Cephalic Neural Crest Cells Research Articles

A Toolbox to Study Tissue Mechanics In Vivo and Ex Vivo.

During vertebrate embryogenesis, tissues interact and influence each other's development to shape an embryo. While communication by molecular components has been extensively explored, the role of mechanical interaction between tissues during embryogenesis is just starting to be revealed. Addressing mechanical involvement in morphogenesis has traditionally been challenging mainly due to the lack of proper tools to measure and modify mechanical environments of cells in vivo. We have recently used atomic force microscopy (AFM) to show that the migration of the Xenopus laevis cephalic neural crest cells is triggered by stiffening of the mesoderm, a tissue that neural crest cells use as a migratory substrate in vivo. Interestingly we showed that the activity of the planar cell polarity (PCP) pathway is required to mediate this novel mechanical interaction between two tissues. In this chapter, we share the toolbox that we developed to study the role of PCP signaling in mesoderm cell accumulation and stiffening (in vivo) as well as the impact of mesoderm stiffness in promoting neural crest cell polarity and migration (ex vivo). We believe that these tools can be of general use for investigators interested in addressing the role of mechanical inputs in vivo and ex vivo.

Cephalic/cardiac neural crest cell and moyamoya disease.

The neural crest is a transient structure present in early embryogenesis. Cephalic neural crest cells migrate into the pharyngeal arches and the frontonasal process that becomes the forehead and midfacial structures. They also contribute to forming the media of the arteries of the circle of Willis and their branches. The cardiac neural crest produces vascular smooth muscle cells in the ascending aorta, cardiac septum and coronary arteries. In this review, we evaluate the role of the neural crest in moyamoya disease and the pathological implications from the concurrence of moyamoya disease and cardiovascular diseases from the point of view of neural crest cell distributions. Midline craniofacial and central nervous system anomalies with eye anomalies, morning glory disc anomaly in patients with moyamoya disease can both be explained as a subtype of cephalic neurocristopathy. Further, the association between moyamoya disease and cardiac manifestations (congenital cardiac defects and coronary artery disease) have also been reported. Both the cephalic neural crest and cardiac neural crest contribute to these concurrent arterial diseases, as cardio-cephalic neurocristopathy. The concept of cephalic/cardio-cephalic neurocristopathy provides a new perspective to understanding the underlying aetiological associations and to developing future therapeutic approaches for concomitant moyamoya disease and cardiovascular diseases.

SNED1: a Novel ECM Protein Regulating Neural Crest Cells Phenotype and Craniofacial Morphogenesis

The extracellular matrix (ECM) is a complex meshwork of proteins that provides structural and biochemical support to cells. We have recently reported that the homozygous ablation of Sned1, which encodes the novel ECM protein SNED1, caused neonatal lethality, and the few knockout (KO) survivors we obtained exhibited craniofacial malformations including shorter snouts and wider neurocrania (Figure 1). Since cranial neural crest cells (NCCs) form the facial skeleton and given the importance of the ECM in guiding NCC phenotype, we hypothesized that SNED1 influences the behavior of this cell population. To test this, we generated an NCC-specific Sned1 KO mouse. While Sned1NCC-/NCC- mice survive, they present craniofacial anomalies resembling the malformations observed in the global Sned1 KOmice (Barqué et al., Dev Dyn, 2020). In order to form craniofacial structures, cephalic NCCs first need to delaminate from the neural tube, then migrate towards the frontonasal region and the pharyngeal arches, and there differentiate into bone and cartilage. Our goal is now to determine precisely which cellular processes along the NCC path are directly controlled by SNED1. To assess this, we are first performing in-vivo assays including in-situ hybridization and immunohistochemical staining on Sned1NCC-/NCC- and control mouse embryos at different embryonic timepoints to assess NCC behaviors. In parallel, we are conducting in-vitro assays using neural tube explants excised from Sned1NCC-/NCC- and control mouse embryos and recapitulating the key steps NCCs take towards the formation of craniofacial structures. These steps include migration, proliferation, and chondrogenic and osteogenic differentiation and can be monitored by tracking the expression of specific molecular markers. Once we identify which cellular processes are controlled by SNED1, we will turn our focus to the identification of the molecular mechanisms by which SNED1 governs NCC phenotype and craniofacial morphogenesis. Sequence analysis of SNED1 revealed two consensus binding sites for integrins, which are known ECM receptors. Thus, we have developed SNED1 constructs containing mutations in the putative integrin-binding sites that we will use to elucidate whether integrins serve as SNED1 receptors. Importantly, deletion of 2q37.3, a region that comprises the SNED1 locus, has been linked to facial dysmorphism in patients. We thus propose that SNED1 contributes to the phenotype of these patients and that our novel mouse model may shed light on some of the features of this syndrome. Moreover, we previously reported SNED1 as a promoter of breast cancer metastasis (Naba et al., eLife, 2014). Since metastatic cells share common features with NCC, our studies will also allow us to better understand the role of SNED1 in metastasis.

Lead exposure affects cephalic morphogenesis and neural crest cells in Gallus gallus embryo

The morphogenesis of the head of vertebrates is a process that involves rapid growth and dynamic movements of various cell populations, including the neural crest cells (NCC). These pluripotent cells generated during neurulation have high proliferative and migratory capacity but xenobiotic agents can affect these migratory periods and cause congenital malformations. Lead (Pb) is the most common toxic metal in the environment and a potent teratogen that can affect growth and induce malformations. Despite the known toxic effects of Pb, there is a gap in knowledge about the impact of realistic concentrations of Pb at critical periods of early development. Here, we evaluated mortality, embryonic morphology, NCC migration, and the amount of Pb deposition in chicken embryos after 3 to 4 days of exposure. One of the most interesting observations in this study is that only about 34% of the injected Pb was present in the embryos after 4 days. We observed that exposure to Pb, even under low concentrations, increased mortality and the occurrence of malformations during embryonic development, especially in the cephalic region (CR). Although Pb was found widely distributed in the CR, no relation between its presence and the migration routes of cephalic NCC was observed. But the number of NCC and their migratory distance were reduced. These changes are consistent and explain the morphological anomalies described in this study, which also correlates with the morphofunctional abnormalities reported in the literature. Therefore, this study highlights the concern of exposure to low concentrations of this metal.

In vivo Neural Crest Cell Migration Is Controlled by "Mixotaxis".

Directed cell migration is essential all along an individual’s life, from embryogenesis to tissue repair and cancer metastasis. Thus, due to its biomedical relevance, directed cell migration is currently under intense research. Directed cell migration has been shown to be driven by an assortment of external biasing cues, ranging from gradients of soluble (chemotaxis) to bound (haptotaxis) molecules. In addition to molecular gradients, gradients of mechanical properties (duro/mechanotaxis), electric fields (electro/galvanotaxis) as well as iterative biases in the environment topology (ratchetaxis) have been shown to be able to direct cell migration. Since cells migrating in vivo are exposed to a challenging environment composed of a convolution of biochemical, biophysical, and topological cues, it is highly unlikely that cell migration would be guided by an individual type of “taxis.” This is especially true since numerous molecular players involved in the cellular response to these biasing cues are often recycled, serving as sensor or transducer of both biochemical and biophysical signals. In this review, we confront literature on Xenopus cephalic neural crest cells with that of other cell types to discuss the relevance of the current categorization of cell guidance strategies. Furthermore, we emphasize that while studying individual biasing signals is informative, the hard truth is that cells migrate by performing a sort of “mixotaxis,” where they integrate and coordinate multiple inputs through shared molecular effectors to ensure robustness of directed cell motion.

Metalloprotease-Dependent Attenuation of BMP Signaling Restricts Cardiac Neural Crest Cell Fate

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. Invitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

Effects of Folic Acid and Homocysteine on the Morphogenesis of Mouse Cephalic Neural Crest Cells In Vitro.

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies-a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exactmechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.

Effects of 13 developmentally toxic chemicals on the migration of rat cephalic neural crest cells in vitro.

The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13-cis-retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.

Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure

Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for potential disorders of this guiding mechanism after ethanol exposure.In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients and to notochord co-cultures (Shh source) or to their conditioned medium, a response inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino (MO). Expression of the Ptch–Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1Δloop2) impaired cephalic NCC migration/distribution, with fewer cells invading the optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs.Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen.The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.

Simplein vitromigration assay for neural crest cells and the opposite effects of all‐trans‐retinoic acid on cephalic‐ and trunk‐derived cells

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all-trans-retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.

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.

Combinatorial activity of Six1-2-4 genes in cephalic neural crest cells controls craniofacial and brain development.

The combinatorial expression of Hox genes is an evolutionarily ancient program underlying body axis patterning in all Bilateria. In the head, the neural crest (NC)--a vertebrate innovation that contributes to evolutionarily novel skeletal and neural features--develops as a structure free of Hox-gene expression. The activation of Hoxa2 in the Hox-free facial NC (FNC) leads to severe craniofacial and brain defects. Here, we show that this condition unveils the requirement of three Six genes, Six1, Six2, and Six4, for brain development and morphogenesis of the maxillo-mandibular and nasofrontal skeleton. Inactivation of each of these Six genes in FNC generates diverse brain defects, ranging from plexus agenesis to mild or severe holoprosencephaly, and entails facial hypoplasia or truncation of the craniofacial skeleton. The triple silencing of these genes reveals their complementary role in face and brain morphogenesis. Furthermore, we show that the perturbation of the intrinsic genetic FNC program, by either Hoxa2 expression or Six gene inactivation, affects Bmp signaling through the downregulation of Bmp antagonists in the FNC cells. When upregulated in the FNC, Bmp antagonists suppress the adverse skeletal and cerebral effects of Hoxa2 expression. These results demonstrate that the combinatorial expression of Six1, Six2, and Six4 is required for the molecular programs governing craniofacial and cerebral development. These genes are crucial for the signaling system of FNC origin, which regulates normal growth and patterning of the cephalic neuroepithelium. Our results strongly suggest that several congenital craniofacial and cerebral malformations could be attributed to Six genes' misregulation.

Neurotrophic factor NT-3 displays a non-canonical cell guidance signaling function for cephalic neural crest cells

Chemotactic cell migration is triggered by extracellular concentration gradients of molecules segregated by target fields. Neural crest cells (NCCs), paradigmatic as an accurately moving cell population, undergo wide dispersion along multiple pathways, invading with precision defined sites of the embryo to differentiate into many derivatives. This report addresses the involvement of NT-3 in early colonization by cephalic NCCs invading the optic vesicle region. The results of in vitro and in vivo approaches showed that NCCs migrate directionally up an NT-3 concentration gradient. We also demonstrated the expression of NT-3 in the ocular region as well as their functional TrkB, TrkC and p75 receptors on cephalic NCCs. On whole-mount embryo, a perturbed distribution of NCCs colonizing the optic vesicle target field was shown after morpholino cancelation of cephalic NT-3 or TrkC receptor on NCCs, as well as in situ blocking of TrkC receptor of mesencephalic NCCs by specific antibody released from inserted microbeads. The present results strongly suggest that, among other complementary cell guidance factor(s), the chemotactic response of NCCs toward the ocular region NT-3 gradient is essential for spatiotemporal cell orientation, amplifying the functional scope of this neurotrophic factor as a molecular guide for the embryo cells, besides its well-known canonical functions.

Protogenin prevents premature apoptosis of rostral cephalic neural crest cells by activating the α5β1-integrin

The bones and connective tissues of the murine jaws and skull are partly derived from cephalic neural crest cells (CNCCs). Here, we report that mice deficient of protogenin (Prtg) protein, an immunoglobulin domain-containing receptor expressed in the developing nervous system, have impairments of the palatine and skull. Data from lineage tracing experiments, expression patterns of neural crest cell (NCC) marker genes and detection of apoptotic cells indicate that the malformation of bones in Prtg-deficient mice is due to increased apoptosis of rostral CNCCs (R-CNCCs). Using a yeast two-hybrid screening, we found that Prtg interacts with Radil, a protein previously shown to affect the migration and survival of NCCs in zebrafish with unknown mechanism. Overexpression of Prtg induces translocation of Radil from cytoplasm to cell membrane in cultured AD293 cells. In addition, overexpression of Prtg and Radil activates α5β1-integrins to high-affinity conformational forms, which is further enhanced by the addition of Prtg ligand ERdj3 into cultured cells. Blockage of Radil by RNA interference abolishes the effect of ERdj3 and Prtg on the α5β1-integrin, suggesting that Radil acts downstream of Prtg. Prtg-deficient R-CNCCs display fewer activated α5β1-integrins in embryos, and these cells show reduced migratory ability in in vitro transwell assay. These results suggest that the inside-out activation of the α5β1-integrin mediated by ERdj3/Prtg/Radil signaling is crucial for proper functions of R-CNCCs, and the deficiency of this pathway causes premature apoptosis of a subset of R-CNCCs and malformation of craniofacial structures.

Control of endothelin-a receptor expression by progesterone is enhanced by synergy with Gata2.

The endothelin-A receptor (Ednra) is involved in several physiological, pathological, and developmental pathways. Known for its function in vasoconstriction after being activated by endothelin-1, Ednra also controls cephalic neural crest cell development and appears to play a role in several pathologies, including cancer and periodontitis. However, the mechanisms regulating Ednra expression have not been identified despite its important functions. In this study, we investigated the role progesterone plays in Ednra gene expression in vivo and in vitro. In mice, pregnancy promotes Ednra expression in the heart, kidney, lung, uterus, and placenta, and the up-regulation is mediated by progesterone. We determined that the conserved region between -5.7 and -4.2 kb upstream of the mouse Ednra gene is necessary for the progesterone response. We also found that progesterone mediates Ednra activation through progesterone receptor B activation by its recruitment to PRE6, one of the 6 progesterone response elements found in that locus. However, gene activation by means of a GATA2 site was also necessary for the progesterone response. The Gata2 transcription factor enhances the progesterone response mediated by the progesterone receptor B. Together these results indicate that progesterone regulates Ednra expression by synergizing with Gata2 activity, a previously unknown mechanism. This mechanism may have an impact on pathologies involving the endothelin signaling.

Dbx1-Expressing Cells Are Necessary for the Survival of the Mammalian Anterior Neural and Craniofacial Structures

Development of the vertebrate forebrain and craniofacial structures are intimately linked processes, the coordinated growth of these tissues being required to ensure normal head formation. In this study, we identify five small subsets of progenitors expressing the transcription factor dbx1 in the cephalic region of developing mouse embryos at E8.5. Using genetic tracing we show that dbx1-expressing cells and their progeny have a modest contribution to the forebrain and face tissues. However, their genetic ablation triggers extensive and non cell-autonomous apoptosis as well as a decrease in proliferation in surrounding tissues, resulting in the progressive loss of most of the forebrain and frontonasal structures. Targeted ablation of the different subsets reveals that the very first dbx1-expressing progenitors are critically required for the survival of anterior neural tissues, the production and/or migration of cephalic neural crest cells and, ultimately, forebrain formation. In addition, we find that the other subsets, generated at slightly later stages, each play a specific function during head development and that their coordinated activity is required for accurate craniofacial morphogenesis. Our results demonstrate that dbx1-expressing cells have a unique function during head development, notably by controlling cell survival in a non cell-autonomous manner.

Trophic and proliferative perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are prevented by Neurotrophin 3

Neural crest cells (NCCs), a transient population that migrates from the developing neural tube, distributes through the embryo and differentiates into many derivatives, are clearly involved in the damage induced by prenatal exposure to ethanol. The aim of this work was to evaluate alterations of trophic parameters of in vivo (in ovo) and in vitro NCCs exposed to teratogenic ethanol doses, and their possible prevention by trophic factor treatment. Chick embryos of 24–30 h of incubation were treated during 10 h with 100 mM ethanol, or 40 ng/ml Neurotrophin 3 (NT3), or 10 ng/ml Ciliary Neurotrophic Factor (CNTF), or ethanol plus NT3 or CNTF, or defined medium; then the topographic distribution of NCC apoptosis was assessed using a whole-mount acridine orange supravital method. Cultures of cephalic NCCs were exposed to the same ethanol or NT3, or CNTF treatments, or ethanol plus one of both trophic factors, or N2 medium. A viability assay was performed using the calcein-ethidium test, apoptosis was evaluated with the TUNEL test, and proliferative capacity after BrdU labeling. After direct exposure of embryos to 100 mM ethanol for 10 h, a high level of NCC apoptosis was coincident with the abnormal closure of the neural tube. These anomalies were prevented in embryos exposed to ethanol plus NT3 but not with CNTF. In NCC cultures, high cell mortality and a diminution of proliferative activity were observed after 3 h of ethanol treatment. Incubation with ethanol plus NT3 (but not with CNTF) prevented NCC mortality as well as a fall in NCC proliferation. The consequences of direct exposure to ethanol expand data from our and other laboratories, supporting current opinion on the potential risk of alcohol ingestion (even at low doses and/or during a short time), in any period of pregnancy or lactation. Our in vivo/in vitro model encourages us to examine the pathogenic mechanism(s) of the ethanol-exposed embryo as well as the use of trophic factors for the treatment and/or prevention of anomalies induced by prenatal alcohol.

Dlx5 and Dlx6 expression in the anterior neural fold is essential for patterning the dorsal nasal capsule

Morphogenesis of the vertebrate facial skeleton depends upon inductive interactions between cephalic neural crest cells (CNCCs) and cephalic epithelia. The nasal capsule is a CNCC-derived cartilaginous structure comprising a ventral midline bar (mesethmoid) overlaid by a dorsal capsule (ectethmoid). Although Shh signalling from the anterior-most region of the endoderm (EZ-I) patterns the mesethmoid, the cues involved in ectethmoid induction are still undefined. Here, we show that ectethmoid formation depends upon Dlx5 and Dlx6 expression in a restricted ectodermal territory of the anterior neural folds, which we name NF-ZA. In both chick and mouse neurulas, Dlx5 and Dlx6 expression is mostly restricted to NF-ZA. Simultaneous Dlx5 and Dlx6 inactivation in the mouse precludes ectethmoid formation, while the mesethmoid is still present. Consistently, siRNA-mediated downregulation of Dlx5 and Dlx6 in the cephalic region of the early avian neurula specifically prevents ectethmoid formation, whereas other CNCC-derived structures, including the mesethmoid, are not affected. Similarly, NF-ZA surgical removal in chick neurulas averts ectethmoid development, whereas grafting a supernumerary NF-ZA results in an ectopic ectethmoid. Simultaneous ablation or grafting of both NF-ZA and EZ-I result, respectively, in the absence or duplication of both dorsal and ventral nasal capsule components. The present work shows that early ectodermal and endodermal signals instruct different contingents of CNCCs to form the ectethmoid and the mesethmoid, which then assemble to form a complete nasal capsule.

Collective cell migration of the cephalic neural crest: The art of integrating information

The cephalic neural crest (NC) cells delaminate from the neuroepithelium in large numbers and undergo collective cell migration under the influence of multiple factors including positive and negative taxis, cell-cell interactions mediating cell sorting, cell cooperation, and Contact-Inhibition of Locomotion. The migration has to be tightly regulated to allow NC cells to reach precise locations in order to contribute to various craniofacial structures such as the skeletal and peripheral nervous systems. Several birth defects, syndromes, and malformations are due to improper cephalic NC (CNC) migration, and NC cell migration bears important similarities to cancer cell invasion and metastasis dissemination. Therefore, understanding how CNC cells interpret multiple inputs to achieve directional collective cell migration will shed light on pathological situations where cell migration is involved.