Vascular endothelial growth factor (VEGF) regulates cranial neural crest migration in vivo

The neural crest is an excellent model to study embryonic cell migration, since cell behaviors can be studied in vivo with advanced optical imaging and molecular intervention. What is unclear is how molecular signals direct neural crest cell (NCC) migration through multiple microenvironments and into specific targets. Here, we tested the hypothesis that the invasion of cranial NCCs, specifically the rhombomere 4 (r4) migratory stream into branchial arch 2 (ba2), is due to chemoattraction through neuropilin-1-vascular endothelial growth factor (VEGF) interactions. We found that the spatio-temporal expression pattern of VEGF in the ectoderm correlated with the NCC migratory front. RT-PCR analysis of the r4 migratory stream showed that ba2 tissue expressed VEGF and r4 NCCs expressed VEGF receptor 2. When soluble VEGF receptor 1 (sVEGFR1) was injected distal to the r4 migratory front, to bind up endogenous VEGF, NCCs failed to completely invade ba2. Time-lapse imaging revealed that cranial NCCs were attracted to ba2 tissue or VEGF sources in vitro. VEGF-soaked beads or VEGF-expressing cells placed adjacent to the r4 migratory stream caused NCCs to divert from stereotypical pathways and move towards an ectopic VEGF source. Our results suggest a model in which NCC entry and invasion of ba2 is dependent on chemoattractive signaling through neuropilin-1-VEGF interactions.     

In Vivo Expression of EphrinA5-Fc in Mice Results in Cephalic Neural Crest Agenesis and Craniofacial Abnormalities

Eph receptors and their ligands ephrins have been implicated in guiding the directed migration of neural crest cells (NCCs). In this study, we found that Wnt1-Cre-mediated expression of ephrinA5-Fc along the dorsal midline of the dien- and mesencephalon resulted in severe craniofacial malformation of mouse embryo. Interestingly, expression of cephalic NCC markers decreased significantly in the frontonasal process and branchial arches 1 and 2, which are target areas for the migratory cephalic NCCs originating in the dien- and mesencephalon. In addition, these craniofacial tissues were much smaller in mutant embryos expressing ephrinA5-Fc. Importantly, EphA7-positive cephalic NCCs were absent along the dorsal dien- and mesencephalon of mutant embryos expressing ephrinA5-Fc, suggesting that the generation of cephalic NCCs is disrupted due to ephrinA5-Fc expression. NCC explant experiments suggested that ephrinA5-Fc perturbed survival of cephalic NCC precursors in the dorsal midline tissue rather than affecting their migratory capacity, which was consistent with our previous report that expression of ephrinA5-Fc in the dorsal midline is responsible for severe neuroepithelial cell apoptotic death. Taken together, our findings strongly suggest that expression of ephrinA5-Fc decreases a population of cephalic NCC precursors in the dorsal midline of the dien- and mesencephalon, thereby disrupting craniofacial development in the mouse embryos.     

Semaphorin 3d promotes cell proliferation and neural crest cell development downstream of TCF in the zebrafish hindbrain

Neural crest cells (NCCs) are pluripotent migratory cells that are crucial to the development of the peripheral nervous system, pigment cells and craniofacial cartilage and bone. NCCs are specified within the dorsal ectoderm and undergo an epithelial to mesenchymal transition (EMT) in order to migrate to target destinations where they differentiate. Here we report a role for a member of the semaphorin family of cell guidance molecules in NCC development. Morpholino-mediated knockdown of Sema3d inhibits the proliferation of hindbrain neuroepithelial cells. In addition, Sema3d knockdown reduces markers of migratory NCCs and disrupts NCC-derived tissues. Similarly, expression of a dominant-repressor form of TCF (DeltaTCF) reduces hindbrain cell proliferation and leads to a disruption of migratory NCC markers. Moreover, expression of DeltaTCF downregulates sema3d RNA expression. Finally, Sema3d overexpression rescues reduced proliferation caused by DeltaTCF expression, suggesting that Sema3d lies downstream of Wnt/TCF signaling in the molecular pathway thought to control cell cycle in NCC precursors.     

In the beginning

Neural crest cells (NCCs) are migratory cells that delaminate from the neural tube early in development and then disseminate throughout the embryo to give rise to a wide variety of cell types that are key to the vertebrate body plan. During their journey from the neural tube to their peripheral targets, NCCs progressively differentiate, raising the question when the fate of an individual NCC is sealed. One hypothesis suggests that the fate of a NCC is specified by target-derived signals emanating from the environment they migrate through, while another hypothesis proposes that NCCs are already specified to differentiate along select lineages at the time they are born in the neural tube, with environmental signals helping them to realize their prespecified fate potential. Alternatively, both mechanisms may cooperate to drive NCC diversity. This review highlights recent advances in our understanding of prespecification during trunk NCC development.     

In the beginning: Generating neural crest cell diversity

Neural crest cells (NCCs) are migratory cells that delaminate from the neural tube early in development and then disseminate throughout the embryo to give rise to a wide variety of cell types that are key to the vertebrate body plan. During their journey from the neural tube to their peripheral targets, NCCs progressively differentiate, raising the question of when the fate of an individual NCC is sealed. One hypothesis suggests that the fate of a NCC is specified by target-derived signals emanating from the environment they migrate through, while another hypothesis proposes that NCCs are already specified to differentiate along select lineages at the time they are born in the neural tube, with environmental signals helping them to realize their prespecified fate potential. Alternatively, both mechanisms may cooperate to drive NCC diversity. This review highlights recent advances in our understanding of prespecification during trunk NCC development.Key words: neural crest cell, multipotent, prespecification, neuropilin, semaphorin, migration, cell fate     

Multiscale mechanisms of cell migration during development: theory and experiment

Long-distance cell migration is an important feature of embryonic development, adult morphogenesis and cancer, yet the mechanisms that drive subpopulations of cells to distinct targets are poorly understood. Here, we use the embryonic neural crest (NC) in tandem with theoretical studies to evaluate model mechanisms of long-distance cell migration. We find that a simple chemotaxis model is insufficient to explain our experimental data. Instead, model simulations predict that NC cell migration requires leading cells to respond to long-range guidance signals and trailing cells to short-range cues in order to maintain a directed, multicellular stream. Experiments confirm differences in leading versus trailing NC cell subpopulations, manifested in unique cell orientation and gene expression patterns that respond to non-linear tissue growth of the migratory domain. Ablation experiments that delete the trailing NC cell subpopulation reveal that leading NC cells distribute all along the migratory pathway and develop a leading/trailing cellular orientation and gene expression profile that is predicted by model simulations. Transplantation experiments and model predictions that move trailing NC cells to the migratory front, or vice versa, reveal that cells adopt a gene expression profile and cell behaviors corresponding to the new position within the migratory stream. These results offer a mechanistic model in which leading cells create and respond to a cell-induced chemotactic gradient and transmit guidance information to trailing cells that use short-range signals to move in a directional manner.     

Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells

Cardiac and cephalic neural crest cells (NCCs) are essential components of the craniofacial and aortic arch mesenchyme. Genetic disruption of the platelet-derived growth factor receptor alpha (PDGFRalpha) results in defects in multiple tissues in the mouse, including neural crest derivatives contributing to the frontonasal process and the aortic arch. Using chimeric analysis, we show that loss of the receptor in NCCs renders them inefficient at contributing to the cranial mesenchyme. Conditional gene ablation in NCCs results in neonatal lethality because of aortic arch defects and a severely cleft palate. The conotruncal defects are first observed at E11.5 and are consistent with aberrant NCC development in the third, fourth and sixth branchial arches, while the bone malformations present in the frontonasal process and skull coincide with defects of NCCs from the first to third branchial arches. Changes in cell proliferation, migration, or survival were not observed in PDGFRalpha NCC conditional embryos, suggesting that the PDGFRalpha may play a role in a later stage of NCC development. Our results demonstrate that the PDGFRalpha plays an essential, cell-autonomous role in the development of cardiac and cephalic NCCs and provides a model for the study of aberrant NCC development.     

Control of neural crest cell behavior and migration: Insights from live imaging

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this Review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.Key words: live imaging, neural crest, EMT, Rho GTPase, ephrin, PCP signaling, cadherin, VEGFNeural crest cells (NCCs) are a pluripotent population of cells that migrate from the dorsal neuroepithelium and give rise to multiple cell types including neurons and glia of the peripheral nervous system, pigment cells and craniofacial bone and cartilage.¹ An important hallmark of NCCs is their remarkable ability to migrate over long distances and along specific pathways through the embryo. NCC migration begins with an epithelial to mesenchymal transition (EMT), in which NCCs lose adhesions with their neighbors and segregate from the neuroepithelium.^2,3 Following EMT, NCCs acquire a polarized morphology and initiate directed migration away from the neural tube. While migrating along their pathways to their target tissues, NCCs are guided by extensive communication with one another and by other cues from the extracellular environment. Each of these aspects of NCC migration requires precise regulation of cell motile behaviors, although the mechanisms controlling them are still not well understood. A critical step toward understanding the molecular control of NCC motility is characterization of NCC behaviors as they migrate in their native environment. In the past 15 years, multiple studies have analyzed specific behaviors associated with NCCs along the various stages of their journey and have begun to identify molecules controlling these behaviors. In this review we will focus specifically on these studies that employ live imaging and will highlight the strength of live imaging to reveal mechanisms regulating NCC motility and migration pathways.     

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition ^1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells ^1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components ³. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration ⁴. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands ^5-8. However, not until recently have any chemoattractants of trunk NCCs been identified ⁹. Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere¹⁰). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.     

Control of neural crest cell behavior and migration

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system, and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.     

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.     

Prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development

Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-β1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol.     

Cranial neural crest migration: New rules for an old road

The neural crest serve as an excellent model to better understand mechanisms of embryonic cell migration. Cell tracing studies have shown that cranial neural crest cells (CNCCs) emerge from the dorsal neural tube in a rostrocaudal manner and are spatially distributed along stereotypical, long distance migratory routes to precise targets in the head and branchial arches. Although the CNCC migratory pattern is a beautifully choreographed and programmed invasion, the underlying orchestration of molecular events is not well known. For example, it is still unclear how single CNCCs react to signals that direct their choice of direction and how groups of CNCCs coordinate their interactions to arrive at a target in an ordered manner. In this review, we discuss recent cellular and molecular discoveries of the CNCC migratory pattern. We focus on events from the time when CNCCs encounter the tissue adjacent to the neural tube and their travel through different microenvironments and into the branchial arches. We describe the patterning of discrete cell migratory streams that emerge from the hindbrain, rhombomere (r) segments r1-r7, and the signals that coordinate directed migration. We propose a model that attempts to unify many complex events that establish the CNCC migratory pattern, and based on this model we integrate information between cranial and trunk neural crest development.     

Neural Crest Cell Fate

The neural crest, the intriguing cell population that gives rise to a panoply of derivatives in the vertebrate embryo, including the mesenchymal structures in the head, melanocytes and most of the peripheral nervous system, still proves to be an important yet enigmatic developmental cell population to study with applications in stem cell biology, cancer biology and clinical medicine. Albeit our knowledge base is rich due to a strong history of experimentation, the fact that we have yet to decipher so many key aspects of neural crest cell (NCC) behavior speaks to the challenging complexity of this transient yet vital cell population. With the advent of new fluorescent tracing techniques, we have reexamined the migratory behaviors and ultimate fate of ventrally migrating avian NCCs within a late wave of emigration and identified a subpopulation of lineally restricted NCCs who migrate to the contralateral dorsal root ganglia (DRG) and therein give rise to mitotically active progenitor cells that ultimately produce the majority of the nociceptive sensory neurons in the DRG. These data provide evidence for the fate prespecification of subsets of NCCs while still resident in the neural tube.     

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.     

Epigenetic regulation in neural crest development

The neural crest (NC) is a multipotent, migratory cell population that arises from the developing dorsal neural fold of vertebrate embryos. Once their fates are specified, neural crest cells (NCCs) migrate along defined routes and differentiate into a variety of tissues, including bone and cartilage of the craniofacial skeleton, peripheral neurons, glia, pigment cells, endocrine cells, and mesenchymal precursor cells (Santagati and Rijli,2003; Dupin et al.,2006; Hall,2009). Abnormal development of NCCs causes a number of human diseases, including ear abnormalities (including deafness), heart anomalies, neuroblastomas, and mandibulofacial dysostosis (Hall,2009). For more than a century, NCCs have attracted the attention of geneticists and developmental biologists for their stem cell-like properties, including self-renewal and multipotent differentiation potential. However, we have only begun to understand the underlying mechanisms responsible for their formation and behavior. Recent studies have demonstrated that epigenetic regulation plays important roles in NC development. In this review, we focused on some of the most recent findings on chromatin-mediated mechanisms for vertebrate NCC development.     

Gene expression in Xenopus laevis embryos after Triadimefon exposure

The triazole derivative Triadimefon (FON) is a systemic fungicide used to control powdery mildews, rusts, and other fungal pests. Some data have already been published about the teratogenic activity of this compound: craniofacial malformations were found in mouse, rat, and Xenopus laevis embryos exposed to FON. These alterations were correlated to defective branchial arch development possibly caused by abnormal neural crest cell (NCC) migration into the branchial mesenchyme. As the migration of NCCs is controlled by the HOX code and by an anteroposterior retinoic acid (RA) gradient, we analyzed the expression of CYP26, a key enzyme in RA metabolism, following FON exposure. The increased expression of this gene and the ability of citral (a RA inhibitor) to reduce the teratogenic effects of the fungicide support the notion that endogenous RA is involved in the mechanism of action of FON. Moreover, by in situ hybridization, we studied the effects of FON exposure at gastrula stage on the expression of some genes involved in craniofacial development, hindbrain patterning, and NCC migration. We observed abnormal localization of xCRABP, Hoxa2 and Xbap signal expression at the level of migrating NCC domains, whereas in the hindbrain, we did not find any alteration in Krox20 and Hoxa2 expression.