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.     

Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells

During embryonic development in vertebrates, the neural crest‐derived melanoblasts migrate along the dorsolateral axis and cross the basal membrane separating the dermis from the epidermis to reach their final location in the interfollicular epidermis and epidermal hair follicles. Neoplastic transformation converts melanocytes into highly invasive and metastatic melanoma cells. In vitro, these cells extend various types of protrusions and adopt two interconvertible modes of migration, mesenchymal and amoeboid, driven by different signalling molecules. In this review, we describe the major contributions of natural mouse mutants, mouse models generated by genetic engineering and in vitro culture systems, to identification of the genes, signalling pathways and mechanisms regulating the migration of normal and pathological cells of the melanocyte lineage, at both the cellular and molecular levels.     

Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte‐specific cell–cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1‐silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1‐silenced keratinocytes. Dsg1‐silenced keratinocytes increased melanocyte‐stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1‐silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1‐silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1‐deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development.     

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.     

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.     

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.     

Melanocytes in development and cancer

Melanocytes are pigment‐producing cells in the skin of humans and other vertebrates. A number of genes involved in melanocyte development and vertebrate pigmentation have been characterized, largely through studies of a diversity of pigment mutations in a variety of species. Embryonic development of the melanocyte initiates with cell fate specification in the neural crest, which is then followed by cell migration and niche localization. Many genes involved in melanocyte development have also been implicated in the development of melanoma, an aggressive and fatal form of skin cancer that originates in the melanocyte. Although early stage melanomas that have not spread to the lymph nodes can be excised with little risk of recurrence, patients diagnosed with metastatic melanoma have a high mortality rate due to the resistance of most tumors to radiotherapy and chemotherapy. Transformed melanocytes that develop into melanomas proliferate abnormally and often begin to grow radially in the skin. Vertical growth can then follow this radial growth, leading to an invasion through the basement membrane into the underlying dermis and subsequent metastasis. It is still unclear, however, how a normal melanocyte becomes a melanoma cell, and how melanoma utilizes the properties of the normal melanocyte and its progenitors in its progression. The goal of this mini‐review is to highlight the role of melanocyte developmental pathways in melanoma, and to discuss recent studies and tools being used to illuminate this connection. J. Cell. Physiol. 222:38–41, 2010. © 2009 Wiley‐Liss, Inc.     

Novel method for isolating human melanoblasts from keratinocyte culture

The characterization of melanoblasts is important for understanding their in vivo development, melanoma formation, and pigment‐related disorders. However, no methods have been reported for the isolation of melanoblasts from human skin. Using a ‘calcium‐pulse’ technique, involving the differentiation of human keratinocytes with high calcium and the subsequent spontaneous separation of the epidermal sheets, we effectively isolated human melanoblasts (keratinocyte‐adapted melanoblasts, KaMBs) from keratinocyte culture. The KaMBs expressed early melanogenesis‐related genes, including BRN2, which is a known melanoblast marker. Moreover, the KaMBs displayed much higher proliferative and growth capacities than the primary melanocytes. Considering that keratinocytes might provide an in vivo‐like environment for KaMBs during isolation and in vitro culture, the ‘calcium‐pulse’ technique offers an unprecedented, easy, and efficient method for the isolation of human melanoblasts, retaining the original characteristics of these cells.     

Vitiligo-related neuropeptides in nerve fibers of the skin

Skin distribution of substance P (SP)-, somatostatin (SOM)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity (LI) in vitiligo patients was studied by an indirect immunofluorescence technique. Immunocytochemical characteristics of the epidermis, dermal-epidermal junction, papillary and reticular dermis and skin appendages were analyzed in lesional and marginal vitiligo areas, as well as in healthy skin. In healthy pigmented skin, SP-, SOM-, CGRP-, and NPY-LI nerve fibers were observed with specific distributional patterns. In uninvolved vitiligo skin, thin SP-containing fibers were evident in dermal papillae, extending into the epidermis, and SP-LI fibers were seen around blood vessels and sweat glands. SOM-LI varicose nerve fibers were associated with Meissner corpuscles in the dermal papillae, while CGRP-LI was demonstrated in the free subepidermal nerve terminals and in sensory nerve fibers around blood vessels, hair follicles and sweat glands. Autonomic NPY-nerve fibers innervated the eccrine sweat glands and blood vessels. The distribution of these neuropeptides in both marginal and lesional areas of vitiliginous skin was the same as in the skin of healthy control subjects, except for an increased immunoreactivity against NPY and, to a lesser extent, against CGRP in the skin depigmentation lesions. The elevated NPY levels in skin affected by vitiligo suggest that this peptide may serve as a neurochemical marker in the pathogenesis of the disease, thus supporting the neuronal theory of vitiligo.     

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.     

MicroRNA‐622 is a novel mediator of tumorigenicity in melanoma by targeting Kirsten rat sarcoma

The network of molecular players is similar when comparing neural crest‐derived, actively migrating melanoblasts to melanoma cells. However, melanoblasts are sensitive to differentiation‐initiating signals at their target site (epidermis), while melanoma cells maintain migratory and undifferentiated features. We aimed at identifying downregulated genes in melanoma that are particularly upregulated in melanoblasts. Loss of such genes could contribute to stabilization of a dedifferentiated, malignant phenotype in melanoma. We determined that microRNA‐622 (miR‐622) expression was strongly downregulated in melanoma cells and tissues compared to melanocytes and melanoblast‐related cells. miR‐622 expression correlated with survival of patients with melanoma. miR‐622 re‐expression inhibited clonogenicity, proliferation, and migration in melanoma. Inhibition of miR‐622 in melanocytes induced enhanced migration. Kirsten rat sarcoma (KRAS) was identified as a major functional target of miR‐622 in melanoma. We conclude that miR‐622 is a novel tumor suppressor in melanoma and identify the miR‐622‐KRAS axis as potential therapeutic target.     

Connecting the dots: Melanoma cell of origin,tumor cell plasticity,trans-differentiation,and drug resistance

Melanoma, a lethal malignancy that arises from melanocytes, exhibits a multiplicity of clinico-pathologically distinct subtypes in sun-exposed and non-sun-exposed areas. Melanocytes are derived from multipotent neural crest cells and are present in diverse anatomical locations, including skin, eyes, and various mucosal membranes. Tissue-resident melanocyte stem cells and melanocyte precursors contribute to melanocyte renewal. Elegant studies using mouse genetic models have shown that melanoma can arise from either melanocyte stem cells or differentiated pigment-producing melanocytes depending on a combination of tissue and anatomical site of origin and activation of oncogenic mutations (or overexpression) and/or the repression in expression or inactivating mutations in tumor suppressors. This variation raises the possibility that different subtypes of human melanomas (even subsets within each subtype) may also be a manifestation of malignancies of distinct cells of origin. Melanoma is known to exhibit phenotypic plasticity and trans-differentiation (defined as a tendency to differentiate into cell lineages other than the original lineage from which the tumor arose) along vascular and neural lineages. Additionally, stem cell-like properties such as pseudo-epithelial-to-mesenchymal (EMT-like) transition and expression of stem cell-related genes have also been associated with the development of melanoma drug resistance. Recent studies that employed reprogramming melanoma cells to induced pluripotent stem cells have uncovered potential relationships between melanoma plasticity, trans-differentiation, and drug resistance and implications for cell or origin of human cutaneous melanoma. This review provides a comprehensive summary of the current state of knowledge on melanoma cell of origin and the relationship between tumor cell plasticity and drug resistance.     

Biological and mathematical modeling of melanocyte development

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function β-catenin mutants in the melanocyte lineage. We found that any alteration of β-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through β-catenin.     

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.