Reversal of a developmental restriction in neural crest-derived cells of avian embryos by a phorbol ester drug

Neural crest cells and some of the crest-derived cells of dorsal root ganglia (DRG) of early avian embryos give rise to pigment cells when placed in culture. DRG from older embryos, however, fail to do so under comparable culture conditions. This age-dependent loss of melanogenic ability might be explained either by the death of a subpopulation of latent melanoblasts within early DRG, or the imposition of additional developmental restrictions in multipotent DRG cells. We show here that 12-O-tetradecanoylphorbol-13-acetate (TPA) causes some DRG cells to undergo pigmentation in cultures from older embryos, indicating that the loss of melanogenic ability in older embryos is not due to cell death. These pigment cells also display morphogenetic properties of normal melanocytes, including the ability to invade feather primordia. In addition to DRG, various other neural crest-derivatives contain cells similarly affected by TPA, including cells within sympathetic ganglia and peripheral nerves. We suggest that TPA reverses the developmental restriction of melanogenic ability that is normally imposed on neural crest-derived cells that migrate to various sites in avian embryos where melanogenesis does not normally occur.     

Neural crest progenitors and stem cells: from early development to adulthood

In the vertebrate embryo, the neural crest forms transiently in the dorsal neural primordium to yield migratory cells that will invade nearly all tissues and later, will differentiate into bones and cartilages, neurons and glia, endocrine cells, vascular smooth muscle cells and melanocytes. Due to the amazingly diversified array of cell types it produces, the neural crest is an attractive model system in the stem cell field. We present here in vivo and in vitro studies of single cell fate, which led to the discovery and the characterization of stem cells in the neural crest of avian and mammalian embryos. Some of the key issues in neural crest cell diversification are discussed, such as the time of segregation of mesenchymal vs. neural/melanocytic lineages, and the origin and close relationships between the glial and melanocytic lineages. An overview is also provided of the diverse types of neural crest-like stem cells and progenitors, recently identified in a growing number of adult tissues in animals and humans. Current and future work, in which in vivo lineage studies and the use of injury models will complement the in vitro culture analysis, should help in unraveling the properties and function of neural crest-derived progenitors in development and disease.     

Restriction of neurogenic ability during neural crest cell differentiation

Multipotent neural crest cells undergo developmental restrictions during embryogenesis and eventually give rise to the neurons and glia of the peripheral nervous system, melanocytes, and pheochromocytes. To understand how neuronal potential is restricted to a subpopulation of crest-derived cells, we have utilized sensitive markers of early neuronal differentiation to assess neurogenesis in crest-derived cell populations subjected to defined experimental conditions in vitro and in vivo. We describe environmental conditions that either (a) result in the irreversible loss of neurogenic potential over a characteristic time course or (b) maintain neurogenic potential among neural crest cells. © 1993 John Wiley & Sons, Inc.     

Colonization of the murine hindgut by sacral crest-derived neural precursors: experimental support for an evolutionarily conserved model

Enteric ganglia in the hindgut are derived from separate vagal and sacral neural crest populations. Two conflicting models, based primarily on avian data, have been proposed to describe the contribution of sacral neural crest cells. One hypothesizes early colonization of the hindgut shortly after neurulation, and the other states that sacral crest cells reside transiently in the extraenteric ganglion of Remak and colonize the hindgut much later, after vagal crest-derived neural precursors arrive. In this study, I show that Wnt1-lacZ-transgene expression, an "early" marker of murine neural crest cells, is inconsistent with the "early-colonization" model. Although Wnt1-lacZ-positive sacral crest cells populate pelvic ganglia in the mesenchyme surrounding the hindgut, they are not found in the gut prior to the arrival of vagal crest cells. Similarly, segments of murine hindgut harvested prior to the arrival of vagal crest cells and grafted under the renal capsule fail to develop enteric neurons, unless adjacent pelvic mesenchyme is included in the graft. When pelvic mesenchyme from DbetaH-nlacZ transgenic embryos is apposed with nontransgenic hindgut, neural precursors from the mesenchyme colonize the hindgut and form intramural ganglion cells that express the transgenic marker. Contribution of sacral crest-derived cells to the enteric nervous system is not affected by cocolonization of grafts by vagal crest-derived neuroglial precursors. The findings complement recent studies of avian chimeras and support an evolutionarily conserved model in which sacral crest cells first colonize the extramural ganglion and secondarily enter the hindgut mesenchyme.     

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.     

Hepatocyte growth factor/scatter factor-MET signaling in neural crest-derived melanocyte development

The mechanisms governing development of neural crest-derived melanocytes, and how alterations in these pathways lead to hypopigmentation disorders, are not completely understood. Hepatocyte growth factor/scatter factor (HGF/SF) signaling through the tyrosine-kinase receptor, MET, is capable of promoting the proliferation, increasing the motility, and maintaining high tyrosinase activity and melanin synthesis of melanocytes in vitro. In addition, transgenic mice that ubiquitously overexpress HGF/SF demonstrate hyperpigmentation in the skin and leptomenigenes and develop melanomas. To investigate whether HGF/ SF-MET signaling is involved in the development of neural crest-derived melanocytes, transgenic embryos, ubiquitously overexpressing HGF/SF, were analyzed. In HGF/SF transgenic embryos, the distribution of melanoblasts along the characteristic migratory pathway was not affected. However, additional ectopically localized melanoblasts were also observed in the dorsal root ganglia and neural tube, as early as 11.5 days post coitus (p.c.). We utilized an in vitro neural crest culture assay to further explore the role of HGF/SF-MET signaling in neural crest development. HGF/SF added to neural crest cultures increased melanoblast number, permitted differentiation into pigmented melanocytes, promoted melanoblast survival, and could replace mast-cell growth factor/Steel factor (MGF) in explant cultures. To examine whether HGF/SF-MET signaling is required for the proper development of melanocytes, embryos with a targeted Met null mutation (Met-/-) were analysed. In Met-/- embryos, melanoblast number and location were not overtly affected up to 14 days p.c. These results demonstrate that HGF/SF-MET signaling influences, but is not required for, the initial development of neural crest-derived melanocytes in vivo and in vitro.     

Characteristic defects in neural crest cell-specific Galphaq/Galpha11- and Galpha12/Galpha13-deficient mice

The endothelin/endothelin receptor system plays a critical role in the differentiation and terminal migration of particular neural crest cell subpopulations. Targeted deletion of the G-protein-coupled endothelin receptors ET(A) and ET(B) was shown to result in characteristic developmental defects of derivatives of cephalic and cardiac neural crest and of neural crest-derived melanocytes and enteric neurons, respectively. Since both endothelin receptors are coupled to G-proteins of the G(q)/G(11)- and G(12)/G(13)-families, we generated mouse lines lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13) in neural crest cells to study their roles in neural crest development. Mice lacking Galpha(q)/Galpha(11) in a neural crest cell-specific manner had craniofacial defects similar to those observed in mice lacking the ET(A) receptor or endothelin-1 (ET-1). However, in contrast to ET-1/ET(A) mutant animals, cardiac outflow tract morphology was intact. Surprisingly, neither Galpha(q)/Galpha(11)- nor Galpha(12)/Galpha(13)-deficient mice showed developmental defects seen in animals lacking either the ET(B) receptor or its ligand endothelin-3 (ET-3). Interestingly, Galpha(12)/Galpha(13) deficiency in neural crest cell-derived cardiac cells resulted in characteristic cardiac malformations. Our data show that G(q)/G(11)- but not G(12)/G(13)-mediated signaling processes mediate ET-1/ET(A)-dependent development of the cephalic neural crest. In contrast, ET-3/ET(B)-mediated development of neural crest-derived melanocytes and enteric neurons appears to involve G-proteins different from G(q)/G(11)/G(12)/G(13).     

The adult hair follicle: cradle for pluripotent neural crest stem cells

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.     

Targeting of endothelin receptor-B to the neural crest

Endothelin receptor B (Ednrb) plays a critical role in the development of melanocytes and neurons and glia of the enteric nervous system. These distinct neural crest-derived cell types express Ednrb and share the property of intercalating into tissues, such as the intestine whose muscle precursor cells also express Ednrb. Such widespread Ednrb expression has been a significant obstacle in establishing precise roles for Ednrb in development. We describe here the production of an Ednrb allele floxed at exon 3 and its use in excising the receptor from mouse neural crest cells by use of Cre-recombinase driven by the Wnt1 promoter. Mice born with neural crest-specific excision of Ednrb possess aganglionic colon, lack trunk pigmentation, and die within 5 weeks due to megacolon. Ednrb receptor expression in these animals is absent only in the neural crest but present in surrounding smooth muscle cells. The absence of Ednrb from crest cells also results in a compensatory upregulation of Ednrb expression in other cells within the gut. We conclude that Ednrb loss only in neural crest cells is sufficient to produce the Hirschsprungs disease phenotype observed with genomic Ednrb mutations.     

Absence of the basement membrane component nidogen 2, but not of nidogen 1, results in increased lung metastasis in mice

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.     

Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis

Avian trunk neural crest cells give rise to a variety of cell types including neurons and satellite glial cells in peripheral ganglia. It is widely assumed that crest cell fate is regulated by environmental cues from surrounding embryonic tissues. However, it is not clear how such environmental cues could cause both neurons and glial cells to differentiate from crest-derived precursors in the same ganglionic locations. To elucidate this issue, we have examined expression and function of components of the NOTCH signaling pathway in early crest cells and in avian dorsal root ganglia. We have found that Delta1, which encodes a NOTCH ligand, is expressed in early crest-derived neuronal cells, and that NOTCH1 activation in crest cells prevents neuronal differentiation and permits glial differentiation in vitro. We also found that NUMB, a NOTCH antagonist, is asymmetrically segregated when some undifferentiated crest-derived cells in nascent dorsal root ganglia undergo mitosis. We conclude that neuron-glia fate determination of crest cells is regulated, at least in part, by NOTCH-mediated lateral inhibition among crest-derived cells, and by asymmetric cell division.     

Developmental potential of neural crest-derived cells migrating from segments of developing quail bowel back-grafted into younger chick host embryos

The technique of back-transplantation was used to investigate the developmental potential of neural crest-derived cells that have migrated to and colonized the avian bowel. Segments of quail bowel (removed at E4) were grafted between the somites and neural tube of younger (E2) chick host embryos. Grafts were placed at a truncal level, adjacent to somites 14-24. Initial experiments, done in vitro, confirmed that crest-derived cells are capable of migrating out of segments of foregut explanted at E4. The foregut, which at E4 has been colonized by cells derived from the vagal crest, served as the donor tissue. Comparative observations were made following grafts of control tissues, which included hindgut, lung primordia, mesonephros and limb bud. Additional experiments were done with chimeric bowel in which only the crest-derived cells were of quail origin. Targets in the host embryos colonized by crest-derived cells from the foregut grafts included the neural tube, spinal roots and ganglia, peripheral nerves, sympathetic ganglia and the adrenals, but not the gut. Donor cells in these target organs were immunostained by the monoclonal antibody, NC-1, indicating that they were crest-derived and developing along neural or glial lineages. Some of the crest-derived cells (NC-1-immunoreactive) that left the bowel and reached sympathetic ganglia, but not peripheral nerves or dorsal root ganglia, co-expressed tyrosine hydroxylase immunoreactivity, a neural characteristic never expressed by crest-derived cells in the avian gut. None of the cells leaving enteric back-grafts produced pigment. Cells of mesodermal origin were also found to leave donor explants and aggregate in dermis and feather germs near the grafts. These observations indicate that crest-derived cells, having previously migrated to the bowel, retain the ability to migrate to distant sites in a younger embryo. The routes taken by these cells appear to reflect, not their previous migratory experience, but the level of the host embryo into which the graft is placed. Some of the population of crest-derived cells that leave the back-transplanted gut remain capable of expressing phenotypes that they do not express within the bowel in situ, but which are appropriate for the site in the host embryo to which they migrate.     

Identification of early neurogenic cells in the neural crest lineage.

Pluripotent neural crest cells are restricted progressively during development. The sequence of restrictions and the time(s) in early development at which such restrictions are imposed on crest-derived cells are largely unknown. We have used a human autoantibody (Anti-Hu) to characterize neurogenic populations of avian neural crest-derived cells. Anti-Hu binds specifically to neurons and neuroendocrine cells in older (greater than E4) quail embryos. Early in development, Anti-Hu also binds a subpopulation of neural crest-derived cells that lack neuronal morphology and do not express other neuronal traits. These cells may represent a putative neurogenic precursor subpopulation within the early crest cell lineage. To test this hypothesis, we have characterized Anti-Hu immunoreactivity within crest-derived populations known to have, or to lack, the ability to give rise to new neurons. We report that the presence of Anti-Hu+ nonneuronal cells is correlated with the neurogenic ability of a given cell population. Moreover, Anti-Hu+ nonneuronal cells are transient and appear to be replaced by Anti-Hu+ neuronal cells. We conclude that Anti-Hu is a very early indicator of neurogenesis among crest-derived cells and that Anti-Hu+ nonneuronal cells are either neurogenic precursors or immature neurons.     

PDGF function in diverse neural crest cell populations

Activation of platelet derived growth factor (PDGF) receptors causes context-dependent cellular responses, including proliferation and migration, and studies in model organisms have demonstrated that this receptor family (PDGFRα and PDGFRβ) is required in many mesenchymal and migratory cell populations during embryonic development. One of these migratory cell populations is the neural crest, which forms cranial bone and mesenchyme, sympathetic neurons and ganglia, melanocytes and smooth muscle. Mice with disruption of PDGF signaling exhibit defects in some of these neural crest derivatives including the palate, aortic arch, salivary gland and thymus. Although many of these neural crest defects were identified many years ago, the mechanism of action of PDGF in neural crest remains controversial. In this review, we examine the current knowledge of PDGF function during neural crest cell (NCC) development, focusing on its role in the formation of different neural crest-derived tissues and the implications for PDGF receptors in NCC-related human birth defects.Key words: neural crest, aortic arch, PDGF, melanocytes, thymus, cleft palate, ventricular septal defects     

Turtle Lung Cells Produce a Melanization-Stimulating Activity That Promotes Melanocytic Differentiation of Avian Neural Crest Cells

We found previously that neural crest cells in turtle embryos migrated into the lung buds and melanocytes were located in the lungs. The finding suggested to us that the lungs provide a stimulatory factor(s) to the differentiation of neural crest cells into melanocytes. We have established lung cell lines to facilitate analysis of the interactions of neural crest cells with the environment in melanocyte development. One cell line, TLC-2, was found to produce a putative melanization-stimulating activity (MSA), which promoted the melanocyte differentiation in vitro of avian neural crest cells. The TLC-2-derived MSA was different from that of basic fibroblast growth factor (bFGF), α-melanocyte stimulating hormone (α-MSH), and steel factor (SLF). Its molecular weight was estimated to be within the range of 150 kD. Our findings suggest that MSA may be a novel factor exercising a positive control over melanocyte differentiation.     

Differentiation of reptilian neural crest cells in Vitro

An attempt was made to culture neural crest cells of the turtle embryo in vitro. Trunk neural tubes from the St. 9/10 embryos were explanted in culture dishes. The developmental potency of the turtle neural crest cells in vitro was shown to be essentially similar to that of avian neural crest cells, although they seem to be more sensitive to melanocyte-stimulating hormone (MSH) stimulation. We describe conditions under which explanted neural tube gives rise to neural crest cells that differentiate into neuronal cells and melanocytes. The potency of melanocyte differentiation was, found to vary according to the concentration of fetal bovine serum (FBS, from 5 to 20%). Melanization of neural crest cells cultured in the medium containing FBS and α-MSH was more extensive than those cultured with FBS alone, combinations of FBS and chick embryo extract, or turtle embryo extract. These culture conditions seem to be useful for the study of the developmental potency of the neural crest cells as well as for investigating local environmental factors.