Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a gamma-secretase inhibitor (GSI) to wild-type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI-treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/-Notch2+/- double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c-kit mutant Wv background, Notch1+/- and Notch2+/- mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/-Notch2+/-Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c-kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.     

SCF/c-kit signaling is required for cyclic regeneration of the hair pigmentation unit.

Hair graying, an age-associated process of unknown etiology, is characterized by a reduced number and activity of hair follicle (HF) melanocytes. Stem cell factor (SCF) and its receptor c-kit are important for melanocyte survival during development, and mutations in these genes result in unpigmented hairs. Here we show that during cyclic HF regeneration in C57BL/6 mice, proliferating, differentiating, and melanin-producing melanocytes express c-kit, whereas presumptive melanocyte precursors do not. SCF overexpression in HF epithelium significantly increases the number and proliferative activity of melanocytes. During the induced hair cycle in C57BL/6 mice, administration of anti-c-kit antibody dose-dependently decreases hair pigmentation and leads to partially depigmented (gray) or fully depigmented (white) hairs, associated with significant decreases in melanocyte proliferation and differentiation, as determined by immunostaining and confocal microscopy. However, in the next hair cycle, the previously treated animals grow fully pigmented hairs with the normal number and distribution of melanocytes. This suggests that melanocyte stem cells are not dependent on SCF/c-kit and when appropriately stimulated can generate melanogenically active melanocytes. Therefore, the blockade of c-kit signaling offers a fully reversible model for hair depigmentation, which might be used for the studies of hair pigmentation disorders.     

Spotlight on spotted mice: a review of white spotting mouse mutants and associated human pigmentation disorders

Mutation of genes that regulate neural crest-derived melanoblast development and survival can result in reduction and/or loss of mature melanocytes. The reduction in melanocyte number in the skin and hair follicles manifests itself as areas of hypopigmentation, commonly described as white spotting in mice. To date ten genes have been identified which are associated with white-spotting phenotypes in mouse. Seven of these genes are associated with neural crest and melanocyte disorders in humans. This review summarizes the phenotypes associated with mutation of these genes in both mouse and man. We describe our current understanding of how these genes function in development, and explore their complex roles regulating the various stages of melanocyte development.     

STX17在不同毛色小鼠皮肤中差异表达与在毛囊中的定位

突触融合蛋白17 (STX17)是一种囊泡蛋白,参与细胞中物质的运输.为研究Stx17在不同毛色皮肤中是否存在差异表达及明确它在毛囊中的定位,进行了普通PCR、real-time PCR、免疫组化和蛋白免疫印迹实验对小鼠皮肤组织和体外培养黑素细胞的Stx17基因及蛋白的检测.普通PCR检测得出小鼠皮肤和黑色素细胞总RNA有Stx17 CDS区序列的表达;荧光定量检测显示,在白、灰、黑3种组织中Stx17均有表达,在灰色腹部表达量最高,是黑色皮肤的1.682倍,昆明鼠白色皮肤中表达量最低,是黑色皮肤的0.115倍;皮肤组织免疫组化结果显示,STX17表达于毛囊的上皮根鞘,且毛囊上段和中段表达量高于下段,黑色素细胞的免疫组化分析得出,STX17在黑色素细胞的细胞质和细胞膜上均有表达;蛋白免疫印迹结果显示,在白色、灰色和黑色皮肤均有STX17蛋白阳性条带且灰色皮肤中表达量最高,黑色皮肤次之,白色皮肤中表达量是最低的,这与荧光定量检测结果一致,体外培养的小鼠黑色素细胞中也有STX17蛋白阳性条带.实验结果表明,小鼠Stx17基因在皮肤组织、毛囊角化细胞以及黑色素细胞中均有表达,Stx17可能参与毛色的形成,且在小鼠腹部毛色变浅中起到了一定的作用.     

In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development.

Previous studies on mice bearing various mutations within the c-kit gene, dominant white spotting (W), indicate the functional role of this tyrosine kinase receptor in the development of melanocytes, germ cells and hematopoietic cells. Despite the availability of mice defective in the c-kit gene and a respectable understanding of the molecular nature of c-kit, however, it is not clear at what stage of gestation c-kit is functionally required for the development of each of these cell lineages. To address this question, we have used a monoclonal anti-c-kit antibody, ACK2, as an antagonistic blocker of c-kit function to interfere with the development of melanocytes during embryonic and postnatal life. ACK2 injected intradermally into pregnant mice entered the embryos where it blocked the proper development of melanocytes. This inhibitory effect was manifested as coat color alteration in the offspring. Furthermore, ACK2 injection also altered the coat color of neonatal and adult mice. Based on the coat color patterns produced by ACK2 administration at various stages before or after birth, the following conclusions are drawn: (i) during mid-gestation, c-kit is functionally required during a restricted period around day 14.5 post-coitum when a sequence of events leading to melanocyte entry into the epidermal layer occurs; (ii) during postnatal life, c-kit is required for melanocyte activation which occurs concomitantly with the hair cycle which continues throughout life after neonatal development of the first hair.     

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.     

RADIATION DEPIGMENTATION OF MOUSE HAIR: A STUDY OF FOLLICULAR MELANOCYTE POPULATIONS

The radiation depigmentation of mouse hair has been studied by a technique enabling melanocyte per follicle counts to be made. Distributions for normal skin show a large peak corresponding to the zigzag hair type. Changes in the frequency distributions of melanocytes per follicle after irradiation are presented for Strong F and DBA-1 mice irradiated in anagen or telogen stages of hair growth. These distributions clearly suggest the existence of some precursor cells, and the dose-response curves obtained by defining radiation survivors as follicles containing more than ten melanocytes gives the sensitivity of these cells to inactivation. D₀ values are 180–220 rads. A melanocyte-melanoblast model is proposed for the follicular melanocyte cycle which can be outlined as follows: The telogen follicle contains a small number of amelanotic melanocytes that survived through catagen. These cells possess the ability to repopulate the follicle with melanocytes. In catagen functional and/or amelanotic melanocytes are lost at random. Genes for dilution (possibly only when modified by other coat colour genes) and radiation both increase the chance of melanocyte loss at catagen by altering the melanocyte-dermal papilla relationship. One way in which this is affected is by a shortening of the dendrites. A feedback may operate in the follicle so that the full complement of melanocytes is achieved whatever number of melanocytes persists in telogen.     

Maintenance of distinct melanocyte populations in the interfollicular epidermis

Hair follicles and sweat glands are recognized as reservoirs of melanocyte stem cells (MSCs). Unlike differentiated melanocytes, undifferentiated MSCs do not produce melanin. They serve as a source of differentiated melanocytes for the hair follicle and contribute to the interfollicular epidermis upon wounding, exposure to ultraviolet irradiation or in remission from vitiligo, where repigmentation often spreads outwards from the hair follicles. It is unknown whether these observations reflect the normal homoeostatic mechanism of melanocyte renewal or whether unperturbed interfollicular epidermis can maintain a melanocyte population that is independent of the skin's appendages. Here, we show that mouse tail skin lacking appendages does maintain a stable melanocyte number, including a low frequency of amelanotic melanocytes, into adult life. Furthermore, we show that actively cycling differentiated melanocytes are present in postnatal skin, indicating that amelanotic melanocytes are not uniquely relied on for melanocyte homoeostasis.     

The Notch pathway: hair graying and pigment cell homeostasis

The Notch signaling pathway is an essential cell-cell interaction mechanism, which regulates processes such as cell proliferation, cell fate decisions, differentiation or stem cell maintenance. Pigmentation in mammals is provided by melanocytes, which are derived from the neural crest, and by the retinal pigment epithelium (RPE), which is part of the optic cup and hence orginates from neuroectoderm. The importance of functional Notch signaling in melanocytes has been unveiled recently. Here, the pathway is essential for the maintenance of proper hair pigmentation. Deletion of Notch1 and Notch2 or RBP-Jkappa in the melanocyte lineage resulted in a gene dosage-dependent precocious hair graying, due to the elimination of melanoblasts and melanocyte stem cells. Expression data support the idea that Notch signaling might equally be involved in development of the RPE. Furthermore, recent analyses indicate a possible role of Notch signaling in the development of melanoma. In this review, we address the essential role of Notch signaling in the regeneration of the melanocyte population during hair follicle cycles, and discuss data supporting the implication of this signaling pathway in RPE development and melanoma.     

Melanocyte homeostasis in vivo tolerates Rb1 loss in a developmentally independent fashion

There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT-Cre mediated loss of exon 19 from a floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi-transformed phenotype. In this study, we show that Rb1-null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre-mediated ablation of the exon occurs. Further, Rb1 loss has no serious long-term ramifications on melanocyte homeostasis in vivo, with Rb1-null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre-mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.     

Endothelin‐1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet‐induced melanocyte homeostasis

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV‐induced melanocyte homeostasis. Selective ablation of endothelin‐1 (EDN1) in murine epidermis (EDN1^ep?/?) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non‐cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte‐derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV‐induced melanocyte activation and recapitulated the phenotype seen in EDN1^ep?/? mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV‐induced melanocyte homeostasis in vivo.     

Coat color dilution in mice because of inactivation of the melanoma antigen MART-1

Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is found only in the pigment cell lineage. However, its exact function and involvement in pigmentation is not clearly understood. Melanoma antigen recognized by T cells 1 has been shown to interact with the melanosomal proteins Pmel17 and OA1. To understand the function of MART-1 in pigmentation, we developed a new knockout mouse model. Mice deficient in MART-1 are viable, but loss of MART-1 leads to a coat color phenotype, with a reduction in total melanin content of the skin and hair. Lack of MART-1 did not affect localization of melanocyte-specific proteins nor maturation of Pmel17. Melanosomes of hair follicle melanocytes in MART-1 knockout mice displayed morphological abnormalities, which were exclusive to stage III and IV melanosomes. In conclusion, our results suggest that MART-1 is a pigmentation gene that is required for melanosome biogenesis and/or maintenance.     

c-Myc is required for the formation of intestinal crypts but dispensable for homeostasis of the adult intestinal epithelium

In self-renewing tissues such as the skin epidermis and the bone marrow, Myc proteins control differentiation of stem cells and proliferation of progenitor cell types. In the epithelium of the small intestine, we show that c-Myc and N-Myc are expressed in a differential manner. Whereas c-Myc is expressed in the proliferating transient-amplifying compartment of the crypts, N-Myc is restricted to the differentiated villus epithelium and a single cell located near the crypt base. c-Myc has been implicated as a critical target of the canonical Wnt pathway, which is essential for formation and maintenance of the intestinal mucosa. To genetically assess the role of c-Myc during development and homeostasis of the mammalian intestine we induced deletion of the c-myc(flox) allele in the villi and intestinal stem cell-bearing crypts of juvenile and adult mice, via tamoxifen-induced activation of the CreER(T2) recombinase, driven by the villin promoter. Absence of c-Myc activity in the juvenile mucosa at the onset of crypt morphogenesis leads to a failure to form normal numbers of crypts in the small intestine. However, all mice recover from this insult to form and maintain a normal epithelium in the absence of c-Myc activity and without apparent compensation by N-Myc or L-Myc. This study provides genetic and molecular evidence that proliferation and expansion of progenitors necessary to maintain the adult intestinal epithelium can unexpectedly occur in a Myc-independent manner.     

Sound needs sound melanocytes to be heard

Intermediate cells in the stria vascularis of the mammalian cochlea are melanocytes, which contain melanin pigments and are capable of synthesizing melanin. These melanocytes are required for normal development of the cochlea, as evidenced by studies of mutant mice with congenital melanocyte anomalies. Melanocytes are also needed for developed cochleae to function normally, as evidenced by studies of mutant mice with late-onset melanocyte anomaly and humans with acquired melanocyte anomaly. Melanin, per se, does not seem to be essential for normal hearing function, but it may protect against traumata to the cochlea, e.g., noise and ototoxic aminoglycosides. Recent electrophysiological studies have revealed that strial intermediate cells are provided with specific ionic channels, such as inwardly rectifying K+ channels (Kir4.1) and voltage-dependent outwardly rectifying K+ channels. These channels may play central roles in strial function and thus in normal hearing.     

Keratinocytes Suppress TRP‐1 Expression and Reduce Cell Number of Co‐cultured Melanocytes – Implications For Grafting of Patients with Vitiligo

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well‐integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co‐cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down‐regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase‐related protein‐1 (TRP‐1) negative in confluent well‐integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP‐1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP‐1 expression.     

Melanocytes: the new Black

Melanocytes, pigment-producing cells residing primarily in the hair follicle, epidermis and eye, are responsible for skin hair and eye pigmentation. Pigmentation is achieved by the highly regulated manufacture of the pigment melanin in specialised organelles, melanosomes that are transported along dendritic processes before being transferred to growing hair, or keratinocytes where melanin protects from UV-induced DNA damage. Because loss of melanocytes gives a clear pigmentation phenotype yet is non-lethal, over 130 genes implicated in the development or function of this cell type have been identified to date, and in humans the loss of melanocytes or their ability to produce pigment, or transport or transfer melanosomes is associated with several diseases such as vitiligo, albinism and Hermansky-Pudlak syndrome. Importantly, the effective combination of genetics, cell and molecular biology possible with this cell type is attracting an increasing number of researchers focussed on understanding how cells coordinate survival, proliferation, differentiation and stem cell maintenance.