Novel expression patterns of Pax3/Pax7 in early trunk neural crest and its melanocyte and non-melanocyte lineages in amniote embryos

Neural crest cells are considered a key vertebrate feature that is studied intensively because of their relevance to development and evolution. Here we report the expression of Pax7 in the dorsal non-neural ectoderm and in the trunk neural crest of the early chick embryo. Pax7 is expressed in the trunk neural crest migrating along the ventral and dorsolateral routes. Pax7 is first downregulated in the neural crest-derived neuronal precursors, secondly in the glial, and finally in the melanocyte precursors. Conserved developmental expression in the melanocyte lineage of both Pax3 and Pax7 was evidenced in chick and quail, but only Pax3 in mouse and rat.     

Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.     

Novel expression patterns of Pax3/Pax7 in early trunk neural crest and its melanocyte and non‐melanocyte lineages in amniote embryos

Neural crest cells are considered a key vertebrate feature that is studied intensively because of their relevance to development and evolution. Here we report the expression of Pax7 in the dorsal non‐neural ectoderm and in the trunk neural crest of the early chick embryo. Pax7 is expressed in the trunk neural crest migrating along the ventral and dorsolateral routes. Pax7 is first downregulated in the neural crest‐derived neuronal precursors, secondly in the glial, and finally in the melanocyte precursors. Conserved developmental expression in the melanocyte lineage of both Pax3 and Pax7 was evidenced in chick and quail, but only Pax3 in mouse and rat.     

Induced Pluripotent Stem Cells Generated from P0-Cre;Z/EG Transgenic Mice

Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases.     

Cellular origin and developmental mechanisms during the formation of skin melanocytes

Melanocytes are derived from the neural crest (NC), which are transient multipotent cells arising by delamination from the developing dorsal neural tube. During recent years, signaling systems and molecular mechanisms of melanocyte development have been studied in detail, but the exact diversification of the NC into melanocytes and how they migrate, expand and disperse in the skin have not been fully understood. The recent finding that Schwann cell precursors (SCPs) of the growing nerve represents a stem cell niche from which various cell types, including Schwann cells, endoneural fibroblasts and melanocytes arise has exposed new knowledge on the cellular basis for melanocyte development. This opens for the identification of new factors and reinterpretation of old data on cell fate instructive, proliferative, survival and cell homing factors participating in melanocyte development.     

Chordate ancestry of the neural crest: new insights from ascidians

This article reviews new insights from ascidians on the ancestry of vertebrate neural crest (NC) cells. Ascidians have neural crest-like cells (NCLC), which migrate from the dorsal midline, express some of the typical NC markers, and develop into body pigment cells. These characters suggest that primordial NC cells were already present in the common ancestor of the vertebrates and urochordates, which have been recently inferred as sister groups. The primitive role of NCLC may have been in pigment cell dispersal and development. Later, additional functions may have appeared in the vertebrate lineage, resulting in the evolution of definitive NC cells.     

Isolation and culture of neural crest cells from embryonic murine neural tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from protocols optimized for the culture of rat NC. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC.     

Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

Distribution and migration pathways of HNK-1-immunoreactive neural crest cells in teleost fish embryos.

Whole mounts and cross-sections of embryos from three species of teleost fish were immunostained with the HNK-1 monoclonal antibody, which recognizes an epitope on migrating neural crest cells. A similar distribution and migration was found in all three species. The crest cells in the head express the HNK-1 epitope after they have segregated from the neural keel. The truncal neural crest cells begin to express the epitope while they still reside in the dorsal region of the neural keel; this has not been observed in other vertebrates. The cephalic and anterior truncal neural crest cells migrate under the ectoderm; the cephalic cells then enter into the gill arches and the anterior truncal cells into the mesentery of the digestive tract where they cease migration. These cephalic and anterior trunk pathways are similar to those described in Xenopus and chick. The neural crest cells of the trunk, after segregation, accumulate in the dorsal wedges between the somites, however, unlike in chick and rat, they do not migrate in the anterior halves of the somites but predominantly between the neural tube and the somites, the major pathway observed in carp and amphibians; some cells migrate over the somites. The HNK-1 staining of whole-mount embryos revealed a structure resembling the Rohon-Beard and extramedullary cells, the primary sensory system in amphibians. Such a system has not been described in fish.     

Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mouse-chick neural chimeras

Embryonic chimera production was used to study the developmental processes of the mouse nervous system. The difficulty of performing in situ transplantation experiments of neural primordium of mouse embryo was overcome by isotopic and isochronic grafting of mouse neural tube fragments into chick embryo. Mouse neural tube cells differentiated perfectly in ovo and neural crest cells associated with the grafted neural tube were able to migrate and reach the normal arrest sites of host neural crests. Cranial neural crest cells penetrated into chick facial areas and entered into the development of dental bud structures, participating in vibrissa formation. Depending on graft level, in ovo implanted mouse neural crest cells formed different components of the peripheral nervous system. At trunk level, they located in spinal ganglia and orthosympathetic chains and gave rise to Schwann cells lining the nerves. When implanted into the lumbosacral region, they penetrated into the enteric nervous system. At the precise 18-24 somite level, they colonized host adrenal gland. Mouse neural tube was involved in the mechanisms required to maintain myogenesis in host somites. Furthermore in ovo grafts of mouse cells from genetically modified embryos, in which many mutations induce early death, are particularly useful to investigate cellular events involved in the development of the nervous system and to identify molecular events of embryogenesis.     

Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest-derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.     

Axolotls with an under- or oversupply of neural crest can regulate the sizes of their dorsal root ganglia to normal levels

How animals adjust the size of their organs is a fundamental, enduring question in biology. Here we manipulate the amount of neural crest (NC) precursors for the dorsal root ganglia (DRG) in axolotl. We produce embryos with an under- or over-supply of pre-migratory NC in order to find out if DRG can regulate their sizes during development. Axolotl embryos are perfectly suitable for this research. Firstly, they are optimal for microsurgical manipulations and tissue repair. Secondly, they possess, unlike most other vertebrates, only one neural crest string located on top of the neural tube. This condition and position enables NC cells to migrate to either side of the embryo and participate in the regulation of NC cell distribution. We show that size compensation of DRG in axolotl occurs in 2 cm juveniles after undersupply of NC (up-regulation) and in 5 cm juveniles after oversupply of NC (down-regulation). The size of DRG is likely to be regulated locally within the DRG and not via adaptations of the pre-migratory NC or during NC cell migration. Ipsi- and contralateral NC cell migration occurs both in embryos with one and two neural folds, and contralateral migration of NC is the only source for contralateral DRG formation in embryos with only one neural fold. Compensatory size increase is accompanied by an increase in cell division of a DRG precursor pool (PCNA+/SOX2−), rather than by DRG neurons or glial cells. During compensatory size decrease, increased apoptosis and reduced proliferation of DRG cells are observed.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro.

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro, although to a lesser extent than do neural crest cells.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro although to a lesser extent than do neural crest cells.     

Wnt11-R signaling regulates a calcium sensitive EMT event essential for dorsal fin development of Xenopus

In the frog embryo, a sub-population of trunk neural crest (NC) cells undergoes a dorsal route of migration to contribute to the mesenchyme in the core of the dorsal fin. Here we show that a second population of cells, originally located in the dorsomedial region of the somite, also contributes to the fin mesenchyme. We find that the frog orthologue of Wnt11 (Wnt11-R) is expressed in both the NC and somite cell populations that migrate into the fin matrix. Wnt11-R is expressed prior to migration and persists in the mesenchymal cells after they have distributed throughout the fin. Loss of function studies demonstrate that Wnt11-R activity is required for an epithelial to mesenchymal transformation (EMT) event that precedes migration of cells into the fin matrix. In Wnt11-R depleted embryos, the absence of fin core cells leads to defective dorsal fin development and to collapse of the fin structure. Experiments using small molecule inhibitors indicate that dorsal migration of fin core cells depends on calcium signaling through calcium/calmodulin-dependent kinase II (CaMKII). In Wnt11-R depleted embryos, normal migration of NC cells and dorsal somite cells into the fin and normal fin development can be rescued by stimulation of calcium release. These studies are consistent with a model in which Wnt11-R signaling, via a downstream calcium pathway, regulates fin cell migration and, more generally, indicates a role for non-canonical Wnt signaling in regulation of EMT.     

Anterior/Posterior Influences on Neural Crest-Derived Pigment Cell Differentiation

The neural crest of vertebrate embryos has been used to elucidate steps involved in early embryonic cellular processes such as differentiation and migration. Neural crest cells form a ridge along the dorsal midline and subsequently they migrate throughout the embryo and differentiate into a wide variety of cell types. Intrinsic factors and environmental cues distributed along the neural tube, along the migratory pathways, and/or at the location of arrest influence the fate of neural crest cells. Although premigratory cells of the cranial and trunk neural crest exhibit differences in their differentiation potentials, premigratory trunk neural crest cells are generally assumed to have equivalent developmental potentials. Axolotl neural crest cells from different regions of origin, different stages of development, and challenged with different culture media have been analyzed for differentiation preferences pertaining to the pigment cell lineages. We report region-dependent differentiation of chromatophores from trunk neural crest at two developmental stages. Also, dosage with guanosine produces region-specific influences on the production of xanthophores from wild-type embryos. Our results support the hypothesis that spatial and temporal differences among premigratory trunk neural crest cells found along the anteroposterior axis influence developmental potentials and diminish the equivalency of axolotl neural crest cells.