EDNRB/EDN3 and Hirschsprung disease type II.

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1-4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah-Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type-B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest-derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest-derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB-mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3-induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post-coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3--a receptor-mediated pathway with pleiotropic effects.     

Cell-autonomous and cell non-autonomous signaling through endothelin receptor B during melanocyte development

The endothelin receptor B gene (Ednrb) encodes a G-protein-coupled receptor that is expressed in a variety of cell types and is specifically required for the development of neural crest-derived melanocytes and enteric ganglia. In humans, mutations in this gene are associated with Waardenburg-Shah syndrome, a disorder characterized by pigmentation defects, deafness and megacolon. To address the question of whether melanocyte development depends entirely on a cell-autonomous action of Ednrb, we performed a series of tissue recombination experiments in vitro, using neural crest cell cultures from mouse embryos carrying a novel Ednrb-null allele characterized by the insertion of a lacZ marker gene. The results show that Ednrb is not required for the generation of early neural crest-derived melanoblasts but is required for the expression of the differentiation marker tyrosinase. Tyrosinase expression can be rescued, however, by the addition of Ednrb wild-type neural tubes. These Ednrb wild-type neural tubes need not be capable of generating melanocytes themselves, but must be capable of providing KIT ligand, the cognate ligand for the tyrosine kinase receptor KIT. In fact, soluble KIT ligand is sufficient to induce tyrosinase expression in Ednrb-deficient cultures. Nevertheless, these tyrosinase-expressing, Ednrb-deficient cells do not develop to terminally differentiated, pigmented melanocytes. Pigmentation can be induced, however, by treatment with tetradecanoyl phorbol acetate, which mimics EDNRB signaling, but not by treatment with endothelin 1, which stimulates the paralogous receptor EDNRA. The results suggest that Ednrb plays a significant role during melanocyte differentiation and effects melanocyte development by both cell non-autonomous and cell-autonomous signaling mechanisms.     

The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.     

Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.     

EDNRB/EDN3 and Hirschsprung Disease Type II

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1–4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah‐Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type‐B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest‐derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest‐derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB‐mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3‐induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post‐coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3 – a receptor‐mediated pathway with pleiotropic effects.     

Dct::lacZ ES cells: a novel cellular model to study melanocyte determination and differentiation

Embryonic stem (ES) cells differentiate into various cell lineages in vitro. A procedure was previously designed to promote the differentiation of ES cells towards the melanocyte lineage and to obtain large and reproducible amounts of melanocytes. To elucidate the main events that lead to the development of melanocytes in vitro, we used transgenic Dct::lacZ mouse blastocysts to establish ES cell lines expressing the lacZ reporter gene under the control of the Dct promoter. Dct, a melanoblast marker, is expressed just after melanoblast determination in vivo. We evaluated the importance of recruitment, proliferation and differentiation during melanocyte ontogeny after the in vitro differentiation of Dct::lacZ ES cells into melanocytes. We showed that bFGF and cholera toxin induce precocious melanoblast determination, associated with early melanocyte differentiation. Edn3 induced melanoblast proliferation and long-term melanoblast recruitment, but not precocious determination. The lack of basic Fibroblast Growth Factor (bFGF) and cholera toxin can be partially compensated by Edn3. Thus, Dct::lacZ ES cells can be used as a model to study determination, proliferation and differentiation in the melanocyte lineage in vitro.     

Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.     

Ednrb2 orients cell migration towards the dorsolateral neural crest pathway and promotes melanocyte differentiation

Endothelin receptors B (Ednrb) are involved in the development of the enteric and melanocytic lineages, which originate from neural crest cells (NCCs). In mice, trunk NCCs and their derivatives express only one Ednrb. In quail, trunk NCCs express two Ednrb: Ednrb and Ednrb2. Quail Ednrb is expressed in NCCs migrating along the ventral pathway, which gives rise to the peripheral nervous system, including enteric ganglia. Ednrb2 is upregulated in NCCs before these cells enter the dorsolateral pathway. The NCCs migrating along the dorsolateral pathway are melanocyte precursors. We analyzed the in vitro differentiation and in ovo migration of mouse embryonic stem (ES) cells expressing and not expressing Ednrb2. We generated a series of transfected ES cell lines expressing Ednrb2. This receptor, like Ednrb, oriented genuine ES cells towards melanocyte lineage differentiation in vitro. The in ovo migration of Ednrb2-expressing ES cells was massively oriented towards the dorsolateral pathway, unlike that of WT or Ednrb-expressing ES cells. Thus, Ednrb2 is involved in melanoblast differentiation and migration.     

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.     

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf

Coordination of cell proliferation and differentiation is crucial for tissue formation, repair and regeneration. Some tissues, such as skin and blood, depend on differentiation of a pluripotent stem cell population, whereas others depend on the division of differentiated cells. In development and in the hair follicle, pigmented melanocytes are derived from undifferentiated precursor cells or stem cells. However, differentiated melanocytes may also have proliferative capacity in animals, and the potential for differentiated melanocyte cell division in development and regeneration remains largely unexplored. Here, we use time-lapse imaging of the developing zebrafish to show that while most melanocytes arise from undifferentiated precursor cells, an unexpected subpopulation of differentiated melanocytes arises by cell division. Depletion of the overall melanocyte population triggers a regeneration phase in which differentiated melanocyte division is significantly enhanced, particularly in young differentiated melanocytes. Additionally, we find reduced levels of Mitf activity using an mitfa temperature-sensitive line results in a dramatic increase in differentiated melanocyte cell division. This supports models that in addition to promoting differentiation, Mitf also promotes withdrawal from the cell cycle. We suggest differentiated cell division is relevant to melanoma progression because the human melanoma mutation MITF(4T)(Δ)(2B) promotes increased and serial differentiated melanocyte division in zebrafish. These results reveal a novel pathway of differentiated melanocyte division in vivo, and that Mitf activity is essential for maintaining cell cycle arrest in differentiated melanocytes.     

Endothelin receptor B is required for the expansion of melanocyte precursors and malignant melanoma

Since embryonic development and tumorigenesis share common characteristics, studying the role of genes during development can identify molecules that have similar functions in both processes. C-kit and Endothelin receptor B (EDNRB or ETRB) are crucial for melanocyte development in mice and humans but have different functions. While c-kit is needed for survival throughout development until late stages of differentiation in the skin, EDNRB promotes early expansion and migration while delaying the differentiation of melanocyte precursors. Transformation of normal melanocytes to melanoma cells is often associated with gradual loss of differentiation and the gain of high autonomous capacity to proliferate. In accordance with their different roles, c-kit expression is gradually lost during melanoma transformation, while that of EDNRB is greatly enhanced and can serve as a marker of melanoma progression. Inhibiting EDNRB function with a specific antagonist (BQ788) in human melanoma cell lines results in inhibition of growth often associated with induced differentiation indicating that, during melanoma transformation also, the function of EDNRB is to promote growth. EDNRB function does not seem to be essential in the adult, as BQ788 administration to healthy people does not result in major effects. This is probably why BQ788 can specifically inhibit the growth of xenograft human melanoma tumors in nude mice, in a way resembling spontaneous human melanoma regression and why it could serve as a potential therapeutic agent for melanoma.     

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.     

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.     

Crosstalk in skin: melanocytes,keratinocytes, stem cells,and melanoma

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.     

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.     

A requirement for <Emphasis Type="Italic">kit</Emphasis> in embryonic zebrafish melanocyte differentiation is revealed by melanoblast delay

Exploring differences in gene requirements between species can allow us to delineate basic developmental mechanisms, provide insight into patterns of evolution, and explain heterochronic differences in developmental processes. One example of differences in gene requirements between zebrafish and mammals is the requirement of the kit receptor tyrosine kinase in melanocyte development. kit is required for migration, survival and differentiation of all neural crest-derived melanocytes in mammals. In contrast, zebrafish kit is not required for differentiation of embryonic melanocytes during normal development. When melanoblast development in zebrafish embryos is delayed by injecting morpholinos targeted to the mitfa gene, we show that these delayed melanoblasts fail to differentiate in kit mutants. Thus, we show that there is a kit requirement for melanocyte differentiation in zebrafish when melanoblast development is delayed. Furthermore, we show that kit is not involved in maintaining melanocyte precursors through the developmental delay, but instead is required for differentiation of melanocytes after the block on their development is removed. Finally, we suggest there is a heterochronic shift in the onset of melanocyte differentiation between fish and mouse, and developmental delay of melanoblast development in zebrafish removes this heterochronic difference.Edited by D. Tautz     

Hear the Wnt Ror: how melanoma cells adjust to changes in Wnt

The interplay between canonical and non‐canonical Wnt pathways in development and tumorigenesis is tightly regulated. In this review we will describe the yin and the yang of canonical and non‐canonical Wnt signaling pathways during melanocyte development, and melanoma genesis. Canonical Wnt signaling, represented by Wnts such as Wnt1 and Wnt3A, signals via β‐catenin to promote melanocyte differentiation and tumor development. Non‐canonical Wnt signaling, specifically Wnt5A, regulates canonical pathways, and signals to induce melanoma metastasis. This review will focus on the role of Wnt5A during melanoma progression, and its relationship to canonical Wnt signaling.