Emigration Of Neural Crest Cells Research Articles

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

Pax3 Hypomorphs Reveal Hidden Pax7 Functional Genetic Compensation in Utero

Pax3 and Pax7 transcription factors are paralogs within the Pax gene family that that are expressed in early embryos in partially overlapping expression domains and have distinct functions. Significantly, mammalian development is largely unaffected by Pax7 systemic deletion but systemic Pax3 deletion results in defects in neural tube closure, neural crest emigration, cardiac outflow tract septation, muscle hypoplasia and in utero lethality by E14. However, we previously demonstrated that Pax3 hypomorphs expressing only 20% functional Pax3 protein levels exhibit normal neural tube and heart development, but myogenesis is selectively impaired. To determine why only some Pax3-expressing cell lineages are affected and to further titrate Pax3 threshold levels required for neural tube and heart development, we generated hypomorphs containing both a hypomorphic and a null Pax3 allele. This resulted in mutants only expressing 10% functional Pax3 protein with exacerbated neural tube, neural crest and muscle defects, but still a normal heart. To examine why the cardiac neural crest appears resistant to very low Pax3 levels, we examined its paralog Pax7. Significantly, Pax7 expression is both ectopically expressed in Pax3-expressing dorsal neural tube cells and is also upregulated in the Pax3-expressing lineages. To test whether this compensatory Pax7 expression is functional, we deleted Pax7 both systemically and lineage-specifically in hypomorphs expressing only 10% Pax3. Removal of one Pax7 allele resulted in partial outflow tract defects, and complete loss of Pax7 resulted in full penetrance outflow tract defects and in utero lethality. Moreover, combinatorial loss of Pax3 and Pax7 resulted in severe craniofacial defects and a total block of neural crest cell emigration from the neural tube. Pax7Cre lineage mapping revealed ectopic labeling of Pax3-derived neural crest tissues and within the outflow tract of the heart, experimentally confirming the observation of ectopic activation of Pax7 in 10% Pax3 hypomorphs. Finally, genetic cell ablation of Pax7Cre-marked cells is sufficient to cause outflow tract defects in hypomorphs expressing only 10% Pax3, confirming that ectopic and induced Pax7 can play an overlapping functional genetic compensational role in both cardiac neural crest lineage and during craniofacial development, which is normally masked by the dominant role of Pax3.

Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling.

Production and emigration of neural crest cells is a transient process followed by the emergence of the definitive roof plate. The mechanisms regulating the end of neural crest ontogeny are poorly understood. Whereas early crest development is stimulated by mesoderm-derived retinoic acid, we report that the end of the neural crest period is regulated by retinoic acid synthesized in the dorsal neural tube. Inhibition of retinoic acid signaling in the neural tube prevents the normal upregulation of BMP inhibitors in the nascent roof plate and prolongs the period of BMP responsiveness which otherwise ceases close to roof plate establishment. Consequently, neural crest production and emigration are extended well into the roof plate stage. In turn, extending the activity of neural crest-specific genes inhibits the onset of retinoic acid synthesis in roof plate suggesting a mutual repressive interaction between neural crest and roof plate traits. Although several roof plate-specific genes are normally expressed in the absence of retinoic acid signaling, roof plate and crest markers are co-expressed in single cells and this domain also contains dorsal interneurons. Hence, the cellular and molecular architecture of the roof plate is compromised. Collectively, our results demonstrate that neural tube-derived retinoic acid, via inhibition of BMP signaling, is an essential factor responsible for the end of neural crest generation and the proper segregation of dorsal neural lineages.

Hyaluronan receptor CD44: developmentally regulated expression and role in the early chick embryo.

CD44 is a membrane glycoprotein and is the main receptor for hyaluronan. We studied CD44 expression and spatio-temporal distribution by RT-PCR and immunofluorescence, and used an anti-CD44 blocking antibody to perturb CD44-depended signalling programs in the early chick embryo. The intense CD44 levels we detected in the morula embryo (XI) were of particular interest, suggestive of a maternally stored transcript. Intriguingly, the early presence of CD44 seemed to be essential for the rapid synthesis of hyaluronan. At stage XIII (blastula), CD44 expression was intense in the epiblast and hypoblast. During gastrulation (HH3-4), the cells ingressing into the primitive groove and migrating, and the blood islands, expressed CD44 intensely. At HH8, the folding neural plate showed polarity regulation of CD44 expression, and expression was also intense in neural crest, notochord, and blood islands. During early organogenesis, CD44 was expressed intensely in the developing cranial and caudal neural tube that showed polarity regulation, in optic stalks, otic vesicles, pre-and migratory neural crest cells, ganglia, notochord, pharynx, gut, liver, aortae, heart, somites, vascular area, amnion and chorion, and was distinct in extracellular matrix of cranial neural tube and otic vesicle lumens. Antibody-mediated perturbation of CD44 function resulted in unorganized extracellular matrix, loss of tissue spaces, grossly abnormal notochord, intermingling of clumped neuroectoderm and mesenchyme, absence of somites and blood vessels and inhibition of neural crest cell emigration. CD44 has various pivotal roles in matrix integrity and tissue patterning, consistent with its known biochemical features and interactions with hyaluronan, growth factors, receptors and other signaling molecules.

Hypoxia promotes production of neural crest cells in the embryonic head.

Hypoxia is encountered in either pathological or physiological conditions, the latter of which is seen in amniote embryos prior to the commencement of a functional blood circulation. During the hypoxic stage, a large number of neural crest cells arise from the head neural tube by epithelial-to-mesenchymal transition (EMT). As EMT-like cancer dissemination can be promoted by hypoxia, we investigated whether hypoxia contributes to embryonic EMT. Using chick embryos, we show that the hypoxic cellular response, mediated by hypoxia-inducible factor (HIF)-1α, is required to produce a sufficient number of neural crest cells. Among the genes that are involved in neural crest cell development, some genes are more sensitive to hypoxia than others, demonstrating that the effect of hypoxia is gene specific. Once blood circulation becomes fully functional, the embryonic head no longer produces neural crest cells in vivo, despite the capability to do so in a hypoxia-mimicking condition in vitro, suggesting that the oxygen supply helps to stop emigration of neural crest cells in the head. These results highlight the importance of hypoxia in normal embryonic development.

Cadherin-6B is required for the generation of Islet-1-expressing dorsal interneurons

Cadherin-6B induces bone morphogenetic protein (BMP) signaling to promote the epithelial mesenchymal transition (EMT) in the neural crest. We have previously found that knockdown of Cadherin-6B inhibits both BMP signaling and the emigration of the early pre-migratory neural crest cells from the dorsal neural tube. In this study, we found that inhibition of BMP signaling in the neural tube, mediated by the ectopic expression of Smad-6 or Noggin, decreased the size of the Islet-1-positive dorsal cell population. Knockdown or loss of function of Cadherin-6B suppressed the generation of Islet-1-expressing cells in the dorsal neural tube, but not the Lim-1/2 positive dorsal cell population. Our results thus indicate that Cadherin-6B is necessary for the generation of Islet-1-positive dorsal interneurons, as well as the initiation of pre-migratory neural crest cell emigration.

Regulation of neural crest cell emigration in turtle embryos (541.9)

Turtle plastron bones develop by intramembranous ossification, suggesting that they are derived, like the facial bones, from neural crest cells. Using cell‐labeling and neural tube explant cultures, we have shown that cells expressing neural crest markers emerge from the trunk neural tube in the turtle Trachemys scripta for a greatly extended period compared other model amniotes. The neural crest cells that emerge in a second wave, well beyond the stage of neural crest emigration in chick or mouse embryos, appear to migrate ventrally to form an ectomesenchymal dermis that gives rise to the bones of the plastron. The specification of premigratory neural crest cells and the epithelial‐mesenchymal transition that produces migratory neural crest cell is controlled by a gene regulatory network including the transcription factors Snail2, FoxD3, Sox9, and Sox10. We are currently examining the expression of markers of premigratory and early migratory neural crest cells to examine whether the premigratory domain persists during the period in between the early and late migratory phases. If the expression of these markers persist throughout this period, it will suggest that the premigratory region is maintained, and that the lack of neural crest cell migration may be due to the lack of a supportive environment. In contrast, if these genes are only expressed during the periods of active neural crest cell emigration, then the second wave of neural crest cell migration would require a second inductive signal not found in chick embryos.Grant Funding Source: NSF

Neural crest αN‐catenin function in cranial ganglia assembly (344.1)

Neural crest cells are crucial for proper vertebrate development, building the craniofacial skeleton and, together with epidermal placode cells, the cranial ganglia of the peripheral nervous system. Defective ganglia function can lead to conditions such as neuralgia and peripheral neuropathies and contribute to other diseases. Migratory neural crest cells are generated from immotile precursors possessing adherens junctions composed of cadherins and αN‐catenin, a protein that also controls formation of central nervous system structures. Although αN‐catenin down‐regulation is critical for initial neural crest cell emigration from the neural tube, a later role for αN‐catenin in the peripheral nervous system is not known. To this end, we evaluated αN‐catenin spatio‐temporal distribution and perturbed αN‐catenin function to elucidate effects on cranial trigeminal ganglia formation in the chick. We find that migratory neural crest cells forming the trigeminal ganglia re‐express αN‐catenin and, as in the trunk, express Cadherin‐7. αN‐catenin perturbation alters trigeminal ganglia assembly through changes in neural crest and placode cell contribution to the ganglia as well as Cadherin‐7 distribution and levels. Importantly, αN‐catenin function is likely conserved in other cranial ganglia. Together, our results highlight a novel role for neural crest cell αN‐catenin in cranial gangliogenesis.Grant Funding Source: Supported by NSF IOS‐0948525

Hif-1α provides a Twist to neural crest migration

![Figure][1] The emigration of neural crest cells (green) seen in control embryos (arrow, left) is inhibited when the cells lack Hif-1α (right). Barriga et al. reveal that a pathway that drives cancer cell metastasis also operates during development to promote the migration of neural

Neural crest and somitic mesoderm as paradigms to investigate cell fate decisions during development

The dorsal domains of the neural tube and somites are transient embryonic epithelia; they constitute the source of neural crest progenitors that generate the peripheral nervous system, pigment cells and ectomesenchyme, and of the dermomyotome that develops into myocytes, dermis and vascular cells, respectively. Based on the variety of derivatives produced by each type of epithelium, a classical yet still highly relevant question is whether these embryonic epithelia are composed of homogeneous multipotent progenitors or, alternatively, of subsets of fate-restricted cells. Growing evidence substantiates the notion that both the dorsal tube and the dermomyotome are heterogeneous epithelia composed of multipotent as well as fate-restricted precursors that emerge as such in a spatio-temporally regulated manner. Elucidation of the state of commitment of the precedent progenitors is of utmost significance for deciphering the mechanisms that regulate fate segregation during embryogenesis. In addition, it will contribute to understanding the nature of well documented neural crest-somite interactions shown to modulate the timing of neural crest cell emigration, their segmental migration, and myogenesis.

Annexin A6 Modulates Chick Cranial Neural Crest Cell Emigration

The vertebrate neural crest is a population of migratory cells that originates in the dorsal aspect of the embryonic neural tube. These cells undergo an epithelial-to-mesencyhmal transition (EMT), delaminate from the neural tube and migrate extensively to generate an array of differentiated cell types. Elucidating the gene regulatory networks involved in neural crest cell induction, migration and differentiation are thus crucial to understanding vertebrate development. To this end, we have identified Annexin A6 as an important regulator of chick midbrain neural crest cell emigration. Annexin proteins comprise a family of calcium-dependent, membrane-binding molecules that mediate a variety of cellular and physiological processes including cell adhesion, migration and invasion. Our data indicate that Annexin A6 is expressed in the proper spatio-temporal pattern in the chick midbrain to play a potential role in neural crest cell ontogeny. To investigate Annexin A6 function, we have depleted or overexpressed Annexin A6 in the developing midbrain neural crest cell population. Our results show that knock-down or overexpression of Annexin A6 reduces or expands the migratory neural crest cell domain, respectively. Importantly, this phenotype is not due to any change in cell proliferation or cell death but can be correlated with changes in the size of the premigratory neural crest cell population and with markers associated with EMT. Taken together, our data indicate that Annexin A6 plays a pivotal role in modulating the formation of cranial migratory neural crest cells during vertebrate development.

The tight junction protein claudin-1 influences cranial neural crest cell emigration

The neural crest is a population of migratory cells that follows specific pathways during development, eventually differentiating to form parts of the face, heart, and peripheral nervous system, the latter of which includes contributions from placodal cells derived from the ectoderm. Stationary, premigratory neural crest cells acquire the capacity to migrate by undergoing an epithelial-to-mesenchymal transition that facilitates their emigration from the dorsal neural tube. This emigration involves, in part, the dismantling of cell-cell junctions, including apically localized tight junctions in the neuroepithelium. In this study, we have characterized the role of the transmembrane tight junction protein claudin-1 during neural crest and placode ontogeny. Our data indicate that claudin-1 is highly expressed in the developing neuroepithelium but is down-regulated in migratory neural crest cells, although expression persists in the ectoderm from which the placode cells arise. Depletion or overexpression of claudin-1 augments or reduces neural crest cell emigration, respectively, but does not impact the development of several cranial placodes. Taken together, our results reveal a novel function for a tight junction protein in the formation of migratory cranial neural crest cells in the developing vertebrate embryo.

SOX2 modulates levels of MITF in normal human melanocytes, and melanoma lines in vitro

SOX2 modulates levels of MITF in normal human melanocytes, and melanoma lines in vitro

The role of cellular junctions in neural crest cell emigration

Neural crest cells are a transient population of migratory cells that differentiate to form a wide array of derivatives in vertebrates, including the craniofacial skeleton, peripheral nervous system and skin pigment cells. As such, defects in neural crest cell formation can have drastic developmental consequences and lead to various human congenital and hereditary malformations, diseases and cancers. Premigratory neural crest cells initially reside in the dorsal region of the embryonic neural tube and become motile by undergoing an epithelial‐to‐mesenchymal transition, a process characterized by cytoskeletal rearrangements and the loss of intercellular contacts in order to facilitate their emigration out of the neuroepithelium. Using the chick embryo as a model system, we have performed depletion and overexpression experiments to examine how perturbation of cellular junction components affects neural crest cell development. Our studies reveal that components of cellular junctions in premigratory neural crest cells must be precisely modulated to permit both appropriate neural crest cell migration and subsequent differentiation into developing cranial ganglia. These results have direct implications for craniofacial development and should provide additional insight into the etiology of various neural crest‐derived diseases, disorders and cancers. This research is supported by grants from the NIH (R00HD055034) and NSF (IOS‐0948525).

Neural crest cells pattern the surface cephalic ectoderm during FEZ formation

Multiple fibroblast growth factor (Fgf) ligands are expressed in the forebrain and facial ectoderm, and vascular endothelial growth factor (VEGF) is expressed in the facial ectoderm. Both pathways activate the MAP kinase cascade and can be suppressed by SU5402. We placed a bead soaked in SU5402 into the brain after emigration of neural crest cells was complete. Within 24 hr we observed reduced pMEK and pERK staining that persisted for at least 48 hr. This was accompanied by significant apoptosis in the face. By day 15, the upper beaks were truncated. Molecular changes in the FNP were also apparent. Normally, Shh is expressed in the frontonasal ectodermal zone and controls patterned growth of the upper jaw. In treated embryos, Shh expression was reduced. Both the structural and molecular deficits were mitigated after transplantation of FNP-derived mesenchymal cells. Thus, mesenchymal cells actively participate in signaling interactions of the face, and the absence of neural crest cells in neurocristopathies may not be merely structural.

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.

The tight junction protein cingulin modulates chick neural crest cell emigration

The tight junction protein cingulin modulates chick neural crest cell emigration

Apoptosis is not required for mammalian neural tube closure

Apoptotic cell death occurs in many tissues during embryonic development and appears to be essential for processes including digit formation and cardiac outflow tract remodeling. Studies in the chick suggest a requirement for apoptosis during neurulation, because inhibition of caspase activity was found to prevent neural tube closure. In mice, excessive apoptosis occurs in association with failure of neural tube closure in several genetic mutants, but whether regulated apoptosis is also necessary for neural tube closure in mammals is unknown. Here we investigate the possible role of apoptotic cell death during mouse neural tube closure. We confirm the presence of apoptosis in the neural tube before and during closure, and identify a correlation with 3 main events: bending and fusion of the neural folds, postfusion remodeling of the dorsal neural tube and surface ectoderm, and emigration of neural crest cells. Both Casp3 and Apaf1 null embryos exhibit severely reduced apoptosis, yet neurulation proceeds normally in the forebrain and spine. In contrast, the mutant embryos fail to complete neural tube closure in the midbrain and hindbrain. Application of the apoptosis inhibitors z-Vad-fmk and pifithrin-alpha to neurulation-stage embryos in culture suppresses apoptosis but does not prevent initiation or progression of neural tube closure along the entire neuraxis, including the midbrain and hindbrain. Remodeling of the surface ectoderm to cover the closed tube, as well as delamination and migration of neural crest cells, also appear to be normal in the apoptosis-suppressed embryos. We conclude that apoptosis is not required for neural tube closure in the mouse embryo.

Neural crest ontogeny during secondary neurulation: a gene expression pattern study in the chick embryo

In the prospective lumbo-sacral region of the chick embryo, neurulation is achieved by cavitation of the medullary cord, a process called secondary neurulation. Neural crest cells (NCC) are generated in this region and they give rise to the same types of derivatives as in more rostral parts of the trunk where neurulation occurs by dorsal fusion of the neural plate borders (primary neurulation). However, no molecular data were available concerning the different steps of their ontogeny. We thus performed a detailed expression study of molecular players likely to participate in the generation of secondary NCC in chick embryos between Hamburger and Hamilton stages 18-20 (HH18-20) at the level of somites 30 to 43. We found that specification of secondary NCC involves, as in primary neurulation, the activity of several transcription factors such as Pax3, Pax7, Snail2, FoxD3 and Sox9, which are all expressed in the dorsal secondary neural tube as soon as full cavitation is achieved. Moreover, once specification has occurred, emigration of NCC from the dorsal neuroepithelium starts facing early dissociating somites and involves a series of changes in cell shape and adhesion, as well as interactions with the extracellular matrix. Furthermore, Bmp4 and Wnt1 expression precedes the detection of migratory secondary NCC and is coincident with maturation of adjacent somites. Altogether, this first study of molecular aspects of secondary NCC ontogeny has revealed that the mechanisms of neural crest generation occurring along the trunk region of the chick embryo are generally conserved and independent of the type of neurulation involved.

A critical role for Cadherin6B in regulating avian neural crest emigration

Neural crest cells originate in the dorsal neural tube but subsequently undergo an epithelial-to-mesenchymal transition (EMT), delaminate, and migrate to diverse locations in the embryo where they contribute to a variety of derivatives. Cadherins are a family of cell–cell adhesion molecules expressed in a broad range of embryonic tissues, including the neural tube. In particular, cadherin6B ( Cad6B) is expressed in the dorsal neural tube prior to neural crest emigration but is then repressed by the transcription factor Snail2, expressed by premigratory and early migrating cranial neural crest cells. To examine the role of Cad6B during neural crest EMT, we have perturbed Cad6B protein levels in the cranial neural crest-forming region and have examined subsequent effects on emigration and migration. The results show that knock-down of Cad6B leads to premature neural crest cell emigration, whereas Cad6B overexpression disrupts migration. Our data reveal a novel role for Cad6B in controlling the proper timing of neural crest emigration and delamination from the neural tube of the avian embryo.