Transforming growth factor-beta control of cell-substratum adhesion during avian neural crest cell emigration in vitro.

It has been proposed that, in higher vertebrates, the onset of neural crest cell migration from the neural tube involves spatially and temporally coordinated changes in cellular adhesiveness that are under the control of external signals released in the extracellular milieu by neighboring tissues. In the present study, we have analyzed the dynamics of changes in cell-substratum adhesiveness during crest cell emigration and searched for regulatory cues using an in vitro model system. This model is based on the fact that, in vivo, crest cell dispersion occurs gradually along a rostrocaudal wave, allowing us to explant portions of the neural axis, termed migratory and premigratory levels, that differ in the time in culture at which neural crest cells initiate migration and in the locomotory behavior of the cells. We found that neural crest cell emigration is not triggered by the main extracellular matrix molecules present in the migratory pathways, as none of these molecules could abolish the intrinsic difference in the timing of emigration between the different axial levels. Using an in vitro adhesion assay, we found that presumptive neural crest cells from premigratory level explants gradually acquired the ability to respond to extracellular matrix material with time in culture, suggesting that acquisition of appropriate, functional integrin receptors was a necessary step for migration. Finally, we showed that members of the transforming growth factor-beta family reduced in a dose-dependent manner the delay of neural crest cell emigration from premigratory level explants and were able to increase significantly the substratum-adhesion properties of crest cells. Our results suggest that acquisition of substratum adhesion by presumptive neural crest cells is a key event during their dispersion from the neural tube in vitro, and that members of the transforming growth factor-beta family may act as potent inducers of crest cell emigration, possibly by increasing the substratum adhesion of the cells.     

Role of adhesion molecules in the genesis of the peripheral nervous system in avians

In vertebrates, the peripheral nervous system arises from the neural crest by a multistep process involving epithelium-mesenchyme interconversions and cell migrations. These successive events are associated with profound and controlled reorganization of the expression of both cell-cell and cell-substratum adhesion molecules responsible for the direct interaction of neural crest cells with their neighbours or the extracellular matrix. Thus, at the onset of emigration of neural crest cells from the neural tube, the cell-cell adhesion systems mediated by N-cadherin and N-CAM are lost by cells. This is accompanied by the complete reorganization of the extracellular matrix in the immediate environment of neural crest cells and by changes in cell shape. Later, as crest cells undergo migration towards their differentiation sites, they are found associated with fibronectin. Cell adhesion molecules are reaquired by neural crest cells following specific sequences as they coalesce into primordia of the various ganglia. In vitro, fibronectin constitutes the most appropriate substrate for migration of neural crest cells. The migration-promoting effect of fibronectin can be specifically inhibited both in vivo and in vitro by antibodies to fibronectin, integrin receptors, or by peptides containing the Arg-Gly-Asp-Ser sequence. Neural crest cells recognize two major adhesion sites along fibronectin molecules; these are the Arg-Gly-Asp-Ser sequence located in the medial part of the molecule and the CS1 site situated in the alternatively spliced IIICS region. These two sequences are required to permit full motile behavior of cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a stem cell for neurons and glia from the mammalian neural crest.

We have isolated mammalian neural crest cells using a monoclonal antibody to the low affinity NGF receptor, and established conditions for the serial propagation of these cells in clonal culture to assess their developmental potential. This analysis indicates that, first, single mammalian neural crest cells are multipotent, able to generate at least neurons and Schwann cells like their avian counterparts. Second, multipotent neural crest cells generate multipotent progeny, indicating that they are capable of self-renewal and therefore are stem cells. Third, multipotent neural crest cells also generate some clonal progeny that form only neurons or glia, suggesting the production of committed neuroblasts and glioblasts. Manipulation of the substrate alters the fate of the multipotent cells. These findings have implications for models of neural crest development in vivo, and establish a system for studying the generation of cellular diversity by a multipotent stem cell in vitro.     

PlexinA2 and semaphorin signaling during cardiac neural crest development.

Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, we use these techniques to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. We demonstrate that PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery.     

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.     

Cell adhesion mechanisms in gangliogenesis studied in avian embryo and in a model system

Neural crest cells undergo rapid changes in their cell-to-cell and cell-to-extracellular matrix adhesion during the ontogeny of the peripheral nervous system. The mechanisms of adhesion have been analyzed to assess the respective roles played by the cell adhesion molecules (CAMs) and the differentiated junctions. Crest cells which lose their terminal bar junctions after emigration from the neural tube contain only very few gap junctions during gangliogenesis. The calcium-dependent cell adhesion molecules, L-CAM, disappear from the neural crest and never reappear in crest cell derivatives. In contrast, the number of calcium-independent cell adhesion molecules, N-CAM, diminishes transiently during the migratory phase. In vitro, N-CAM is expressed de novo either just before or at the onset of aggregation into autonomic ganglion rudiments, whereas it is delayed in the dorsal root ganglion cells. In vitro, N-CAM mediates the calcium-independent aggregation mechanism; the rate of aggregation is, however, similar whether crest cells are derived from well-spread cultures or from two- and three-dimensional clusters. Crest cells also exhibit a calcium-dependent mechanism of adhesion controlled by molecules differing from N-CAM but which may codistribute on many different cell types during embryogenesis. These two classes of cell adhesion molecules are present on the surface of neural precursors prior to their differentiation into neurons and glial cells.     

Connexin 43 expression reflects neural crest patterns during cardiovascular development

We used transgenic mice in which the promoter sequence for connexin 43 linked to a lacZ reporter was expressed in neural crest but not myocardial cells to document the pattern of cardiac neural crest cells in the caudal pharyngeal arches and cardiac outflow tract. Expression of lacZ was strikingly similar to that of cardiac neural crest cells in quail-chick chimeras. By using this transgenic mouse line to compare cardiac neural crest involvement in cardiac outflow septation and aortic arch artery development in mouse and chick, we were able to note differences and similarities in their cardiovascular development. Similar to neural crest cells in the chick, lacZ-positive cells formed a sheath around the persisting aortic arch arteries, comprised the aorticopulmonary septation complex, were located at the site of final fusion of the conal cushions, and populated the cardiac ganglia. In quail-chick chimeras generated for this study, neural crest cells entered the outflow tract by two pathways, submyocardially and subendocardially. In the mouse only the subendocardial population of lacZ-positive cells could be seen as the cells entered the outflow tract. In addition lacZ-positive cells completely surrounded the aortic sac prior to septation, while in the chick, neural crest cells were scattered around the aortic sac with the bulk of cells distributed in the bridging portion of the aorticopulmonary septation complex. In the chick, submyocardial populations of neural crest cells assembled on opposite sides of the aortic sac and entered the conotruncal ridges. Even though the aortic sac in the mouse was initially surrounded by lacZ-positive cells, the two outflow vessels that resulted from its septation showed differential lacZ expression. The ascending aorta was invested by lacZ-positive cells while the pulmonary trunk was devoid of lacZ staining. In the chick, both of these vessels were invested by neural crest cells, but the cells arrived secondarily by displacement from the aortic arch arteries during vessel elongation. This may indicate a difference in derivation of the pulmonary trunk in the mouse or a difference in distribution of cardiac neural crest cells. An independent mouse neural crest marker is needed to confirm whether the differences are indeed due to species differences in cardiovascular and/or neural crest development. Nevertheless, with the differences noted, we believe that this mouse model faithfully represents the location of cardiac neural crest cells. The similarities in location of lacZ-expressing cells in the mouse to that of cardiac neural crest cells in the chick suggest that this mouse is a good model for studying mammalian cardiac neural crest and that the mammalian cardiac neural crest performs functions similar to those shown for chick.     

Development of the mammalian enteric nervous system.

The mammalian enteric nervous system is derived from neural crest cells which invade the foregut and hindgut mesenchyme. It has been established that signalling molecules produced by the mesenchyme of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have characterised further the role of such molecules and have identified novel extracellular and intracellular signals that are critical for enteric ganglia formation.     

Extracellular matrix and embryonal morphogenesis: role of fibronectin in cell migration

The neural crest provides a useful paradigm for cell migration and modulations in cell adhesion during morphogenesis. In the present review, we describe the major findings on the role of the extracellular matrix glycoprotein fibronectin and its corresponding integrin receptor in the locomotory behavior of neural crest cells. In vivo, fibronectin is associated with the migratory routes of neural crest cells and, in some cases, it disappears from the environment of the cells as they stop migrating. In vitro, neural crest cells show a great preference for fibronectin substrates as compared to other matrix molecules. Both in vivo and in vitro, neural crest cell migration can be specifically inhibited by antibodies or peptides that interfere with the binding of fibronectin to its integrin receptor. However, the migratory behavior of neural crest cells cannot result solely from the interaction with fibronectin. Thus, neural crest cells exhibit a particular organization of integrin receptors on their surface and develop a cytoskeletal network which differs from that of non-motile cells. These properties are supposed to permit rapid changes in the shape of cells and to favor a transient adhesion to the substratum. Recent findings have established that different forms of fibronectin may occur, which differ by short sequences along the molecule. The functions of most of these sequences are not known, except for 1 of them which carries a binding site for integrin receptors. We have demonstrated that this site is recognized by neural crest cells and plays a crucial role in their displacement. It is therefore possible that the forms of fibronectin carrying this sequence are not evenly distributed in the embryo, thus allowing migrating neural crest cells to orientate in the embryo. Fibronectin would then not only play a permissive role in embryonic cell motility, but have an instructive function in cell behavior.     

Abnormalities in neural crest cell migration in laminin alpha5 mutant mice

Although numerous in vitro experiments suggest that extracellular matrix molecules like laminin can influence neural crest migration, little is known about their function in the embryo. Here, we show that laminin alpha5, a gene up-regulated during neural crest induction, is localized in regions of newly formed cranial and trunk neural folds and adjacent neural crest migratory pathways in a manner largely conserved between chick and mouse. In laminin alpha5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin alpha5 in vitro show a reduction by half in the number of migratory neural crest cells. During gangliogenesis, laminin alpha5 mutants exhibit defects in condensing cranial sensory and trunk sympathetic ganglia. However, ganglia apparently recover at later stages. These data suggest that the laminin alpha5 subunit functions as a cue that restricts neural crest cells, focusing their migratory pathways and condensation into ganglia. Thus, it is required for proper migration and timely differentiation of some neural crest populations.     

Analysis of high PSA N-CAM expression during mammalian spinal cord and peripheral nervous system development.

Using a monoclonal antibody that recognizes specifically a high polysialylated form of N-CAM (high PSA N-CAM), the temporal and spatial expression of this molecule was studied in developing spinal cord and neural crest derivatives of mouse truncal region. Temporal expression was analyzed on immunoblots of spinal cord and dorsal root ganglia (DRGs) extracts microdissected at different developmental stages. Analysis of the ratio of high PSA N-CAM to total N-CAM indicated that sialylation and desialylation are independently regulated from the expression of polypeptide chains of N-CAM. Motoneurons, dorsal root ganglia cells and commissural neurons present a homogeneous distribution of high PSA N-CAMs on both their cell bodies and their neurites. Sialylation of N-CAM can occur in neurons after their aggregation in peripheral ganglia as demonstrated for dorsal root ganglia at E12. Furthermore, peripheral ganglia express different levels of high PSA N-CAM. With in vitro models using mouse neural crest cells, we found that expression of high PSA N-CAM was restricted to cells presenting an early neuronal phenotype, suggesting a common regulation for the expression of high PSA N-CAM molecules, neurofilament proteins and sodium channels. Using perturbation experiments with endoneuraminidase, we confirmed that high PSA N-CAM molecules are involved in fasciculation and neuritic growth when neurons derived from neural crest grow on collagen substrata. However, we demonstrated that these two parameters do not appear to depend on high PSA N-CAM molecules when cells were grown on a fibronectin substratum, indicating the existence of a hierarchy among adhesion molecules.     

Neural crest contribution to mammalian tooth formation

The cranial neural crest cells, which are specialized cells of neural origin, are central to the process of mammalian tooth development. They are the only source of mesenchyme able to sustain tooth development, and give rise not only to most of the dental tissues, but also to the periodontium, the surrounding tissues that hold teeth in position. Tooth organogenesis is regulated by a series of interactions between cranial neural crest cells and the oral epithelium. In the development of a tooth, the epithelium covering the inside of the developing oral cavity provides the first instructive signals. Signaling molecules secreted by the oral epithelium 1) establish large cellular fields competent to form a specific tooth shape (mono- or multicuspid) along a proximodistal axis; 2) define an oral (capable of forming teeth) and non-oral mesenchyme along a rostrocaudal axis; and 3) position the sites of future tooth development. The critical information to model tooth shape resides later in the neural crest-derived mesenchyme. Cranial neural crest cells ultimately differentiate into highly specialized cell types to produce mature dental organs. Some cranial neural crest cells located in the dental pulp, however, maintain plasticity in their developmental potential up to postnatal life, offering new prospects for regeneration of dental tissues.     

Cadherins in neural crest cell development and transformation

Cadherins constitute a superfamily of cell adhesion molecules involved in cell-cell interaction, histogenesis and cellular transformation. They have been implicated in the development of various lineages, including derivatives of the neural crest. Neural crest cells (NCC) emerge from the dorsal part of the neural tube after an epithelio-mesenchymal transition (EMT) and migrate through the embryo. After homing and differentiation, NCC give rise to many cell types, such as neurons, Schwann cells and melanocytes. During these steps, the pattern of expression of the various cadherins studied is very dynamic. Cadherins also display plasticity of expression during the transformation of neural crest cell derivatives. Here, we review the pattern of expression and the role of the main cadherins involved in the development and transformation of neural crest cell derivatives.     

Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules.

Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell-binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively-spliced type III homology-connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.     

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.     

Molecular mechanisms of neural crest induction

The neural crest is an embryonic cell population that originates at the border between the neural plate and the prospective epidermis. Around the time of neural tube closure, neural crest cells emigrate from the neural tube, migrate along defined paths in the embryo and differentiate into a wealth of derivatives. Most of the craniofacial skeleton, the peripheral nervous system, and the pigment cells of the body originate from neural crest cells. This cell type has important clinical relevance, since many of the most common craniofacial birth defects are a consequence of abnormal neural crest development. Whereas the migration and differentiation of the neural crest have been extensively studied, we are just beginning to understand how this tissue originates. The formation of the neural crest has been described as a classic example of embryonic induction, in which specific tissue interactions and the concerted action of signaling pathways converge to induce a multipotent population of neural crest precursor cells. In this review, we summarize the current status of knowledge on neural crest induction. We place particular emphasis on the signaling molecules and tissue interactions involved, and the relationship between neural crest induction, the formation of the neural plate and neural plate border, and the genes that are upregulated as a consequence of the inductive events.     

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.