Division of labor during trunk neural crest development

Neural crest cells, the migratory precursors of numerous cell types including the vertebrate peripheral nervous system, arise in the dorsal neural tube and follow prescribed routes into the embryonic periphery. While the timing and location of neural crest migratory pathways has been well documented in the trunk, a comprehensive collection of signals that guides neural crest migration along these paths has only recently been established. In this review, we outline the molecular cascade of events during trunk neural crest development. After describing the sequential routes taken by trunk neural crest cells, we consider the guidance cues that pattern these neural crest trajectories. We pay particular attention to segmental neural crest development and the steps and signals that generate a metameric peripheral nervous system, attempting to reconcile conflicting observations in chick and mouse. Finally, we compare cranial and trunk neural crest development in order to highlight common themes.     

Genomic analysis of neural crest induction

The vertebrate neural crest is a migratory stem cell population that arises within the central nervous system. Here, we combine embryological techniques with array technology to describe 83 genes that provide the first gene expression profile of a newly induced neural crest cell. This profile contains numerous novel markers of neural crest precursors and reveals previously unrecognized similarities between neural crest cells and endothelial cells, another migratory cell population. We have performed a secondary screen using in situ hybridization that allows us to extract temporal information and reconstruct the progression of neural crest gene expression as these cells become different from their neighbors and migrate. Our results reveal a sequential 'migration activation' process that reflects stages in the transition to a migratory neural crest cell and suggests that migratory potential is established in a pool of cells from which a subset are activated to migrate.     

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.     

Plasticity and predetermination of mesencephalic and trunk neural crest transplanted into the region of the cardiac neural crest

The cardiac neural crest contains ectomesenchymal and neural anlagen that are necessary for normal heart development. It is not known whether other regions of the neural crest are capable of supporting normal heart development. In the experiments reported herein, quail donor embryos provided cardiac, trunk, or mesencephalic neural crest to replace or add to the chick host cardiac neural crest. Neither trunk nor mesencephalic neural crest was capable of generating ectomesenchyme competent to effect truncal septation. Addition of mesencephalic neural crest resulted in a high incidence of persistent truncus arteriosus, suggesting that ectomesenchyme derived from the mesencephalic region interferes with ectomesenchyme derived from the cardiac neural crest. Derivatives from the trunk neural crest, on the other hand, did not result in abnormal development of the truncal septum. While mesencephalic neural crest seeded the cardiac ganglia with both neurons and supporting cells, this capability was limited in the trunk neural crest to the more mature regions. These studies indicate a predetermination of the ectomesenchymal derivatives of the cranial neural crest and a possible competition of neural anlagen to form neurons and supporting cells in the cardiac ganglia.     

A chemotactic model of trunk neural crest cell migration

Trunk neural crest cells follow a common ventral migratory pathway but are distributed into two distinct locations to form discrete sympathetic and dorsal root ganglia along the vertebrate axis. Although fluorescent cell labeling and time‐lapse studies have recorded complex trunk neural crest cell migratory behaviors, the signals that underlie this dynamic patterning remain unclear. The absence of molecular information has led to a number of mechanistic hypotheses for trunk neural crest cell migration. Here, we review recent data in support of three distinct mechanisms of trunk neural crest cell migration and develop and simulate a computational model based on chemotactic signaling. We show that by integrating the timing and spatial location of multiple chemotactic signals, trunk neural crest cells may be accurately positioned into two distinct targets that correspond to the sympathetic and dorsal root ganglia. In doing so, we honor the contributions of Wilhelm His to his identification of the neural crest and extend the observations of His and others to better understand a complex question in neural crest cell biology.     

How to become neural crest: from segregation to delamination

The development of the neural crest up to the stage where they leave the neural tube can be observed as a series of concatenated but independent events that involve dorsalization of the neural plate/neural tube, neural crest induction, segregation and stabilization, epithelial to mesenchymal transition and delamination. During all these processes, the nascent neural crest cells are subjected to the influence of different signals and have to overcome competition for cell fate and apoptotic signals. In addition, striking rostrocaudal differences unveil how the regulatory cascades are somehow different but still can lead to the production of bona fide neural crest cells.     

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.     

The flavonoids hesperidin and rutin promote neural crest cell survival

The neural crest (NC) corresponds to a collection of multipotent and oligopotent progenitors endowed with both neural and mesenchymal potentials. The derivatives of the NC at trunk level include neurons and glial cells of the peripheral nervous system in addition to melanocytes, smooth muscle cells and some endocrine cells. Environmental factors control the fate decisions of NC cells. Despite the well-known influence of flavonoids on the central nervous system, the issue of whether they also influence NC cells has not been yet addressed. Flavonoids are polyphenolic compounds that are integral components of the human diet. The biological activities of these compounds cover a very broad spectrum, from anticancer and antibacterial activities to inhibition of bone reabsorption and modulation of inflammatory response. In the present work, we have investigated the actions of the flavonoids hesperidin, rutin and quercetin on NC cells of quail, in vitro. We show for the first time, that hesperidin and rutin increase the viability of trunk NC cells in culture, without affecting cell differentiation and proliferation. The molecular mechanism of this action is dependent on ERK2 and PI3K pathways. Quercetin had no effect on NC progenitors. Taken together, these results suggest that flavonoids hesperidin and rutin increase NC cell survival, which may be useful against the toxicity of some chemicals during embryonic development.     

Developmental potential of avian trunk neural crest cells in situ

To analyze the developmental potential of individual neural crest cells or their precursors, we have microinjected a vital dye, lysinated rhodamine dextran (LRD), into single cells in the dorsal neural tube. The phenotypes of the descendants that inherited the LRD from the injected cells were evaluated based upon their position, morphology, and neurofilament expression. Individual neural crest cells labeled before or as they emigrated from the neural tube gave rise to both sensory and sympathetic neurons as well as nonneuronal cells, some of which had the morphological characteristics of Schwann cells or pigment cells. In numerous cases, the descendants of a single cell included both neural crest- and neural tube-derived neurons, suggesting that some cells of the peripheral and central nervous systems share a common lineage. Our data demonstrate definitively that both emigrating and premigratory trunk neural crest cells can be multipotent, giving rise not only to cells in multiple neural crest derivatives, but also to both neuronal and nonneuronal elements within a given derivative.     

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.     

Pathways and mechanisms of avian trunk neural crest cell migration and localization

Crest cells individualized at the dorsal border of the neural tube, while they became surrounded by a fibronectin-rich matrix. Crest cells initiated their migration between the basement membranes of the neural tube and the ectoderm. In the vagal region, crest cells migrated in a fibronectin-rich environment between the ectoderm and the dermomyotome, very rapidly reaching the apex of the pharynx. In the trunk region, crest cells opposite the bulk of the somite accumulated at the junction between the somite, the neural tube, and the ectoderm; they resumed their migration at the onset of the dissociation of the somite into dermomyotome and sclerotome. Migration occurred more ventrally along the neural tube; nevertheless, the formation of the rapidly expanding sclerotome prevented crest cells from reaching the paranotochordal region. Thereafter, crest cells accumulated between the neural tube, the dermomyotome, and the sclerotome, where ultimately they formed the dorsal root ganglia. In contrast, cells opposite the intersomitic space did not encounter these obstacles and utilized a narrow pathway formed between the basement membranes of the two adjacent somites. This pathway allowed crest cells to reach the most ventral regions of the embryo very rapidly; they accumulated along the aorta to form the aortic plexuses, the adrenal medulla, and the sympathetic ganglia. The basic features of the migration pathways are (1) a strict delimitation by the fibronectin-rich basement membranes of the surrounding tissues, (2) a formation of space concomitant with the migration of crest cells, (3) a transient existence: continued migration is correlated with the presence of fibronectin, whereas cessation is correlated with its focal disappearance. The crest cells are characterized by their inability to traverse basement membranes and penetrate within tissues. We propose that the combination of active proliferation, unique motility properties, and the presence of narrow pathways are the major mechanisms ensuring correct directionality. Morphologically defined transient routes of migration along with developmentally regulated changes in the extracellular matrix and in the adhesive properties of crest cells are most probably involved in their stabilization in defined territories and their aggregation into ganglia.     

FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk

Coordination between functionally related adjacent tissues is essential during development. For example, formation of trunk neural crest cells (NCCs) is highly influenced by the adjacent mesoderm, but the molecular mechanism involved is not well understood. As part of this mechanism, fibroblast growth factor (FGF) and retinoic acid (RA) mesodermal gradients control the onset of neurogenesis in the extending neural tube. In this paper, using gain- and loss-of-function experiments, we show that caudal FGF signaling prevents premature specification of NCCs and, consequently, premature epithelial-mesenchymal transition (EMT) to allow cell emigration. In contrast, rostrally generated RA promotes EMT of NCCs at somitic levels. Furthermore, we show that FGF and RA signaling control EMT in part through the modulation of elements of the bone morphogenetic protein and Wnt signaling pathways. These data establish a clear role for opposition of FGF and RA signaling in control of the timing of NCC EMT and emigration and, consequently, coordination of the development of the central and peripheral nervous system during vertebrate trunk elongation.     

Head and trunk neural crest in vitro: Autonomic neuron differentiation

Isolated cultures of premigratory neural crest cells were used to study the initial stages of autonomic neuron development. Autonomic neurons are phenotypically characterized on the basis of their neurotransmitter synthetic enzymes, dopamine β-hydroxylase (DBH) and choline acetyltransferase (CAT). DBH converts dopamine to norepinephrine in noradrenergic neurons while CAT synthesizes acetylcholine from choline in cholinergic neurons. Activities of both enzymes were detected in isolated cultures of trunk neural crest and head neural crest. DBH was detected at all culture ages examined (from 1 to 20 days) whereas CAT activity was first detected only after 5 days in vitro. While specific enzyme activity of DBH peaks on Day 6 and specific enzyme activity of CAT peaks on Day 10, absolute activity for both enzymes increases throughout the 20-day culture period. DBH and CAT develop in vitro without any spinal presynaptic input, without typical target tissue interactions (such as blood vascular elements or heart tissue), and without addition of conditioned medium factors.     

Lunatic fringe causes expansion and increased neurogenesis of trunk neural tube and neural crest populations

Both neurons and glia of the PNS are derived from the neural crest. In this study, we have examined the potential function of lunatic fringe in neural tube and trunk neural crest development by gain-of-function analysis during early stages of nervous system formation. Normally lunatic fringe is expressed in three broad bands within the neural tube, and is most prominent in the dorsal neural tube containing neural crest precursors. Using retrovirally-mediated gene transfer, we find that excess lunatic fringe in the neural tube increases the numbers of neural crest cells in the migratory stream via an apparent increase in cell proliferation. In addition, lunatic fringe augments the numbers of neurons and upregulates Delta-1 expression. The results indicate that, by modulating Notch/Delta signaling, lunatic fringe not only increases cell division of neural crest precursors, but also increases the numbers of neurons in the trunk neural crest.