Cultured quail neural crest cells attain competence for terminal differentiation into melanocytes before competence to terminal differentiation into adrenergic neurons

At the onset of migration the quail neural crest contains pluripotent progenitor cells that give rise to both melanocytes and adrenergic neurons as well as progenitor cells that are already committed to the melanogenic or the neuronal pathway. In this paper we show that melanogenic progenitors attain the competence for terminal differentiation prior to adrenergic progenitors. The adrenergic phenotype was only expressed when the crest cells were allowed to proliferate in vitro for at least 3 days. Differentiation into melanocytes, however, occurred even when proliferation was blocked with cytosine arabinoside immediately after explantation of the neural tube.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: (1) what establishes the pathways of migration? And (2), what controls the final destination and differentiation of various neural crest subpopulations? These questions will be addressed in this Review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk.The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: (1) the vagal-level neural crest cells exhibit modest developmental bias; (2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively and (3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but take the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.Key words: neural crest, morphogenesis, cell migration, chicken embryo, fate restriction, vagal neural crest, pathways     

Spatio‐temporal control of neural epithelial cell migration and epithelium‐to‐mesenchyme transition during avian neural tube development

As opposed to the neural crest, the neural epithelium is generally viewed as a static and cohesive structure. Here, using an ex vivo system free of the environmental influences and physical constraints encountered in the embryo, we show that neural epithelial cells are on the contrary intrinsically motile, although they do not undergo spontaneous epithelium‐to‐mesenchyme transition and display molecular and cellular characteristics distinct from those of neural crest cells. However, they can be instructed to undergo epithelium‐to‐mesenchyme conversion independently of the acquisition of neural crest traits. Migration potentialities of neural epithelial cells are transient and are progressively restricted during neural tube development. Restriction of cell migration is irreversible and can be in part accounted for by increase in N‐cadherin in cellular junctions and in cell polarity. In conclusion, our study reveals that the neural epithelium is a highly flexible tissue in which cells are maintained cohesive under the control of a combination of extrinsic factors and physical constraints.     

Defective ALK5 signaling in the neural crest leads to increased postmigratory neural crest cell apoptosis and severe outflow tract defects

Background  

Congenital cardiovascular diseases are the most common form of birth defects in humans. A substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. While TGF-β-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood.     

Distribution of pluripotent neural crest cells in the embryo and the role of brain-derived neurotrophic factor in the commitment to the primary sensory neuron lineage

Many early migratory neural crest cells are pluripotent in the sense that their progeny are able to generate more than one differentiated phenotype (Sieber-Blum and Cohen, 1980, Dev. Biol. 80:95–106; Baroffio, Dupin, and Le Douarin, 1988, Proc. Natl. Acad. Sci. USA 85:5325–5329; Bronner-Fraser and Fraser, 1988, Nature 335:161–164; Sieber-Blum, 1989a, Science 243:1608–1611; Ito and Sieber-Blum, 1991, Dev. Biol. 148:95–106). At trunk levels, the neural crest contains two classes (Sieber-Blum and Cohen, 1980) and at posterior rhombencephalic levels, three different classes of pluripotent cells (Ito and Sieber-Blum, 1991). We investigated cell differentiation by in vitro clonal analysis to determine when in development the pool of pluripotent neural crest cells becomes exhausted. The data suggest that different classes of pluripotent cells, precursor cells with more restricted developmental potentials, and apparently committed cells, exist at sites of advanced migration (posterior branchial arches) and even at target sites of neural crest cell differentiation [posterior branchial arches, dorsal root ganglia (DRG), sympathetic ganglia (SG), and epidermal ectoderm]. Some putative classes of pluripotent cells persist well into the second half of embryonic development. These observations have implications for our understanding of the mechanisms that control neural crest cell migration and differentiation. They support the idea that cues originating from the microenvironment affect differentiation of pluripotent neural crest cells. One such signal appears to be brain-derived neurotrophic factor (BDNF). In the presence of BDNF, but not nerve growth factor (NGF), there is a significant increase in the number of neural crest cells per colony that express a sensory neuron-specific marker. Because this increase is not accompanied by a corresponding increase in the total number of cells per colony, this suggests that BDNF plays a role in cell type specification. © 1993 John Wiley & Sons, Inc.     

Multiple lineage-specific roles of Smad4 during neural crest development

During vertebrate development, neural crest cells are exposed to multiple extracellular cues that drive their differentiation into neural and non-neural cell lineages. Insights into the signals potentially involved in neural crest cell fate decisions in vivo have been gained by cell culture experiments that have allowed the identification of instructive growth factors promoting either proliferation of multipotent neural crest cells or acquisition of specific fates. For instance, members of the TGFβ factor family induce neurogenesis and smooth muscle cell formation at the expense of other fates in culture. In vivo, conditional ablation of various TGFβ signaling components resulted in malformations of non-neural derivatives of the neural crest, but it is unclear whether these phenotypes involved aberrant fate decisions. Moreover, it remains to be shown whether neuronal determination indeed requires TGFβ factor activity in vivo. To address these issues, we conditionally deleted Smad4 in the neural crest, thus inactivating all canonical TGFβ factor signaling. Surprisingly, neural crest cell fates were not affected in these mutants, with the exception of sensory neurogenesis in trigeminal ganglia. Rather, Smad4 regulates survival of smooth muscle and proliferation of autonomic and ENS neuronal progenitor cells. Thus, Smad signaling plays multiple, lineage-specific roles in vivo, many of which are elicited only after neural crest cell fate decision.     

Clonal analysis of quail neural crest cells: They are pluripotent and differentiate in vitro in the absence of noncrest cells

To determine if neural crest cells are pluripotent and establish whether differentiation occurs in the absence of noncrest cells, a cell culture method was devised in which differentiation could be examined in clones derived from single, isolated neural crest cells. Single neural crest cells, which were isolated before the onset of in vivo migration, gave rise to three types of clones: pigmented, unpigmented, and mixed. Pigmented clones consisted of melanocytes only, whereas some unpigmented cells in mixed and unpigmented clones contained catecholamines, identifying them as adrenergic cells. Extracellular matrix derived from quail somite or chick skin fibroblast cultures stimulated adrenergic differentiation and axon formation. These results demonstrate for the first time the existence of pluripotent quail neural crest cells that give rise to at least two progeny, melanocytes and neuronal cells. They also suggest that continuous direct interactions with noncrest cells are not required for the differentiation of these two cell types. However, components of the extracellular matrix derived from noncrest cells may play an important role in expression of the adrenergic phenotype.     

Transplantation as a tool to study progenitors within the vertebrate nervous system

In recent years, many studies have focused on the fate and potential of neural progenitors in vertebrates. While much progress has been made, many questions remain about the mechanisms which lead to neural diversity, in terms of both the regionalization of the nervous system and specification of cell fates within those regions. Studies aimed at addressing these questions have fallen into three main categories: in vivo lineage tracings, in vitro differentiation analyses, and in vivo cell transplantation studies. This body of work has pointed to the existence of both pluripotent and unipotent neural progenitors, and has suggested that both cell intrinsic and extrinsic cues play a role in the determination of neural cell fate. In addition, the existence of neural “stem cells” maintained into adulthood has been suggested. This review will focus on transplantation studies in mammals, and will emphasize how this method has been useful as a means of determining the changing potential of neural precursors and their environments within the developing nervous system. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 152–161, 1998     

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively, and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: 1) what establishes the pathways of migration and 2) what controls the final destination and differentiation of various neural crest subpopulations. These questions will be addressed in this review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube, and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted, or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk. The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: 1) the vagal-level neural crest cells exhibit modest developmental bias; 2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively; 3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.     

Early stages of neural crest ontogeny: formation and regulation of cell delamination

Long standing research of the Neural Crest embodies the most fundamental questions of Developmental Biology. Understanding the mechanisms responsible for specification, delamination, migration and phenotypic differentiation of this highly diversifying group of progenitors has been a challenge for many researchers over the years and continues to attract newcomers into the field. Only a few leaps were more significant than the discovery and successful exploitation of the quail-chick model by Nicole Le Douarin and colleagues from the Institute of Embryology at Nogent-sur-Marne. The accurate fate mapping of the neural crest performed at virtually all axial levels was followed by the determination of its developmental potentialities as initially analysed at a population level and then followed by many other significant findings. Altogether, these results paved the way to innumerable questions which brought us from an organismic view to mechanistic approaches. Among them, elucidation of functions played by identified genes is now rapidly underway. Emerging results lead the way back to an integrated understanding of the nature of interactions between the developing neural crest and neighbouring structures. The Nogent Institute thus performed an authentic "tour de force" in bringing the Neural Crest to the forefront of Developmental Biology. The present review is dedicated to the pivotal contributions of Nicole Le Douarin and her collaborators and to unforgettable memories that one of the authors bears from the time spent in the Nogent Institute. We summarize here recent advances in our understanding of early stages of crest ontogeny that comprise specification of epithelial progenitors to a neural crest fate and the onset of neural crest migration. Particular emphasis is given to signaling by BMP and Wnt molecules, to the role of the cell cycle in generating cell movement and to possible interactions between both mechanisms.     

Draxin,an axon guidance protein,affects chick trunk neural crest migration

The neural crest is a multipotent population of migratory cells that arises in the central nervous system and subsequently migrates along defined stereotypic pathways. In the present work, we analyzed the role of a repulsive axon guidance protein, draxin, in the migration of neural crest cells. Draxin is expressed in the roof plate of the chick trunk spinal cord and around the early migration pathway of neural crest cells. Draxin modulates chick neural crest cell migration in vitro by reducing the polarization of these cells. When exposed to draxin, the velocity of migrating neural crest cells was reduced, and the cells changed direction so frequently that the net migration distance was also reduced. Overexpression of draxin also caused some early migrating neural crest cells to change direction to the dorsolateral pathway in the chick trunk region, presumably due to draxin’s inhibitory activity. These results demonstrate that draxin, an axon guidance protein, can also affect trunk neural crest migration in the chick embryo.     

Adipose Stromal Cells Contain Phenotypically Distinct Adipogenic Progenitors Derived from Neural Crest

Recent studies have shown that adipose-derived stromal/stem cells (ASCs) contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs). This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2) and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta). NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives.     

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.     

Restrictions of developmental capabilities in neural crest cell derivatives as tested by in vivo transplantation experiments

The behavior of neural crest cells from various origins and of neural crest derivatives were investigated when they were transplanted into a host embryo as supernumerary crest structures. They were inserted into the dorsal trunk between the neural primordium and the somites. In this situation the grafted cells (of quail origin) migrated in the chick host and could be recognized any time after the graft by the structure of their nucleus. After a migration phase, they became exclusively localized in the various sites of arrest of neural crest cells and were found mixed with host crest cells. Their localizations varied according to their origin and their developmental stages at grafting time. Cells of autonomic ganglia (ciliary and sympathetic) had a definitive localization restricted to the autonomic structures of the host. They differentiated into adrenergic or cholinergic cells irrespective of their sympathetic or parasympathetic origin, according exclusively to their localization in the host. They were practically never found in the host dorsal root ganglion (DRG). In contrast, the developmental potentialities of DRG cells are broader and, as far as peripheral nervous system potentialities are concerned, they behave like neural crest cells, i.e., they gave rise to both sensory and autonomic neurones plus adrenomedullary cells. A model for cell line segregation from the neural crest is proposed. Several aspects of this model need further analysis, others are based on well-established experimental data.     

Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates

Neural crest (NC) progenitors generate a wide array of cell types, yet molecules controlling NC multipotency and self-renewal and factors mediating cell-intrinsic distinctions between multipotent versus fate-restricted progenitors are poorly understood. Our earlier work demonstrated that Foxd3 is required for maintenance of NC progenitors in the embryo. Here, we show that Foxd3 mediates a fate restriction choice for multipotent NC progenitors with loss of Foxd3 biasing NC toward a mesenchymal fate. Neural derivatives of NC were lost in Foxd3 mutant mouse embryos, whereas abnormally fated NC-derived vascular smooth muscle cells were ectopically located in the aorta. Cranial NC defects were associated with precocious differentiation towards osteoblast and chondrocyte cell fates, and individual mutant NC from different anteroposterior regions underwent fate changes, losing neural and increasing myofibroblast potential. Our results demonstrate that neural potential can be separated from NC multipotency by the action of a single gene, and establish novel parallels between NC and other progenitor populations that depend on this functionally conserved stem cell protein to regulate self-renewal and multipotency.     

A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.     

Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull

Neural crest cells that form the vertebrate head skeleton migrate and interact with surrounding tissues to shape the skull, and defects in these processes underlie many human craniofacial syndromes. Signals at the midline play a crucial role in the development of the anterior neurocranium, which forms the ventral braincase and palate, and here we explore the role of Hedgehog (Hh) signaling in this process. Using sox10:egfp transgenics to follow neural crest cell movements in the living embryo, and vital dye labeling to generate a fate map, we show that distinct populations of neural crest form the two main cartilage elements of the larval anterior neurocranium: the paired trabeculae and the midline ethmoid. By analyzing zebrafish mutants that disrupt sonic hedgehog (shh) expression, we demonstrate that shh is required to specify the movements of progenitors of these elements at the midline, and to induce them to form cartilage. Treatments with cyclopamine, to block Hh signaling at different stages, suggest that although requirements in morphogenesis occur during neural crest migration beneath the brain, requirements in chondrogenesis occur later, as cells form separate trabecular and ethmoid condensations. Cell transplantations indicate that these also reflect different sources of Shh, one from the ventral neural tube that controls trabecular morphogenesis and one from the oral ectoderm that promotes chondrogenesis. Our results suggest a novel role for Shh in the movements of neural crest cells at the midline, as well as in their differentiation into cartilage, and help to explain why both skeletal fusions and palatal clefting are associated with the loss of Hh signaling in holoprosencephalic humans.