Perturbation of cranial neural crest migration by the HNK-1 antibody

The HNK-1 antibody recognizes a carbohydrate moiety that is shared by a family of cell adhesion molecules and is also present on the surface of migrating neural crest cells. Here, the effects of the HNK-1 antibody on neural crest cells were examined in vitro and in vivo. When the HNK-1 antibody was added to neural tube explants in tissue culture, neural crest cells detached from laminin substrates but were unaffected on fibronectin substrates. In order to examine the effects of the HNK-1 antibody in vivo, antibody was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. The injected antibody persisted for approximately 16 hr on the injected side of the embryo and appeared to be most prevalent on the surface of neural crest cells. Embryos fixed within the first 24 hr after injection of HNK-1 antibodies (either whole IgMs or small IgM fragments) showed one or more of the following abnormalities: (1) ectopic neural crest cells external to the neural tube, (2) an accumulation of neural crest cell volume on the lumen of the neural tube, (3) some neural tube anomalies, or (4) a reduction in the neural crest cell volume on the injected side. The ectopic cells and neural tube anomalies persisted in embryos fixed 2 days postinjection. Only embryos having 10 or less somites at the time of injection were affected, suggesting a limited period of sensitivity to the HNK-1 antibody. Control embryos injected with a nonspecific antibody or with a nonblocking antibody against the neural cell adhesion molecule (N-CAM) were unaffected. Previous experiments from this laboratory have demonstrated than an antibody against integrin, a fibronectin and laminin receptor caused defects qualitatively similar to those resulting from HNK-1 antibody injection (M. Bronner-Fraser, J. Cell Biol., 101, 610, 1985). Coinjection of the HNK-1 and integrin antibodies resulted in a greater percentage of affected embryos than with either antibody alone. The additive nature of the effects of the two antibodies suggests that they act at different sites. These results demonstrate that the HNK-1 antibody causes abnormalities in cranial neural crest migration, perhaps by perturbing interactions between neural crest cells and laminin substrates.     

Deltex/Dtx mediates NOTCH signaling in regulation of Bmp4 expression in cranial neural crest formation during avian development

NOTCH signaling plays a key role in cell fate determination in both vertebrates and invertebrates. It is well known that Su(H)/RBP-J is a major mediator of NOTCH signaling. In a previous study, it was shown that NOTCH signaling was involved in cranial neural crest formation in avian embryos. However, Su(H)/RBP-J activity did not appear to be required in this process. In this study, the Deltex/Dtx gene was focussed on as a potential mediator of NOTCH signaling in neural crest formation. At the time of neural crest formation, quail Deltex2 was expressed throughout the ectoderm. Misexpression of a dominant-negative form of Deltex in the ectoderm caused reduced expression of Slug, a neural crest marker. Dominant-negative Deltex expression reduced the expression of Bmp4, a neural crest inducer, whereas co-transfection of Bmp4 with dominant-negative Deltex rescued Slug expression. In parallel, Hairy2 expression in the epidermis was regulated by a Su(H)-dependent pathway. These results indicate that NOTCH signaling has dual functions mediated by either Su(H) or Deltex in the avian embryonic ectoderm.     

Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.

Cranial neural crest (CNC) cells migrate extensively, typically in a pattern of cell streams. In Xenopus, these cells express the adhesion molecule Xcadherin-11 (Xcad-11) as they begin to emigrate from the neural fold. In order to study the function of this molecule, we have overexpressed wild-type Xcad-11 as well as Xcad-11 mutants with cytoplasmic (deltacXcad-11) or extracellular (deltaeXcad-11) deletions. Green fluorescent protein (GFP) was used to mark injected cells. We then transplanted parts of the fluorescent CNC at the premigratory stage into non-injected host embryos. This altered not only migration, but also the expression of neural crest markers. Migration of transplanted cranial neural crest cells was blocked when full-length Xcad-11 or its mutant lacking the beta-catenin-binding site (deltacXcad-11) was overexpressed. In addition, the expression of neural crest markers (AP-2, Snail and twist) diminished within the first four hours after grafting, and disappeared completely after 18 hours. Instead, these grafts expressed neural markers (2G9, nrp-I and N-Tubulin). Beta-catenin co-expression, heterotopic transplantation of CNC cells into the pharyngeal pouch area or both in combination failed to prevent neural differentiation of the grafts. By contrast, deltaeXcad-11 overexpression resulted in premature emigration of cells from the transplants. The AP-2 and Snail patterns remained unaffected in these migrating grafts, while twist expression was strongly reduced. Co-expression of deltaeXcad-11 and beta-catenin was able to rescue the loss of twist expression, indicating that Wnt/beta-catenin signalling is required to maintain twist expression during migration. These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/beta-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.     

Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis

Neural crest cells are multipotential stem cells that contribute extensively to vertebrate development and give rise to various cell and tissue types. Determination of the fate of mammalian neural crest has been inhibited by the lack of appropriate markers. Here, we make use of a two-component genetic system for indelibly marking the progeny of the cranial neural crest during tooth and mandible development. In the first mouse line, Cre recombinase is expressed under the control of the Wnt1 promoter as a transgene. Significantly, Wnt1 transgene expression is limited to the migrating neural crest cells that are derived from the dorsal CNS. The second mouse line, the ROSA26 conditional reporter (R26R), serves as a substrate for the Cre-mediated recombination. Using this two-component genetic system, we have systematically followed the migration and differentiation of the cranial neural crest (CNC) cells from E9.5 to 6 weeks after birth. Our results demonstrate, for the first time, that CNC cells contribute to the formation of condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel's cartilage, mandible, the articulating disc of temporomandibular joint and branchial arch nerve ganglia. More importantly, there is a dynamic distribution of CNC- and non-CNC-derived cells during tooth and mandibular morphogenesis. These results are a first step towards a comprehensive understanding of neural crest cell migration and differentiation during mammalian craniofacial development. Furthermore, this transgenic model also provides a new tool for cell lineage analysis and genetic manipulation of neural-crest-derived components in normal and abnormal embryogenesis.     

Mechanism of Xenopus cranial neural crest cell migration

This Review focuses on recent advances in the field of cranial neural crest cell migration in Xenopus laevis with specific emphasis on cell adhesion and the regulation of cell migration. Our goal is to combine the understanding of cell adhesion to the extracellular matrix with the regulation of cell-cell adhesion and the involvement of the planar cell polarity signaling-pathway in guiding the migration of cranial neural crest cells during embryogenesis.Key words: neural crest, cell migration, extracellular matrix, cell adhesion, Wnt, planar cell polarity     

Mechanism of Xenopus cranial neural crest cell migration

This review focuses on recent advances in the field of cranial neural crest cell migration in Xenopus laevis with specific emphasis on cell adhesion and the regulation of cell migration. Our goal is to combine the understanding of cell adhesion to the extracellular matrix with the regulation of cell-cell adhesion and the involvement of the planar cell polarity signaling-pathway in guiding the migration of cranial neural crest cells during embryogenesis.     

Xenopus Teashirt1 regulates posterior identity in brain and cranial neural crest

We have isolated two related Xenopus homologues of the homeotic zinc finger protein Teashirt1 (Tsh1), XTsh1a and XTsh1b. While Drosophila teashirt specifies trunk identity in the fly, the developmental relevance of vertebrate Tsh homologues is unknown. XTsh1a/b are expressed in prospective trunk CNS throughout early neurula stages and later in the migrating cranial neural crest (CNC) of the third arch. In postmigratory CNC, XTsh1a/b is uniformly activated in the posterior arches. Gain- and loss-of-function experiments reveal that reduction or increase of XTsh1 levels selectively inhibits specification of the hindbrain and mid/hindbrain boundary in Xenopus embryos. In addition, both overexpression and depletion of XTsh1 interfere with the determination of CNC segment identity. In transplantation assays, ectopic XTsh1a inhibits the routing of posterior, but not of mandibular CNC streams. The loss of function phenotype could be rescued with low amounts either of XTsh1a or murine Tsh3. Our results demonstrate that proper expression of XTsh1 is essential for segmentally restricted gene expression in the posterior brain and CNC and suggest for the first time that teashirt genes act as positional factors also in vertebrate development.     

Fate of cranial neural crest cells during craniofacial development in endothelin-A receptor-deficient mice

Most of the bone, cartilage and connective tissue of the lower jaw is derived from cranial neural crest cells (NCCs) arising from the posterior midbrain and hindbrain. Multiple factors direct the patterning of these NCCs, including endothelin-1-mediated endothelin A receptor (Edn1/Ednra) signaling. Loss of Ednra signaling results in multiple defects in lower jaw and neck structures, including homeotic transformation of lower jaw structures into upper jaw-like structures. However, since the Ednra gene is expressed by both migrating and post-migrating NCCs, the actual function of Ednra in cranial NCC development is not clear. Ednra signaling could be required for normal migration or guidance of NCCs to the pharyngeal arches or in subsequent events in post-migratory NCCs, including proliferation and survival. To address this question, we performed a fate analysis of cranial NCCs in Ednra-/- embryos using the R26R;Wnt1-Cre reporter system, in which Cre expression within NCCs results in permanent beta-galactosidase activity in NCCs and their derivatives. We find that loss of Ednra does not detectably alter either migration of most cranial NCCs into the mandibular first arch and second arch or their subsequent proliferation. However, mesenchymal cell apoptosis is increased two fold in both E9.5 and E10.5 Ednra-/- embryos, with apoptotic cells being present in and just proximal to the pharyngeal arches. Based on these studies, Ednra signaling appears to be required by most cranial NCCs after they reach the pharyngeal arches. However, a subset of NCCs appear to require Ednra signaling earlier, with loss of Ednra signaling likely leading to premature cessation of migration into or within the arches and subsequent cell death.     

Heterochronic shifts during early cranial neural crest cell migration in two ranid frogs

We describe the development of the cranial neural crest cell streams relative to embryonic events such as neural tube formation and somite appearance in two Eurasian frog species belonging to the Ranidae, Rana temporaria and Sylvirana nigrovittata, and demonstrate developmental heterochronies. The mandibular stream appeared well developed in R. temporaria at a time when the embryo was still spherical, the neural folds were elevated, and the neural plate was wide open, thus earlier than known from any frog species so far. The appearance of the second stream and its division into hyoid and branchial portions was clearly accelerated in R. temporaria relative to other embryonic events when compared to S. nigrovittata. For example, in R. temporaria, the hyoid and branchial portions of the cranial neural crest cell streams were separated before the neural folds had started to fuse, whereas in S. nigrovittata this event took place only after the neural folds had fused completely. Such ostentatious heterochronies related to the characters used herein have formerly only been reported from comparisons between species belonging to different higher taxa. Our results re‐confirm that to understand the full dynamics of the evolution of development, studies need to implement comparative embryological approaches, and include phylogenetically relatively closely related taxa.     

Excess lunatic fringe causes cranial neural crest over-proliferation.

Lunatic fringe is a vertebrate homologue of Drosophila fringe, which plays an important role in modulating Notch signaling. This study examines the distribution of chick lunatic fringe at sites of neural crest formation and explores its possible function by ectopic expression. Shortly after neural tube closure, lunatic fringe is expressed in most of the neural tube, with the exception of the dorsal midline containing presumptive neural crest. Thus, there is a fringe/non-fringe border at the site of neural crest production. Expression of excess lunatic fringe in the cranial neural tube and neural crest by retrovirally mediated gene transfer resulted in a significant increase ( approximately 60%) in the percentage of cranial neural crest cells 1 day after infection. This effect was mediated by an increase in cell division as assayed by BrdU incorporation. Infected embryos had an up-regulation of Delta-1 in the dorsal neural tube and redistribution of Notch-1 to the lumen of the neural tube, confirming that excess fringe modulates Notch signaling. These findings point to a novel role for lunatic fringe in regulating cell division and/or production of neural crest cells by the neural tube.     

Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.     

FGF2 promotes skeletogenic differentiation of cranial neural crest cells

The cranial neural crest gives rise to most of the skeletal tissues of the skull. Matrix-mediated tissue interactions have been implicated in the skeletogenic differentiation of crest cells, but little is known of the role that growth factors might play in this process. The discovery that mutations in fibroblast growth factor receptors (FGFRs) cause the major craniosynostosis syndromes implicates FGF-mediated signalling in the skeletogenic differentiation of the cranial neural crest. We now show that, in vitro, mesencephalic neural crest cells respond to exogenous FGF2 in a dose-dependent manner, with 0.1 and 1 ng/ml causing enhanced proliferation, and 10 ng/ml inducing cartilage differentiation. In longer-term cultures, both endochondral and membrane bone are formed. FGFR1, FGFR2 and FGFR3 are all detectable by immunohistochemistry in the mesencephalic region, with particularly intense expression at the apices of the neural folds from which the neural crest arises. FGFRs are also expressed by subpopulations of neural crest cells in culture. Collectively, these findings suggest that FGFs are involved in the skeletogenic differentiation of the cranial neural crest.     

Semaphorin signaling guides cranial neural crest cell migration in zebrafish

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.     

Roles of collagen and periostin expression by cranial neural crest cells during soft palate development

The tissue in the palatal region can be divided into the hard and the soft palates, each having a specialized function such as occlusion, speech, or swallowing. Therefore, an understanding of the mechanism of palatogenesis in relation to the function of each region is important. However, in comparison with the hard palate, there is still a lack of information about the mechanisms of soft palate development. In this study, the authors investigated the contribution of cranial neural crest (CNC) cells to development of both hard and soft palates. They also demonstrated a unique pattern of periostin expression during soft palate development, which was closely related to that of collagen type I (Col I) in palatine aponeurosis. Furthermore, organ culture analysis showed that exogenous transforming growth factor-β (TGF-β) induced the expression of both periostin and Col I. These novel patterns of expression in the extracellular matrix (ECM) induced by CNC cells suggest that these cells may help to determine the character of both the hard and soft palates through ECM induction. TGF-β signaling appears to be one of the mediators of Col I and periostin expression in the formation of functional structures during soft palate development.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.     

Isotretinoin embryopathy and the cranial neural crest: an in vivo and in vitro study

Severe congenital malformations have been associated with the inadvertant use in early pregnancy of a new dermatological drug, isotretinoin. We present proposals for the pathogenesis of this embryopathy based on the study of animal models. The characteristic malformations of the face, thymus, and great vessels were induced in mice by prenatal exposure to the drug during the early somite stages of development. From histological examination of mouse embryos it was shown that the drug directly interferes with the development of cranial neural crest cells. Subsequent deficiency of crest cell-derived mesenchyme adequately explains most of the observed malformations. Rat embryo culture studies showed that, when used at concentrations of 500 ng/ml, both isotretinoin and its main metabolite in the human, 4-oxo-isotretinoin, induce malformations similar to those seen in vivo. Since during normal repetitive dosing in the human the mean trough blood concentration of isotretinoin ranges from 132 to 196 ng/ml, while 4-oxo-isotretinoin ranges from 610 to 791 ng/ml, it is likely that the metabolite plays a major role in the induction of the isotretinoin embryopathy.