follicular sensory nerve. Long-term Gelfoam® whisker histoculture enabled hair follicle nestin-expressing stem cells to promote the extensive elongation of the whisker sensory nerve, which contained axon fibers. Transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP) were used as the source of the whiskers allowing imaging of the nestin-expressing stem cells as they formed the follicular sensory nerve. In the present report, we show that Gelfoam®-histocultured whisker follicles produced growing pigmented and unpigmented hair shafts. Hair-shaft length increased rapidly by day-4 and continued growing until at least day-12 after which the hair-shaft length was constant. By day-63 in histoculture, the number of ND-GFP hair follicle stem cells increased significantly and the follicles were intact. The present study shows that Gelfoam® histoculture can support extensive hair-shaft growth as well as hair follicle sensory-nerve growth from isolated hair follicles which were maintained over very long periods of time. Gelfoam® histoculture of hair follicles can provide a very long-term period for evaluating novel agents to promote hair growth.     

Changes in the Proliferative Activity of Epidermal Melanocytes in Serum‐Free Primary Culture During the Development of Ultraviolet Radiation B‐Induced Pigmented Spots in Hairless Mice

Long‐term exposure to ultraviolet radiation B (UVB) induced pigmented spots in the dorsal skin of hairless mice of strain (HR‐1 X HR/De)F₁. To clarify the cellular mechanism for the development of these UVB‐induced pigmented spots, we investigated changes in the proliferative activity of epidermal melanoblasts and melanocytes in the dorsal skin at various weeks after UVB irradiation. Epidermal cell suspensions from the dorsal skin of hairless mice were cultured in a serum‐free medium supplemented with dibutyryl adenosine 3′:5′‐cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). The suspensions were prepared from dorsal skins of mice exposed to UVB for 4 weeks (the stage of hyperpigmentation). Suspensions were also prepared from mice at 3 (the stage of depigmentation), 8 (the stage of appearance of pigmented spots), 20 (the stage of development of small‐sized pigmented spots) and 37 (the stage of development of medium‐sized pigmented spots) weeks after the cessation of 8‐week UVB exposure. At the stage of hyperpigmentation the proliferative activity of melanoblasts and melanocytes was suppressed. With the development of pigmented spots, the proliferative activity of undifferentiated melanoblasts gradually increased, and then followed the increase in the proliferative activity of differentiated melanocytes. These results suggest that the proliferative activity of epidermal melanoblasts and melanocytes in UVB‐irradiated skin increases with the development of pigmented spots.     

Melanocyte stem cells express receptors for canonical Wnt-signaling pathway on their surface

It has been reported that melanocytes play important roles in skin and hair pigmentation and are differentiated from melanocyte stem cells (MSCs) residing in the bulge area of hair follicles. Recently, interest has been growing in MSCs because regulation of the upstream of differentiated melanocytes is essential for the determination of skin and hair pigmentation; however, their precise characteristics remain to be elucidated. The aim of this study is to explore cell-surface markers expressed on MSCs in order to understand their characteristics.To explore genes specifically expressed in the bulge region, we classified a hair follicle into four areas, hair bulb, hair bulb to bulge (lower bulge), bulge, and epidermis to bulge (upper bulge), and collected these areas from back skin sections of C57BL/6 mice by laser microdissection. Real-time RT-PCR performed on these areas revealed that Frizzled (Fzd)-4, Fzd7, low density lipoprotein receptor-related protein 5 (Lrp5), and Lrp6, receptors for Wnt molecules, were expressed higher in the bulge area than other areas. Furthermore, FACS analysis showed that populations of Fzd4⁺ cells and Fzd7⁺ cells were different from those of Kit⁺ cells (precursor of melanocytes: melanoblasts). Fzd4⁺ and Fzd7⁺ cells isolated by FACS required a longer culture period to differentiate into mature melanocytes than Kit⁺ cells. Up-regulation of mRNA expressions of melanocyte markers (dopa chrometautomerase: Dct, tyrosinase: Tyr, tyrosinase-related protein 1: Tyrp1) was observed in Fzd4⁺ and Fzd7⁺ cells following Kit⁺ cells during differentiation. These results suggested that Fzd4⁺ and Fzd7⁺ cells were more immature than melanoblasts, therefore raising the possibility that Fzd4⁺ and Fzd7⁺ cells are MSCs.