Neural Crest-derived Pigment Cells Research Articles

Cannabinoid receptor type 1 regulates sequential stages of migration and morphogenesis of neural crest cells and derivatives in chicken and frog embryos.

The main cannabinoid receptor CB1R first shows expression during early neurula stage in chicken (Gallus gallus) embryos, and at early tailbud stage in the frog (Xenopus laevis) embryos. This raises the question of whether CB1R regulates similar or distinct processes during the embryonic development of these two species. Here, we examined whether CB1R influences the migration and morphogenesis of neural crest cells and derivatives in both chicken and frog embryos. Early neurula stage chicken embryos were exposed to arachidonyl-2'-chloroethylamide (ACEA; a CB1R agonist), N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; a CB1R inverse agonist) or Blebbistatin (nonmuscle Myosin II inhibitor) in ovo and examined during migration of neural crest cells and at condensing cranial ganglia stage. Early tailbud stage frog embryos were bathed in ACEA, AM251 or Blebbistatin, and analyzed at late tailbud stage for changes in craniofacial and eye morphogenesis, and in patterning and morphology of melanophores (neural crest-derived pigment cells). In chicken embryos exposed to ACEA and Myosin II inhibitor, cranial neural crest cells migrated erratically from the neural tube, and the right, but not the left, ophthalmic nerve of the trigeminal ganglia was affected in ACEA- and AM251-treated embryos. In frog embryos with inactivation or activation of CB1R, or inhibition of Myosin II, the craniofacial and eye regions were smaller and/or less developed, and the melanophores overlying the posterior midbrain were more dense, and stellate in morphology, than the same tissues and cells in control embryos. This data suggests that despite differences in the time of onset of expression, normal activity of CB1R is required for sequential steps in migration and morphogenesis of neural crest cells and derivatives in both chicken and frog embryos. In addition, CB1R may signal through Myosin II to regulate migration and morphogenesis of neural crest cells and derivatives in chicken and frog embryos.

Two induced pluripotent stem cell (iPSC) lines derived from patients affected by Waardenburg syndrome type 1 retain potential to activate neural crest markers

Waardenburg syndrome type 1 (WS1), a rare genetic disease characterized by pigmentation defects and mild craniofacial anomalies often associated with congenital deafness is caused by heterozygous mutations in the PAX3 gene (2q36.1). We have generated two induced pluripotent stem cell lines (PCli029-A and PCli031-A) from two patients from the same family both carrying the same heterozygous deletion in PAX3 exon 1 (c.-70_85 + 366del). These cells are pluripotent as they can differentiate into ectoderm, mesoderm and endoderm. They also can activate the early neural crest marker SNAI2. These cells will be useful for studying the human neural crest-derived pigment cells.

Comparative embryogenesis in ungulate domesticated species.

We compared embryogenesis of five species of domesticated even-toed and one odd-toed ungulate and used a phylogenetic framework to contextualize such comparison. Organ systems that occur relatively earlier in embryogenesis generally have more time to develop and therefore are found to be more mature at birth when compared to structures that appear later in development. We hypothesized that the less mature the animals' organs are at birth, the more they are susceptible to artificial selection. The horse had the most mature organs at birth, followed by cattle, reindeer, sheep/goat, and pig. This pattern of maturity could be observed almost during the entire development. Heterochronic shifts among species were observed only after fur starts to develop. Changes in the fur coloration are one of the first observable signs of domestication and the heterochrony of this trait may be related to the effects on neural crest-derived pigment cells by artificial selection. The six ungulate species also differ in the relative duration of their weaning period and the potential extent of its artificial shortening. We put all these traits in the context of their inherited evolutionary characteristics and artificial domestication process. Related to their altriciality, carnivoran domesticates, which also belong to Scrotifera, are less mature at birth than all domesticated ungulates. Although we detected clear character correlations to life history traits, it is impossible based on the present data, to trace specific exaptations to the domestication process. We hypothesize a deep time developmental penetration of adult characters into embryogenesis.

The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor.

Understanding how fate specification of distinct cell-types from multipotent progenitors occurs is a fundamental question in embryology. Neural crest stem cells (NCSCs) generate extraordinarily diverse derivatives, including multiple neural, skeletogenic and pigment cell fates. Key transcription factors and extracellular signals specifying NCSC lineages remain to be identified, and we have only a little idea of how and when they function together to control fate. Zebrafish have three neural crest-derived pigment cell types, black melanocytes, light-reflecting iridophores and yellow xanthophores, which offer a powerful model for studying the molecular and cellular mechanisms of fate segregation. Mitfa has been identified as the master regulator of melanocyte fate. Here, we show that an Mitf-related transcription factor, Tfec, functions as master regulator of the iridophore fate. Surprisingly, our phenotypic analysis of tfec mutants demonstrates that Tfec also functions in the initial specification of all three pigment cell-types, although the melanocyte and xanthophore lineages recover later. We show that Mitfa represses tfec expression, revealing a likely mechanism contributing to the decision between melanocyte and iridophore fate. Our data are consistent with the long-standing proposal of a tripotent progenitor restricted to pigment cell fates. Moreover, we investigate activation, maintenance and function of tfec in multipotent NCSCs, demonstrating for the first time its role in the gene regulatory network forming and maintaining early neural crest cells. In summary, we build on our previous work to characterise the gene regulatory network governing iridophore development, establishing Tfec as the master regulator driving iridophore specification from multipotent progenitors, while shedding light on possible cellular mechanisms of progressive fate restriction.

Regulation of zebrafish melanocyte development by ligand-dependent BMP signaling.

Preventing terminal differentiation is important in the development and progression of many cancers including melanoma. Recent identification of the BMP ligand GDF6 as a novel melanoma oncogene showed GDF6-activated BMP signaling suppresses differentiation of melanoma cells. Previous studies have identified roles for GDF6 orthologs during early embryonic and neural crest development, but have not identified direct regulation of melanocyte development by GDF6. Here, we investigate the BMP ligand gdf6a, a zebrafish ortholog of human GDF6, during the development of melanocytes from the neural crest. We establish that the loss of gdf6a or inhibition of BMP signaling during neural crest development disrupts normal pigment cell development, leading to an increase in the number of melanocytes and a corresponding decrease in iridophores, another neural crest-derived pigment cell type in zebrafish. This shift occurs as pigment cells arise from the neural crest and depends on mitfa, an ortholog of MITF, a key regulator of melanocyte development that is also targeted by oncogenic BMP signaling. Together, these results indicate that the oncogenic role ligand-dependent BMP signaling plays in suppressing differentiation in melanoma is a reiteration of its physiological roles during melanocyte development.

Thyroid hormone regulates distinct paths to maturation in pigment cell lineages.

Thyroid hormone (TH) regulates diverse developmental events and can drive disparate cellular outcomes. In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers. To learn how TH elicits seemingly opposite responses in cells having a common embryological origin, we analyzed individual transcriptomes from thousands of neural crest-derived cells, reconstructed developmental trajectories, identified pigment cell-lineage specific responses to TH, and assessed roles for TH receptors. We show that TH promotes maturation of both cell types but in distinct ways. In melanophores, TH drives terminal differentiation, limiting final cell numbers. In xanthophores, TH promotes accumulation of orange carotenoids, making the cells visible. TH receptors act primarily to repress these programs when TH is limiting. Our findings show how a single endocrine factor integrates very different cellular activities during the generation of adult form.

ALKALs are in vivo ligands for ALK family receptor tyrosine kinases in the neural crest and derived cells

Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

Fishful thinking: the rise and fall of MITF in melanoma

Fishful thinking: the rise and fall of <scp>MITF</scp> in melanoma

Old before their time: the ancient origins of the neural crest.

Old before their time: the ancient origins of the neural crest.

Stem cells and pluripotency: emerging themes and tools

The Minisymposium on Stem Cells and Pluripotency was broad in scope, covering a variety of mechanisms regulating stem cell behavior during regeneration, aging, and tumorigenesis, as well as factors involved in regulating the conversion of differentiated cells back to a pluripotent state (reprogramming). In addition, one talk described the development of tools to assay functionality of cells differentiated in vitro from pluripotent stem cells. Altogether, the session provided a broad overview of a number of active areas in the field of stem cell biology. Leanne Jones (Salk Institute for Biological Studies) presented data describing a potential mechanism that regulates aging of the stem cell microenvironment, also known as the stem cell “niche.” In the Drosophila testis, aging results in a decrease in self-renewal factors emanating from key niche support cells. The Jones laboratory has characterized an RNA-binding protein that binds to the mRNA for a key self-renewal factor and protects it from degradation. However, with age, expression of the RNA-binding protein also decreases, leaving the self-renewal factor unprotected, resulting in decreased signaling, loss of stem cells, and decreased tissue homeostasis. Hebao Yuan (Yamashita laboratory, University of Michigan) proposed a model for how stem cells monitor spindle orientation to ensure an asymmetric division in the Drosophila male germ line. His work has demonstrated that germ-line stem cells (GSCs) possess a checkpoint (“the centrosome orientation checkpoint”) that monitors correct centrosome orientation prior to mitosis to ensure an oriented spindle and thus asymmetric outcome of the division. Par-1, a serine/threonine kinase that regulates polarity in many systems, is involved in this checkpoint. Par-1 shows a cell cycle–dependent localization to a germ line–specific, endoplasmic reticulum–like organelle, which in turn leads to localization of cyclin A. He proposes that the regulation of cyclin A localization via Par-1 function plays a critical role in the centrosome orientation checkpoint. Adam Cohen (Department of Chemistry and Chemical Biology, Harvard University) described experiments to incorporate fluorescent voltage-indicating proteins (VIPs), which are based on a modified microbial rhodopsin protein, into high-throughput assays to screen differentiated cells for functionality. The fluorescence of VIPs sensitively and accurately reflects membrane potential; therefore, VIPs would be very useful for detecting action potentials in neurons or membrane potential of cardiomyocytes differentiated in vitro from human pluripotent stem cells. Having such tools in place would greatly facilitate the use of such cell types in screens for drugs or small molecules that could be used to treat patients suffering from diseases that affect such tissues. Jizhou Yan (College of Fisheries and Life Sciences, Shanghai Ocean University) is studying the cellular and molecular processes that underlie restoration of the lower jaw after amputation in zebrafish. Zebrafish possesses the remarkable ability to regenerate the complicated structures by formation of the characteristic blastema; however, the mechanism by which blastemal cells arise and reorganize is debated. Yan and colleagues characterized the stages of jaw regeneration and found that the blastema is formed from dedifferentiation of mesodermal cells and neural crest–derived pigment cells into two types of precursor cells to restore the original tissue structures, including bone, muscle, pigment, and connective tissue. Further time point–based RNA sequencing revealed that specific signaling pathways are spatiotemporally activated in response to successive steps of cell type conversions. These data indicate that epithelial–mesenchymal interactions and local microenvironmental cues induce transdifferentiation and dedifferentiation of preblastemal cells, and subsequent redifferentiation. Jennifer Brady (Blau laboratory, Stanford University) described experiments aimed at uncovering the molecular mechanisms underlying the process by which induced pluripotent stem cells (iPS cells) can be generated from essentially any somatic cell. A synchronous, reprogramming approach consisting of heterokaryons (interspecies, multinucleate, fused cells) was developed in which activation of human pluripotency genes occurs rapidly (<24 h) and efficiently (70% of single heterokaryons), enabling early mechanistic studies. High-throughput RNA sequencing revealed transcriptome-wide changes during heterokaryon reprogramming, including the induction of key human pluripotency genes and chromatin remodelers, validating this approach as a discovery tool. Fernando Camargo (Children's Hospital, Boston; Harvard Stem Cell Institute, Cambridge) is studying the function of the highly conserved Hippo signaling pathway and its effects on tissue size and development and cancer. Growth suppression by Hippo signaling occurs by the inactivation of the transcriptional coactivator YAP. Camargo described experiments in mice that demonstrate that YAP, a protein known for its powerful growth-inducing and oncogenic properties, has an unexpected tumor-suppressive function in the mammalian intestine. Consistent with these findings, Camargo also presented data that indicate YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas. Altogether, the data he presented describe a novel mechanistic paradigm for how stem cell expansion and proliferative signals are counterbalanced by the Hippo pathway during organ growth.

Zebrafish: A Model System to Study Heritable Skin Diseases

Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in ∼500 different genes. A number of model systems have been developed to advance our understanding of the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5-6 days post-fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphological and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provide a useful model system to study the molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases.

Tracing blastomere fate choices of early embryos in single cell culture

AbstractBlastomeres of early vertebrate embryos undergo numerous fate choices for division, motility, pluripotency maintenance and restriction culminating in various cell lineages. Tracing blastomere fate choices at the single cell level in vitro has not been possible because of the inability to isolate and cultivate early blastomeres as single cells. Here we report the establishment of single cell culture system in the fish medaka, enabling the isolation and cultivation of individual blastomeres from 16- to 64-cell embryos for fate tracing at the single cell level in vitro. Interestingly, these blastomeres immediately upon isolation exhibit motility, lose synchronous divisions and even stop dividing in &amp;8805;50% cases, suggesting that the widely accepted nucleocytoplasmic ratio controlling synchronous divisions in entire embryos does not operate on individual blastomeres. We even observed abortive division, endomitosis and cell fusion. Strikingly, ~5% of blastomeres in single cell culture generated extraembryonic yolk syncytial cells, embryonic stem cells and neural crest-derived pigment cells with timings mimicking their appearance in embryos. We revealed the maternal inheritance of key lineage regulators and their differential expression in cleavage embryos. Therefore, medaka blastomeres possess the accessibility for single cell culture, previously unidentified heterogeneity in motility, division, gene expression and intrinsic ability to generate major extraembryonic and embryonic lineages without positioning cues. Our data demonstrate the fidelity and potential of the single cell culture system for tracking blastomere fate decisions under defined conditions in vitro.

Morphogenesis of post-embryonic neural crest-derived pigment cell precursors revealed by time lapse imaging during zebrafish adult pigment pattern formation

Morphogenesis of post-embryonic neural crest-derived pigment cell precursors revealed by time lapse imaging during zebrafish adult pigment pattern formation

Development and regeneration of the zebrafish maxillary barbel: a novel study system for vertebrate tissue growth and repair.

BackgroundBarbels are integumentary sense organs found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops paired nasal and maxillary barbels approximately one month post fertilization. Small in diameter and optically clear, these adult appendages offer a window on the development, maintenance and function of multiple cell types including skin cells, neural-crest derived pigment cells, circulatory vessels, taste buds and sensory nerves. Importantly, barbels in other otophysan fishes (e.g., catfish) are known to regenerate; however, this capacity has not been tested in zebrafish.Methodology/Principal FindingsWe describe the development of the maxillary barbel in a staged series of wild type and transgenic zebrafish using light microscopy, histology and immunohistochemistry. By imaging transgenic zebrafish containing fluorescently labeled endothelial cells (Tg(fli1a:EGFP)), we demonstrate that the barbel contains a long (∼2–3 mm) closed-end vessel that we interpret as a large lymphatic. The identity of this vessel was further supported by live imaging of the barbel circulation, extending recent descriptions of the lymphatic system in zebrafish. The maxillary barbel can be induced to regenerate by proximal amputation. After more than 750 experimental surgeries in which approximately 85% of the barbel's length was removed, we find that wound healing is complete within hours, followed by blastema formation (∼3 days), epithelial redifferentiation (3–5 days) and appendage elongation. Maximum regrowth occurs within 2 weeks of injury. Although superficially normal, the regenerates are shorter and thicker than the contralateral controls, have abnormally organized mesenchymal cells and extracellular matrix, and contain prominent connective tissue “stumps” at the plane of section—a mode of regeneration more typical of mammalian scarring than other zebrafish appendages. Finally, we show that the maxillary barbel can regenerate after repeated injury and also in senescent fish (>2 years old).Conclusions/SignificanceAlthough the teleost barbel has no human analog, the cell types it contains are highly conserved. Thus “barbology” may be a useful system for studying epithelial-mesenchymal interactions, angiogenesis and lymphangiogenesis, neural pathfinding, wound healing, scar formation and other key processes in vertebrate physiology.

Basonuclin-2 requirements for zebrafish adult pigment pattern development and female fertility.

Relatively little is known about the generation of adult form. One complex adult trait that is particularly amenable to genetic and experimental analysis is the zebrafish pigment pattern, which undergoes extensive remodeling during post-embryonic development to form adult stripes. These stripes result from the arrangement of three classes of neural crest-derived pigment cells, or chromatophores: melanophores, xanthophores, and iridophores. Here, we analyze the zebrafish bonaparte mutant, which has a normal early pigment pattern but exhibits a severe disruption to the adult stripe pattern. We show that the bonaparte mutant phenotype arises from mutations in basonuclin-2 (bnc2), encoding a highly conserved, nuclear-localized zinc finger protein of unknown function. We show that bnc2 acts non-autonomously to the melanophore lineage and is expressed by hypodermal cells adjacent to chromatophores during adult pigment pattern formation. In bonaparte (bnc2) mutants, all three types of chromatophores differentiate but then are lost by extrusion through the skin. We further show that while bnc2 promotes the development of two genetically distinct populations of melanophores in the body stripes, chromatophores of the fins and scales remain unaffected in bonaparte mutants, though a requirement of fin chromatophores for bnc2 is revealed in the absence of kit and colony stimulating factor-1 receptor activity. Finally, we find that bonaparte (bnc2) mutants exhibit dysmorphic ovaries correlating with infertility and bnc2 is expressed in somatic ovarian cells, whereas the related gene, bnc1, is expressed within oocytes; and we find that both bnc2 and bnc1 are expressed abundantly within the central nervous system. These findings identify bnc2 as an important mediator of adult pigment pattern formation and identify bonaparte mutants as an animal model for dissecting bnc2 functions.

LiCl에 의해 유도되는 phosphoprotein이 embryonic zebrafish (Danio rerio)의 pigmentation에 미치는 영향

Neural crest는 신경계의 발생과정에서 생긴 특정화된 외배엽으로서 말초신경계(peripheral nervous system)의 모든 sensory cells과 fibers, 자율신경계의 대부분의 peripheral cells, unipolar spinal ganglion cell, cranial sensory ganglia, peripheral nerve의 neurolemmal sheath cells, ganglia의 capsule cells, sympathetic ganglia, chromaffin cells, pigment cell 등이 분화한다. Fish의 경우는 melanin을 가지고 있는 melanophores, yellow pigment를 가지고 있는 xanthopores, reflecting platelets를 가지고 있는 iridophores등 3가지의 pigment-producing cell을 가지고 있다. 다양한 pigement들의 deposition, distribution에 의해 Fish와 amphibian에서 볼 수 있는 수많은 color와 pattern이 만들어지게 된다. Embryonic neural crest가 patterning을 연구하기에 아주 좋은 모델임에도 불구하고, choromatophores의 cell-signaling mechanism에 관한 연구는 거의 없는 실정이다. 본 연구에서는 melanosomes의 melanocyt로의 이동기작과 이들의 dentiritic processe를 밝히기 밝히기 위해 phosphorylaion assay와 투과형 전자 현미경(transmission electron microscope)등을 이용한 다양한 실험들을 토대로, Lithium에 의해 유도되는 morphological alteration에 IP cell signaling pathway에 의해 조절되는 단백질의 하나인 55-kDa단백질의 인산화가 중요한 역할을 함을 밝혔다. The embryonic zebrafish (Danio rerio) is rapidly becoming an important model organism for studies of early events in vertebrate development. Neural crest-derived pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores, and/or iridophores. Cell-signaling mechanisms related to the development of pigmentation and pigment pattern formation remain obscure. In this study, zebrafish embryos were treated with various signaling-related molecules - LiCl (an inositol-phosphatase inhibitor), forskolin (a protein kinase-A activator), a combination of LiCl/forskolin, and LiCl/heparin (an IP3 inhibitor) in order to identify the mechanisms involved in pigmentation. LiCl treatment resulted in ultrastructural and morphological alterations of melanophores. To identify the possible proteins responsible for this ultrastructural and morphological change, phosphorylation patterns in vitro and in vivo were analyzed. LiCl and LiCl/forskolin treatment elicited dramatic increases in the phosphorylation of a 55-kDa protein which was inhibited by heparin treatment. LiCl treatment also induced phosphorylation of a 55-kDa protein in melanophores purified from adult zebrafish. Collectively these results suggest that a LiCl-induced 55-kDa phosphoprotein plays a role in melanophore morphology and ultrastructure and ultimately effects gross pigmentation.

Zebrafish Endzone Regulates Neural Crest-Derived Chromatophore Differentiation and Morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.

Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes

The signaling of stem cell factor (SCF) and its receptor KIT (membrane-bound KIT; m-KIT) plays an important role in melanocyte development, survival, proliferation, and melanogenesis. It has been demonstrated in other systems that a soluble form of m-KIT released from the cell surface (s-KIT) regulates SCF signaling, although there have been no reports pertaining to the existence and the biological role of s-KIT in melanocytes. In this study, we therefore examined the involvement of s-KIT in melanogenesis. Western blotting analysis revealed that treatment with phorbol 12-myristate-13-acetate (PMA) or 4-aminophenylmercuric acetate (APMA) induced s-KIT production in cultured human melanocytes. Inhibitors of tumor necrosis factor-alpha-converting enzyme (TACE) and metalloproteinases (MMPs) muted this release of s-KIT into the media. Human recombinant s-KIT added to melanocytes inhibited SCF-induced phosphorylation of m-KIT, resulting in suppression of SCF-induced melanogenesis. Additionally, APMA-induced s-KIT production abolished SCF-induced melanogenesis as effectively as a KIT-neutralizing antibody. Concomitantly, APMA and TACE inhibitors significantly decreased and increased melanin synthesis, respectively, in an in vitro skin model. Taken together, these findings provided an insight into the elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggested that production of s-KIT contributes to the regulation of human skin pigmentation. Journal of Investigative Dermatology (2008) 128, 1763-1772; doi:10.1038/jid.2008.9; published online 31 January 2008.

Colgate/hdac1 repression of foxd3 expression is required to permit mitfa-dependent melanogenesis

Neural crest-derived pigment cell development has been used extensively to study cell fate specification, migration, proliferation, survival and differentiation. Many of the genes and regulatory mechanisms required for pigment cell development are conserved across vertebrates. The zebrafish mutant colgate ( col) /histone deacetylase1 ( hdac1) has reduced numbers, delayed differentiation and decreased migration of neural crest-derived melanophores and their precursors. In hdac1 col mutants normal numbers of premigratory neural crest cells are induced. Later, while there is only a slight reduction in the number of neural crest cells in hdac1 col mutants, there is a severe reduction in the number of mitfa-positive melanoblasts suggesting that hdac1 is required for melanoblast specification. Concomitantly, there is a significant increase in and prolonged expression of foxd3 in neural crest cells in hdac1 col mutants. We found that partially reducing Foxd3 expression in hdac1 col mutants rescues mitfa expression and the melanophore defects in hdac1 col mutants. Furthermore, we demonstrate the ability of Foxd3 to physically interact at the mitfa promoter. Because mitfa is required for melanoblast specification and development, our results suggest that hdac1 is normally required to suppress neural crest foxd3 expression thus de-repressing mitfa resulting in melanogenesis by a subset of neural crest-derived cells.

Frizzled7 mediates canonical Wnt signaling in neural crest induction

The neural crest is a multipotent cell population that migrates from the dorsal edge of the neural tube to various parts of the embryo where it differentiates into a remarkable variety of different cell types. Initial induction of neural crest is mediated by a combination of BMP, Wnt, FGF, Retinoic acid and Notch/Delta signaling. The two-signal model for neural crest induction suggests that BMP signaling induces the competence to become neural crest. The second signal involves Wnt acting through the canonical pathway and leads to expression of neural crest markers such as slug. Wnt signals from the neural plate, non-neural ectoderm and paraxial mesoderm have all been suggested to play a role in neural crest induction. We show that Xenopus frizzled7 (Xfz7) is expressed in the dorsal ectoderm including early neural crest progenitors and is a key mediator of the Wnt inductive signal. We demonstrate that Xfz7 expression is induced in response to a BMP antagonist, noggin, and that Xfz7 can induce neural crest specific genes in noggin-treated ectodermal explants (animal caps). Morpholino-mediated or dominant negative inhibition of Xfz7 inhibits Wnt induced Xslug expression in the animal cap assay and in the whole embryo leading to a loss of neural crest derived pigment cells. Full-length Xfz7 rescues the morpholino-induced phenotype, as does activated β-catenin, suggesting that Xfz7 is signaling through the canonical pathway. We therefore demonstrate that Xfz7 is regulated by BMP antagonism and is required for neural crest induction by Wnt in the developing vertebrate embryo.