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.     

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.     

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.     

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.     

Constitutive expression of preproendothelin in the cardiac neural crest selectively promotes expansion of the adventitia of the great vessels in vivo

Cardiac neural crest cells are essential for normal development of the great vessels and the heart, giving rise to a range of cell types, including both neuronal and non-neuronal adventitial cells and smooth muscle. Endothelin (ET) signaling plays an important role in the development of cardiac neural crest cell lineages, yet the underlying mechanisms that act to control their migration, differentiation, and proliferation remain largely unclear. We examined the expression patterns of the receptor, ET(A), and the ET-specific converting enzyme, ECE-1, in the pharyngeal arches and great vessels of the developing chick embryo. In situ hybridization analysis revealed that, while ET(A) is expressed in the pharyngeal arch mesenchyme, populated by cardiac neural crest cells, ECE-1 expression is localized to the outermost ectodermal cells of the arches and then to the innermost endothelial cells of the great vessels. This dynamic pattern of expression suggests that only a subpopulation of neural crest cells in these regions is responsive to ET signaling at particular developmental time points. To test this, retroviral gene delivery was used to constitutively express preproET-1, a precursor of mature ET-1 ligand, in the cardiac neural crest. This resulted in a selective expansion of the outermost, adventitial cell population in the great vessels. In contrast, neither differentiation nor proliferation of neural crest-derived smooth muscle cells was significantly affected. These results suggest that constitutive expression of exogenous preproET-1 in the cardiac neural crest results in expansion restricted to an adventitial cell population of the developing great vessels.     

Roles of endothelin signaling in melanocyte development and melanoma

Endothelin (Edn) signaling via the G-coupled, Edn receptor type B (Ednrb) is essential for the development of melanocytes from the neural crest (NC) and has been associated with melanoma progression. Edn3 plays varying roles during melanocyte development, promoting the proliferation and self-renewal of NC-derived multi- and bi-potential precursors as well as the survival, proliferation, differentiation and migration of committed melanocyte precursors. Melanocyte differentiation is achieved via the interaction of Ednrb and Kit signaling, with Ednrb being specifically required in the final differentiation step, rather than in the initial specification of melanocytic fate. Ednrb has also been implicated in the de-differentiation of mature melanocytes, a process that takes place during the malignant transformation of these cells. Ednrb was found to be upregulated in melanoma metastases and was shown to alter tumor–host interactions leading to melanoma progression. Antagonists to this receptor were shown to inhibit melanoma cell growth and increase the apoptotic rate of these cells, and to lead to disease stabilization in melanoma patients. Thus, Edn signaling inhibition may prove useful in the treatment of certain types of melanoma.     

Receptor tyrosine kinase-dependent neural crest migration in response to differentially localized growth factors

How different neural crest derivatives differentiate in distinct embryonic locations in the vertebrate embryo is an intriguing issue. Many attempts have been made to understand the underlying mechanism of specific pathway choices made by migrating neural crest cells. In this speculative review we suggest a new mechanism for the regulation of neural crest cell migration patterns in avian and mammalian embryos, based on recent progress in understanding the expression and activity of receptor tyrosine kinases during embryogenesis. Distinct subpopulations of crest-derived cells express specific receptor tyrosine kinases while residing in a migration staging area. We postulate that the differential expression of receptor tyrosine kinases by specific subpopulations of neural crest cells allows them to respond to localized growth factor ligand activity in the embryo. Thus, the migration pathway taken by neural crest subpopulations is determined by their receptor tyrosine kinase response to the differential localization of their cognate ligand.     

Cardiovascular malformations with normal smooth muscle differentiation in neural crest-specific type II TGFbeta receptor (Tgfbr2) mutant mice

Previous studies have demonstrated that TGFbeta induces a smooth muscle fate in primary neural crest cells in culture. By crossing a conditional allele of the type II TGFbeta receptor with the neural crest-specific Wnt1cre transgene, we have addressed the in vivo requirement for TGFbeta signaling in smooth muscle specification and differentiation. We find that elimination of the TGFbeta receptor does not alter neural crest cell specification to a smooth muscle fate in the cranial or cardiac domains, and that a smooth muscle fate is not realized by trunk neural crest cells in either control or mutant embryos. Instead, mutant embryos exhibit with complete penetrance two very specific and mechanistically distinct cardiovascular malformations--persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA-B). Pharyngeal organ defects such as those seen in models of DiGeorge syndrome were not observed, arguing against an early perturbation of the cardiac neural crest cell lineage. We infer that TGFbeta is an essential morphogenic signal for the neural crest cell lineage in specific aspects of cardiovascular development, although one that is not required for smooth muscle differentiation.     

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.     

Eph/ephrin interactions modulate muscle satellite cell motility and patterning

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. We have recently shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. We show here that, in vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, we propose that it might also play a role in satellite cell-mediated muscle repair. Therefore, we investigated whether Eph-ephrin signaling would produce changes in satellite cell directional motility. Using a classical ephrin 'stripe' assay, we found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. We hypothesize that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle.     

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages

Clonal and lineage analyses have demonstrated that although some neural crest cells have the ability to generate multiple cell types and display self-renewal ability, other crest cells generate a single or limited repertoire of cell types. However, it is not yet clear when, and in what order, crest cells become specified to adopt a particular fate. We report that the receptor tyrosine kinases TrkC and C-Kit are expressed by distinct neural crest subpopulations in vitro. We then analyzed the lineages of individual receptor-expressing crest cells and found that TrkC-expressing cells that have just emerged from the neural tube give rise to clones containing neurons or glial cells, or both, but never produce melanocytes. A short time later, TrkC-expressing cells only generate pure neuronal clones. By contrast, from their earliest appearance in neural tube outgrowths, C-Kit-expressing cells invariably give rise to clones containing only melanocytes. Our results directly demonstrate that distinct neurogenic and melanogenic sublineages diverge before or soon after crest cells emerge from the neural tube, that fate-restricted precursors are present in nascent neural crest populations and that these sublineages can be distinguished by their cell type-specific expression of receptor tyrosine kinases.     

The effects of embryonic retinal neurons on neural crest cell differentiation into Schwann cells

Amongst the many cell types that differentiate from migratory neural crest cells are the Schwann cells of the peripheral nervous system. While it has been demonstrated that Schwann cells will not fully differentiate unless in contact with neurons, the factors that cause neural crest cells to enter the differentiative pathway that leads to Schwann cells are unknown. In a previous paper (Development 105: 251, 1989), we have demonstrated that a proportion of morphologically undifferentiated neural crest cells express the Schwann cell markers 217c and NGF receptor, and later, as they acquire the bipolar morphology typical of Schwann cells in culture, express S-100 and laminin. In the present study, we have grown axons from embryonic retina on neural crest cultures to see whether this has an effect on the differentiation of neural crest cells into Schwann cells. After 4 to 6 days of co-culture, many more cells had acquired bipolar morphology and S-100 staining than in controls with no retinal explant, and most of these cells were within 200 microns of an axon, though not necessarily in contact with axons. However, the number of cells expressing the earliest Schwann cell markers 217c and NGF receptor was not affected by the presence of axons. We conclude that axons produce a factor, which is probably diffusible, and which makes immature Schwann cells differentiate. The factor does not, however, influence the entry of neural crest cells into the earliest stages of the Schwann cell differentiative pathway.     

Involvement of endothelin receptors in normal and pathological development of neural crest cells

Endothelin receptors (Ednr) are G-protein-coupled receptors with seven membrane-spanning domains and are involved in various physiological processes in adults. We review here the function of these receptors during the development and transformation of the neural crest cell-specific lineage. Neural crest cells (NCC) may be classified according to their location in the body. In particular, there are clear differences between the neural crest cells arising from the cephalic part of the embryo and those arising from the vagal and truncal part. The development of cranial and cardiac NCC requires the endothelin-1/Ednra system to be fully functional whereas the development of more posterior NCC requires full functionality of the endothelin-3/Ednrb system. Mutations have been found in the genes corresponding to these systems in mammals. These mutations principally impair pigmentation and enteric ganglia development. The precise patterns of expression of these receptors and their ligands have been determined in avian and mammalian models. Data obtained in vitro and in vivo have provided insight into the roles of these proteins in cell proliferation, migration, differentiation and transformation.     

Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro

During development neural crest cells give rise to a wide variety of specialized cell types in response to cytokines from surrounding tissues. Depending on the cranial-caudal level of their origin, different populations of neural crest cells exhibit differential competence to respond to these signals as exemplified by the unique ability of cranial neural crest to form skeletal cell types. We show that in addition to differences in whether they respond to particular signals, cranial neural crest cells differ dramatically from the trunk neural crest cells in how they respond to specific extracellular signals, such that under identical conditions the same signal induces dissimilar cell fate decisions in the two populations in vitro. Conversely, the same differentiated cell types are induced by different signals in the two populations. These in vitro differences in neural crest response are consistent with in vivo manipulations. We also provide evidence that these differences in responsiveness are modulated, at least in part, by differential expression of Hox genes within the neural crest.     

Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.     

Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development

Background

In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/Principal Findings

Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1^−/− mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/Significance

This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.     

Temporally distinct requirements for endothelin receptor B in the generation and migration of gut neural crest stem cells

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.     

Expression of Schwann cell markers by mammalian neural crest cells in vitro

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.