Cranial Neural Crest Migration Research Articles

Vascular endothelial growth factor regulates cranial neural crest migration in vivo

Vascular endothelial growth factor regulates cranial neural crest migration in vivo

Novel insight into the function and regulation of αN-catenin by Snail2 during chick neural crest cell migration

The neural crest is a transient population of migratory cells that differentiates to form a variety of cell types in the vertebrate embryo, including melanocytes, the craniofacial skeleton, and portions of the peripheral nervous system. These cells initially exist as adherent epithelial cells in the dorsal aspect of the neural tube and only later become migratory after an epithelial-to-mesenchymal transition (EMT). Snail2 plays a critical role in mediating chick neural crest cell EMT and migration due to its expression by both premigratory and migratory cranial neural crest cells and its ability to down-regulate intercellular junctions components. In an attempt to delineate the role of cellular junction components in the neural crest, we have identified the adherens junction molecule neural alpha-catenin ( αN-catenin) as a Snail2 target gene whose repression is critical for chick neural crest cell migration. Knock-down and overexpression of αN-catenin enhances and inhibits neural crest cell migration, respectively. Furthermore, our results reveal that αN-catenin regulates the appropriate movement of neural crest cells away from the neural tube into the embryo. Collectively, our data point to a novel function of an adherens junction protein in facilitating the proper migration of neural crest cells during the development of the vertebrate embryo.

Jaw muscularization requires Dlx expression by cranial neural crest cells

The origin of active predation in vertebrates is associated with the rise of three major, uniquely derived developmental characteristics of the head: (i) migratory cranial neural crest cells (CNCCs) giving rise to most skeletal skull elements; (ii) expression of Dlx genes by CNCCs in the Hox-free first pharyngeal arch (PA1); and (iii) muscularization of PA1 derivatives. Here we show that these three innovations are tightly linked. Expression of Dlx genes by CNCCs is not only necessary for head skeletogenesis, but also for the determination, differentiation, and patterning of cephalic myogenic mesoderm leading to masticatory muscle formation. In particular, inactivation of Dlx5 and Dlx6 in the mouse results in loss of jaw muscles. As Dlx5/6 are not expressed by the myogenic mesoderm, our findings imply an instructive role for Dlx5/6-positive CNCCs in muscle formation. The defect in muscularization does not result from the loss of mandibular identity observed in Dlx5/6(-/-) mice because masticatory muscles are still present in EdnRA(-/-) mutants, which display a similar jaw transformation. The genesis of jaws and their muscularization should therefore be seen as an integrated Dlx-dependent developmental process at the origin of the vertebrate head. The role of Dlx genes in defining gnathostome jaw identity could, therefore, be secondary to a more primitive function in the genesis of the oral skeletomuscular system.

Tgfβ3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest

Zebrafish tgfβ3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfβ3 in head skeletal formation, we knocked down tgfβ3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfβ3 in embryos resulted in smaller craniofacial cartilages without any gross malformations. These defects suggest that tgfβ3 is required for normal chondrogenesis. To address the cellular mechanisms that lead to the observed malformations, we analyzed cranial neural crest development in morphant and tgfβ3 over-expressing fish. We observed reduced pre-migratory and migratory cranial neural crest, the precursors of the neurocranial cartilage and pharyngeal arches, in tgfβ3 knockdown embryos. In contrast, only the migratory neural crest was reduced in embryos over-expressing tgfβ3. This raised the possibility that the reduced number of cranial neural crest cells is a result of increased apoptosis. Consistent with this, markedly elevated TUNEL staining in the midbrain and hindbrain, and developing pharyngeal arch region was observed in morphants, while tgfβ3 over-expressing embryos showed marginally increased apoptosis in the developing pharyngeal arch region. We propose that both Tgfβ3 suppression and over-expression result in reduced chondrocyte and osteocyte formation, but to different degrees and through different mechanisms. In Tgfβ3 suppressed embryos, this is due to impaired formation and survival of a subpopulation of cranial neural crest cells through markedly increased apoptosis in regions containing the cranial neural crest cells, while in Tgfβ3 over-expressing embryos, the milder phenotype is also due to a slightly elevated apoptosis in these regions. Therefore, proper cranial neural crest formation and survival, and ultimately craniofacial chondrogenesis and osteogenesis, are dependent on tight regulation of Tgfβ3 protein levels in zebrafish.

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.

Myosin‐X is required for cranial neural crest cell migration in Xenopus laevis

Myosin-X (MyoX) belongs to a large family of unconventional, nonmuscle, actin-dependent motor proteins. We show that MyoX is predominantly expressed in cranial neural crest (CNC) cells in embryos of Xenopus laevis and is required for head and jaw cartilage development. Knockdown of MyoX expression using antisense morpholino oligonucleotides resulted in retarded migration of CNC cells into the pharyngeal arches, leading to subsequent hypoplasia of cartilage and inhibited outgrowth of the CNC-derived trigeminal nerve. In vitro migration assays on fibronectin using explanted CNC cells showed significant inhibition of filopodia formation, cell attachment, spreading and migration, accompanied by disruption of the actin cytoskeleton. These data support the conclusion that MyoX has an essential function in CNC migration in the vertebrate embryo.

Myosin-X is critical for migratory ability of Xenopus cranial neural crest cells

The neural crest is a highly migratory cell population, unique to vertebrates, that forms much of the craniofacial skeleton and peripheral nervous system. In exploring the cell biological basis underlying this behavior, we have identified an unconventional myosin, myosin-X (Myo10) that is required for neural crest migration. Myo10 is highly expressed in both premigratory and migrating cranial neural crest (CNC) cells in Xenopus embryos. Disrupting Myo10 expression using antisense morpholino oligonucleotides leads to impaired neural crest migration and subsequent cartilage formation, but only a slight delay in induction. In vivo grafting experiments reveal that Myo10-depleted CNC cells migrate a shorter distance and fail to segregate into distinct migratory streams. Finally, in vitro cultures and cell dissociation-reaggregation assays suggest that Myo10 may be critical for cell protrusion and cell-cell adhesion. These results demonstrate an essential role for Myo10 in normal cranial neural crest migration and suggest a link to cell-cell interactions and formation of processes.

ADAM13 is Required for Xenopus Head Development by Regulating Class B Ephrin and Wnt Signaling

Protein ectodomain shedding by disintegrin metalloproteinases (ADAMs) has emerged as a key regulatory event in cell signaling. We previously showed that an ADAM metalloproteinase, ADAM13, is expressed in cranial neural crest (CNC) cells in Xenopus laevis, and that overexpression of a protease dead mutant of ADAM13 prevents CNC migration. To further understand the roles of this gene in vertebrate development, we carried out loss‐of‐function studies in X. tropicalis, a closely related frog species with a diploid genome. Knockdown of ADAM13 by antisense morpholinos (MOs) resulted in abnormal head development, including reduced CNC induction and perturbed eye field patterning. The metalloproteinase activity of ADAM13 was required for these functions in vivo. Using cultured cells, we identified membrane‐bound ephrins (Efns) B1 and B2 as substrates for ADAM13. Knockdown of ADAM13 protected endogenous EfnB1/B2 in developing embryos, indicating that these two ephrins are in vivo substrates for ADAM13. The defects caused by ADAM13 MO could be rescued by blocking forward EfnB signaling, restoring canonical Wnt signaling, or by ectopic expression of the transcription factor Slug. Our data suggest a novel EfnB‐Wnt‐Slug signaling cascade through which ADAM13 controls CNC induction and eye field patterning.

Extracellular Cleavage of Cadherin-11 by ADAM Metalloproteases Is Essential forXenopusCranial Neural Crest Cell Migration

Cell adhesion molecules such as cadherins alternate their expression throughout cranial neural crest (CNC) development, yet our understanding of the role of these molecules during CNC migration remains incomplete. The "mesenchymal" cadherin-11 is expressed in the CNC during migration yet prevents migration when overexpressed in the embryo, suggesting that a defined level of cadherin-11-mediated cell adhesion is required for migration. Here we show that members of the meltrin subfamily of ADAM metalloproteases cleave the extracellular domain of cadherin-11 during CNC migration. We show that a fragment corresponding to the putative shed form of cadherin-11 retains biological activity by promoting CNC migration in vivo, in a non-cell-autonomous manner. Additionally, cleavage of cadherin-11 does not affect binding to beta-catenin and downstream signaling events. We propose that ADAM cleavage of cadherin-11 promotes migration by modifying its ability to support cell-cell adhesion while maintaining the membrane-bound pool of beta-catenin associated with the cadherin-11 cytoplasmic domain.

Wnt11r is required for cranial neural crest migration

wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co-injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non-cell-autonomous manner to control neural crest migration.

Gli3‐deficient mice exhibit cleft palate associated with abnormal tongue development

Palatogenesis depends on appropriate growth, elevation, and fusion of the palatal shelves and aberration in these processes can lead to palatal clefting. We observed a high incidence of palate clefting in mice deficient in Gli3, known for its role as a repressor in the absence of Shh signaling. In contrast with several current mouse models of cleft palate, Meckel's cartilage extension, cranial neural crest migration, palatal shelf proliferation, apoptosis, and key signaling components mediated by Shh, Bmp, Fgf, and Tgfbeta, appeared unaffected in Gli3-/- mice. Palatal clefting in Gli3-/- mice was consistently associated with tongue abnormalities such as failure to flatten and improper positioning, implicating a critical role of Gli3 and normal tongue morphogenesis for timely palatal shelf elevation and joining. Furthermore, Gli3-/- palatal shelves grown in roller cultures without tongue can fuse suggesting that the abnormal tongue is likely an impediment for palatal shelf joining in Gli3-/- mutants.

Maintaining the balance: Regulation of Cadherin-11 by ADAM13 during cranial neural crest migration in Xenopus laevis

Maintaining the balance: Regulation of Cadherin-11 by ADAM13 during cranial neural crest migration in Xenopus laevis

Divergent roles for Eph and Ephrin in Avian Cranial Neural Crest

BackgroundAs in other vertebrates, avian hindbrain neural crest migrates in streams to specific branchial arches. Signalling from Eph receptors and ephrins has been proposed to provide a molecular mechanism that guides the cells restricting them to streams. In mice and frogs, cranial neural crest express a combination of Eph receptors and ephrins that appear to exclude cells from adjacent tissues by forward and reverse signalling. The objective of this study was to provide comparative data on the distribution and function of Eph receptors and ephrins in avian embryos.ResultsTo distinguish neural crest from bordering ectoderm and head mesenchyme, we have co-labelled embryos for Eph or ephrin RNA and a neural crest marker protein. Throughout their migration avian cranial neural crest cells express EphA3, EphA4, EphA7, EphB1, and EphB3 and move along pathways bordered by non-neural crest cells expressing ephrin-B1. In addition, avian cranial neural crest cells express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Thus, the distribution of avian Eph receptors and ephrins differs from those reported in other vertebrates. In stripe assays when explanted cranial neural crest were given the choice between FN or FN plus clustered ephrin-B1 or EphB2 fusion protein, the cells strongly localize to lanes containing only FN. This preference is mitigated in the presence of soluble ephrin-B1 or EphB2 fusion protein.ConclusionThese findings show that avian cranial neural crest use Eph and ephrin receptors as other vertebrates in guiding migration. However, the Eph receptors are expressed in different combinations by neural crest destined for each branchial arch and ephrin-B1 and ephrin-B2 appear to have opposite roles to those reported to guide cranial neural crest migration in mice. Unlike many of the signalling, specification, and effector pathways of neural crest, the roles of Eph receptors and ephrins have not been rigorously conserved. This suggests diversification of receptor and ligand expression is less constrained, possibly by promiscuous binding and use of common downstream pathways.

Tissue‐specific requirement for TGF‐β signaling during craniofacial development

Members of the transforming growth factor‐β (TGF‐β) superfamily mediate a wide range of biological activities, including cell proliferation, differentiation, extracellular matrix formation, and induction of homeobox genes, suggesting that TGF‐β signaling is important in pattern formation and organogenesis. During craniofacial development, TGF‐β is expressed in the craniofacial epithelium and the cranial neural crest (CNC) derived mesenchyme, suggesting tissue‐specific functions in regulating craniofacial development. To investigate the tissue‐specific requirement of TGF‐β signaling, we generated animal models with epithelim or CNC‐derived mesenchyme specific inactivation of Tgfbr2 gene and investigated the molecular mechanism of TGF‐β signaling in regulating the fate of craniofacial epithelial or the CNC mesenchyme cells, respectively. Using mice with conditional inactivation of Tgfbr2 in the epithelium, we show that TGF‐β signaling is specifically required for regulating palate fusion and skin development. At the cellular level, TGF‐β signaling is critical for mediating apoptosis of MEE cells during palatal fusion. On the other hand, conditional Tgfbr2 gene ablation in the CNC resulted in complete cleft secondary palate, calvaria agenesis, and other skull defects with complete phenotype penetrance. Significantly, disruption of the TGF‐β signaling does not adversely affect CNC migration. Cleft palate in Tgfbr2 mutant mice results from a cell proliferation defect within the CNC‐derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Our data suggests that TGF‐β IIR plays a critical, cell autonomous role in regulating the fate of MEE and CNC cells during palatogenesis. To explore the molecular mechanism of TGF‐β signaling in regulating craniofacial development, we analyzed downstream target genes affected by altered TGF‐β signaling in either the epithelium or the CNC‐derived mesenchyme. Comprehensive analysis reveals specific TGF‐β signaling network in regulating craniofacial development. Collectively, our study demonstrates that TGF‐β signaling plays an essential role in cell fate determination of the craniofacial epithelium and the CNC‐derived mesenchyme and provides a model for the study of abnormal craniofacial development.

The fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.

Barx1 is necessary for zebrafish cranial neural crest patterning and pharyngeal arch chondrogenesis

BARX1, most prominently expressed in the pharyngeal arches is a candidate gene implicated in human craniofacial anomalies, and syndromic malformations; however, little is understood of its regulation or role in patterning the arches. We examined the zebrafish barx1 gene during craniofacial development and performed a functional analysis using morpholino oligonucleotides. Embryos microinjected with barx1 morpholinos exhibit reduced crestin and dlx2a expression. By 2.5 days post fertilization barx1 morphants exhibit poor facial outgrowth and micrognathia. Histological analysis reveals reductions in chondrocyte differentiation and condensation within the arches. Alcian blue stained morphants have small and dysmorphic arch cartilage elements, and chondrogenic marker col2a1 arch expression is perturbed. We investigated barx1 regulation within the arches by implanting beads imbued with growth factors FGF8 and BMP2. The bead experiments show the influence of the growth factors on barx1 expression, and its role in modulating chondrocyte condensation. Together these results suggest a necessary role for barx1 at early stages of cranial neural crest patterning and chondrogenesis within the developing zebrafish viscerocranium. Investigating the zebrafish barx1 provides new insights into the human BARX1 ortholog that may be implicated in agnathia, micrognathia or other craniofacial syndromes. To ascertain its role we utilized a morpholino mediated knock-down approach. Morphants exhibited micrognathia. Alcian blue staining indicates abnormal formation and/or complete loss of pharyngeal arch cartilage elements suggesting that barx1 is necessary for organization of the prechondrogenic ectomesenchyme. Examination of arch markers, sox9a, and dlx2a as well as fli1 transgenic zebrafish indicate that cranial neural crest differentiation and migration remains unperturbed. However, we observed a loss of collagen type II expression in the mandible.

Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation

Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation

Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation

In the head of vertebrate embryos, neural crest cells migrate from the neural tube into the presumptive facial region and condense to form cranial ganglia and skeletal elements in the branchial arches. We show that newly formed neural folds and migrating neural crest cells express the neuropilin 2 (npn2) receptor in a manner that is highly conserved in amniotes. The repulsive npn2 ligand semaphorin (sema) 3F is expressed in a complementary pattern in the mouse. Furthermore, mice carrying null mutations for either npn2 or sema3F have abnormal cranial neural crest migration. Most notably, "bridges" of migrating cells are observed crossing between neural crest streams entering branchial arches 1 and 2. In addition, trigeminal ganglia fail to form correctly in the mutants and are improperly condensed and loosely organized. These data show that npn2/sema3F signaling is required for proper cranial neural crest development in the head.

CYP26A1 and CYP26C1 cooperatively regulate anterior–posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse

The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A–P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A–P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1 −/− Cyp26c1 −/− mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A–P patterning and production of migratory cranial neural crest cells in the developing mammalian brain.

PCNS: A novel protocadherin required for cranial neural crest migration and somite morphogenesis in Xenopus

Protocadherins (Pcdhs), a major subfamily of cadherins, play an important role in specific intercellular interactions in development. These molecules are characterized by their unique extracellular domain (EC) with more than 5 cadherin-like repeats, a transmembrane domain (TM) and a variable cytoplasmic domain. PCNS ( Proto cadherin in Neural crest and Somites), a novel Pcdh in Xenopus, is initially expressed in the mesoderm during gastrulation, followed by expression in the cranial neural crest (CNC) and somites. PCNS has 65% amino acid identity to Xenopus paraxial protocadherin (PAPC) and 42–49% amino acid identity to Pcdh 8 in human, mouse, and zebrafish genomes. Overexpression of PCNS resulted in gastrulation failure but conferred little if any specific adhesion on ectodermal cells. Loss of function accomplished independently with two non-overlapping antisense morpholino oligonucleotides resulted in failure of CNC migration, leading to severe defects in the craniofacial skeleton. Somites and axial muscles also failed to undergo normal morphogenesis in these embryos. Thus, PCNS has essential functions in these two important developmental processes in Xenopus.