Cranial Neural Crest Migration Research Articles

ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing.

Cranial neural crest (CNC) cells are key stem cells that contribute to most of the facial structures in all vertebrates. Previous work has shown that multiple ADAM (A Disintegrin And Metalloprotease) cell surface metalloproteases are essential for the induction and migration of the CNC in multiple vertebrate animal models. In Xenopus, we have shown that Adam13 associates with the transcription factor Arid3a to regulate gene expression. Here we show that Adam13 regulates gene expression by modulating Histone modifications globally in the CNC. Furthermore, we show that Arid3a binding to the tfap2a; promoter depends on the presence of Adam13. This association promotes the expression of one tfap2a; variant predominantly expressed in the CNC that uniquely activates the expression of calpain-8, a gene critical for CNC migration. This tfap2a; variant selectively associates with proteins involved with mRNA splicing, a function that is critically affected by the loss of Adam13 in the CNC. Our results suggest that ADAM metalloproteases may act as sensors of the environment that can modulate chromatin availability, leading to changes in gene expression and splicing.

ADAM11 a novel regulator of Wnt and BMP4 signaling in neural crest and cancer.

Introduction: Cranial neural crest (CNC) cells are induced at the border of the neural plate by a combination of FGF, Wnt, and BMP4 signaling. CNC then migrate ventrally and invade ventral structures where they contribute to craniofacial development. Methods: We used loss and gain of function experiments to determine phenotypes associated with the perturbation of Adam11 expression in Xenopus Laevis. Mass spectrometry to identify partners of Adam11 and changes in protein expression in CNC lacking Adam11. We used mouse B16 melanoma to test the function of Adam11 in cancer cells, and published database analysis to study the expression of ADAM11 in human tumors. Results: Here we show that a non-proteolytic ADAM, Adam11, originally identified as a putative tumor suppressor binds to proteins of the Wnt and BMP4 signaling pathway. Mechanistic studies concerning these non-proteolytic ADAM lack almost entirely. We show that Adam11 positively regulates BMP4 signaling while negatively regulating β-catenin activity. In vivo, we show that Adam11 influences the timing of neural tube closure and the proliferation and migration of CNC. Using both human tumor data and mouse B16 melanoma cells, we further show that ADAM11 levels similarly correlate with Wnt or BMP4 activation levels. Discussion: We propose that ADAM11 preserves naïve cells by maintaining low Sox3 and Snail/Slug levels through stimulation of BMP4 and repression of Wnt signaling, while loss of ADAM11 results in increased Wnt signaling, increased proliferation and early epithelium to mesenchyme transition.

The Tumor Suppressor Adenomatous Polyposis Coli (apc) Is Required for Neural Crest-Dependent Craniofacial Development in Zebrafish.

Neural crest (NC) is a unique vertebrate cell type arising from the border of the neural plate and epidermis that gives rise to diverse tissues along the entire body axis. Roberto Mayor and colleagues have made major contributions to our understanding of NC induction, delamination, and migration. We report that a truncating mutation of the classical tumor suppressor Adenomatous Polyposis Coli (apc) disrupts craniofacial development in zebrafish larvae, with a marked reduction in the cranial neural crest (CNC) cells that contribute to mandibular and hyoid pharyngeal arches. While the mechanism is not yet clear, the altered expression of signaling molecules that guide CNC migration could underlie this phenotype. For example, apcmcr/mcr larvae express substantially higher levels of complement c3, which Mayor and colleagues showed impairs CNC cell migration when overexpressed. However, we also observe reduction in stroma-derived factor 1 (sdf1/cxcl12), which is required for CNC migration into the head. Consistent with our previous work showing that APC directly enhances the activity of glycogen synthase kinase 3 (GSK-3) and, independently, that GSK-3 phosphorylates multiple core mRNA splicing factors, we identify 340 mRNA splicing variations in apc mutant zebrafish, including a splice variant that deletes a conserved domain in semaphorin 3f (sema3f), an axonal guidance molecule and a known regulator of CNC migration. Here, we discuss potential roles for apc in CNC development in the context of some of the seminal findings of Mayor and colleagues.

The insufficiency of Dlx5 for ventral patterning in post-migratory neural crest cells reveals a loss of plasticity in early jaw-forming tissue

The articulated jaws of vertebrates arise from the first pharyngeal arch, the most rostral of several transient ventral structures in pharyngeal stage embryos. Migratory cranial neural crest cells from the caudal midbrain and rostral hindbrain populate the first arch as ectomesenchyme and supply the progenitors of skeletal and soft tissues that form the upper (maxillary) and lower (mandibular) jaws. Dlx genes encode key transcriptional regulators that profoundly influence jaw development through their actions in first pharyngeal arch patterning. The broadly conserved nested expression of Dlx paralogues in vertebrate embryos points to a retained ancestral role in patterning first arch tissue. Loss-of-function experiments consistently highlight the necessity of Dlx gene function for jaw morphogenesis. Specifically, the combined effects of Dlx5 and Dlx6 are required to specify ventral/mandibular fate and forced expression of Dlx5 in migrating neural crest cells results in the ectopic upregulation of ventral markers in the maxillary arch. Here, we ask whether Dlx5 is also sufficient to respecify post-migratory ectomesenchyme in the maxillary branch as mandibular. Unexpectedly, we show that Dlx5 is not sufficient to activate mandibular marker genes in the maxillary branch of PA1, highlighting a loss of plasticity in post-migratory first arch ectomesenchyme.

Mcrs1 is required for branchial arch and cranial cartilage development

Mcrs1 is a multifunctional protein that is critical for many cellular processes in a wide range of cell types. Previously, we showed that Mcrs1 binds to the Six1 transcription factor and reduces the ability of the Six1-Eya1 complex to upregulate transcription, and that Mcrs1 loss-of-function leads to the expansion of several neural plate genes, reduction of neural border and pre-placodal ectoderm (PPR) genes, and pleiotropic effects on various neural crest (NC) genes. Because the affected embryonic structures give rise to several of the cranial tissues affected in Branchio-otic/Branchio-oto-renal (BOR) syndrome, herein we tested whether these gene expression changes subsequently alter the development of the proximate precursors of BOR affected structures - the otic vesicles (OV) and branchial arches (BA). We found that Mcrs1 is required for the expression of several OV genes involved in inner ear formation, patterning and otic capsule cartilage formation. Mcrs1 knockdown also reduced the expression domains of many genes expressed in the larval BA, derived from either NC or PPR, except for emx2, which was expanded. Reduced Mcrs1 also diminished the length of the expression domain of tbx1 in BA1 and BA2 and interfered with cranial NC migration from the dorsal neural tube; this subsequently resulted in defects in the morphology of lower jaw cartilages derived from BA1 and BA2, including the infrarostral, Meckel's, and ceratohyal as well as the otic capsule. These results demonstrate that Mcrs1 plays an important role in processes that lead to the formation of craniofacial cartilages and its loss results in phenotypes consistent with reduced Six1 activity associated with BOR.

TFAP2 Paralogs Regulate Alx Genes During Midface Cranial Neural Crest Development

Craniofacial anomalies are the 2nd leading structural malformation present at birth. Those in the midface, such as orofacial clefts and hypertelorism, can arise from mutations in the Transcription Factor Activating Protein 2 genes TFAP2A and TFAP2B (abbreviated as TFAP2). These mutations do not reduce their gene expression, but instead generate mutant proteins that disrupt the activity of other wild‐type and functionally redundant paralogs. Previous studies showed that both paralogs are co‐expressed in the embryonic midface, hinting at their cooperative role during face development. Yet, how TFAP2 regulate midface development at the cellular and molecular level is poorly understood. To this end, we leveraged CRE‐based approaches in mice to delete both Tfap2 members in the neural crest (NC), a cell lineage that gives rise to craniofacial bone and cartilage. We show that NC‐specific knockout of Tfap2 (Tfap2NCKO) results in a fully penetrant midface cleft compared to controls and single paralog mutants. Lineage tracing analyses in Tfap2NCKO embryos suggest that cranial NC migration into the embryonic face is not severely impaired. Corroborating this result, we find that Tfap2 deletion in either pre‐migratory or late‐migratory NC cells still results in a midface cleft. This suggests this phenotype stems from disrupted TFAP2 activity in post‐migratory NC cells. Moreover, when examining Tfap2NCKO craniofacial skeletons, we observe severe bone malformations and ectopic structures in the frontonasal regions when compared to controls. Single‐cell RNA sequencing analysis reveals that loss of Tfap2 results in reduced cranial NC cell numbers and dysregulated Alx1/3/4 (Alx) gene expression. This is supported by the literature, as the Tfap2NCKO midface clefts phenocopy those with loss of various Alxgene combinations. Our ongoing efforts aim to test how TFAP2 regulates Alx genes, if loss of Tfap2 compromises midface NC survival or proliferation, and whether Tfap2 and Alx genetically interact. In sum, we propose the model that TFAP2 regulates Alx gene expression in NC cells during midface development.

Selective disruption of trigeminal sensory neurogenesis and differentiation in a mouse model of 22q11.2 deletion syndrome.

ABSTRACT22q11.2 Deletion Syndrome (22q11DS) is a neurodevelopmental disorder associated with cranial nerve anomalies and disordered oropharyngeal function, including pediatric dysphagia. Using the LgDel 22q11DS mouse model, we investigated whether sensory neuron differentiation in the trigeminal ganglion (CNgV), which is essential for normal orofacial function, is disrupted. We did not detect changes in cranial placode cell translocation or neural crest migration at early stages of LgDel CNgV development. However, as the ganglion coalesces, proportions of placode-derived LgDel CNgV cells increase relative to neural crest cells. In addition, local aggregation of placode-derived cells increases and aggregation of neural crest-derived cells decreases in LgDel CNgV. This change in cell-cell relationships was accompanied by altered proliferation of placode-derived cells at embryonic day (E)9.5, and premature neurogenesis from neural crest-derived precursors, reflected by an increased frequency of asymmetric neurogenic divisions for neural crest-derived precursors by E10.5. These early differences in LgDel CNgV genesis prefigure changes in sensory neuron differentiation and gene expression by postnatal day 8, when early signs of cranial nerve dysfunction associated with pediatric dysphagia are observed in LgDel mice. Apparently, 22q11 deletion destabilizes CNgV sensory neuron genesis and differentiation by increasing variability in cell-cell interaction, proliferation and sensory neuron differentiation. This early developmental divergence and its consequences may contribute to oropharyngeal dysfunction, including suckling, feeding and swallowing disruptions at birth, and additional orofacial sensory/motor deficits throughout life.

MAPRE2 mutations result in altered human cranial neural crest migration, underlying craniofacial malformations in CSC-KT syndrome

Circumferential skin creases (CSC-KT) is a rare polymalformative syndrome characterised by intellectual disability associated with skin creases on the limbs, and very characteristic craniofacial malformations. Previously, heterozygous and homozygous mutations in MAPRE2 were found to be causal for this disease. MAPRE2 encodes for a member of evolutionary conserved microtubule plus end tracking proteins, the end binding (EB) family. Unlike MAPRE1 and MAPRE3, MAPRE2 is not required for the persistent growth and stabilization of microtubules, but plays a role in other cellular processes such as mitotic progression and regulation of cell adhesion. The mutations identified in MAPRE2 all reside within the calponin homology domain, responsible to track and interact with the plus-end tip of growing microtubules, and previous data showed that altered dosage of MAPRE2 resulted in abnormal branchial arch patterning in zebrafish. In this study, we developed patient derived induced pluripotent stem cell lines for MAPRE2, together with isogenic controls, using CRISPR/Cas9 technology, and differentiated them towards neural crest cells with cranial identity. We show that changes in MAPRE2 lead to alterations in neural crest migration in vitro but also in vivo, following xenotransplantation of neural crest progenitors into developing chicken embryos. In addition, we provide evidence that changes in focal adhesion might underlie the altered cell motility of the MAPRE2 mutant cranial neural crest cells. Our data provide evidence that MAPRE2 is involved in cellular migration of cranial neural crest and offers critical insights into the mechanism underlying the craniofacial dysmorphisms and cleft palate present in CSC-KT patients. This adds the CSC-KT disorder to the growing list of neurocristopathies.

Transcriptomic Identification of Draxin-Responsive Targets During Cranial Neural Crest EMT.

Canonical Wnt signaling plays an essential role in proper craniofacial morphogenesis, at least partially due to regulation of various aspects of cranial neural crest development. In an effort to gain insight into the etiology of craniofacial abnormalities resulting from Wnt signaling and/or cranial neural crest dysfunction, we sought to identify Wnt-responsive targets during chick cranial neural crest development. To this end, we leveraged overexpression of a canonical Wnt antagonist, Draxin, in conjunction with RNA-sequencing of cranial neural crest cells that have just activated their epithelial–mesenchymal transition (EMT) program. Through differential expression analysis, gene list functional annotation, hybridization chain reaction (HCR), and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we validated a novel downstream target of canonical Wnt signaling in cranial neural crest – RHOB – and identified possible signaling pathway crosstalk underlying cranial neural crest migration. The results reveal novel putative targets of canonical Wnt signaling during cranial neural crest EMT and highlight important intersections across signaling pathways involved in craniofacial development.

Cranial Neural Crest Cells and Their Role in the Pathogenesis of Craniofacial Anomalies and Coronal Craniosynostosis.

Craniofacial anomalies are among the most common of birth defects. The pathogenesis of craniofacial anomalies frequently involves defects in the migration, proliferation, and fate of neural crest cells destined for the craniofacial skeleton. Genetic mutations causing deficient cranial neural crest migration and proliferation can result in Treacher Collins syndrome, Pierre Robin sequence, and cleft palate. Defects in post-migratory neural crest cells can result in pre- or post-ossification defects in the developing craniofacial skeleton and craniosynostosis (premature fusion of cranial bones/cranial sutures). The coronal suture is the most frequently fused suture in craniosynostosis syndromes. It exists as a biological boundary between the neural crest-derived frontal bone and paraxial mesoderm-derived parietal bone. The objective of this review is to frame our current understanding of neural crest cells in craniofacial development, craniofacial anomalies, and the pathogenesis of coronal craniosynostosis. We will also discuss novel approaches for advancing our knowledge and developing prevention and/or treatment strategies for craniofacial tissue regeneration and craniosynostosis.

Characterizing the Craniofacial Morphological Changes in Neural Crest Cell Specific Conditional Connexin‐43 Knock‐Out Mice

The craniofacial skeleton is an intricate structure that forms through complex cell‐cell interactions across neural crest and mesoderm derived cell populations. Proper cell‐cell communication during the migration and differentiation of cranial neural crest cells is critical for normal craniofacial development and this intercellular communication is thought to be facilitated through gap junctions – membrane channels composed of connexin proteins. In particular, connexin‐43 (Cx43) has been shown to be expressed in pre‐migratory and migratory cranial neural crest cells and plays a role in neural crest epithelial‐to‐mesenchyme transition. Additionally, within the skull, neural crest‐derived regions show the greatest morphological changes in mice wherein Cx43 has been globally ablated providing further evidence that Cx43 is important for proper neural crest function and ultimately proper development of the skull. To date, there have been no studies that have directly looked at the role of Cx43 in cranial neural crest cells in a mouse model. Thus, we have developed a neural crest‐specific Cx43 knockout mouse to address this gap in knowledge. In this study we (1) verify that Cx43 ablation is limited to neural crest cell and (2) characterize and compare craniofacial size and shape among wildtype, heterozygous and homozygous knockout mice.Heterozygous Wnt1‐Cre2 recombinase mice were bred with homozygous floxed Cx43 mice to create offspring which were bred to express the genotype, Wnt1‐Cre2/+:Cx43fl/+. These mice were bred to create: control mice (Wnt‐1Cre2/+:Cx43+/+), heterozygous conditional knock‐outs (Wnt1‐Cre2/+:Cx43fl/fl) and homozygous conditional knock‐outs (Wnt1‐Cre2+/+:Cx43fl/fl). To verify the knockout, paraffin embedded sagittal sections of embryonic and post‐natal mouse hearts and skulls were tagged with Wnt1 and Cx43 antibodies. RT‐PCR, and Western blotting were performed on neural crest and mesoderm derived newborn skull bones, to verify the expression levels of Cx43 mRNA and protein respectively. Newborn skulls from each genotype were imaged using high resolution micro‐CT and these images were used to quantify and compare skull morphology among genotypes using statistical analyses of shape.We have confirmed that Cx43 ablation is limited to neural crest cells in our mice. Analyses of skull shape comparisons among genotypes is ongoing with our initial findings indicating that mice homozygous for the knockout demonstrate overt changes to skull morphology, heterozygous mouse skulls are qualitatively similar to control mouse skulls. We predict that statistical analyses will uncover that only the bones derived from neural crest cells will display morphological differences among genotypes.Our study addresses a fundamental gap of the role connexin‐43 plays in cranial neural crest function and subsequent development of much of the skull.Support or Funding InformationFunding: Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (R5211A02)

The gap junction protein connexin 43 controls multiple aspects of cranial neural crest cell development.

Gap junctions are intercellular channels between cells that facilitate cell-cell communication. Connexin 43 (Cx43; also known as GJA1), the predominant gap junction protein in vertebrates, is expressed in premigratory cranial neural crest cells and is maintained throughout the neural crest cell epithelial-to-mesenchymal transition (EMT), but its function in these cells is unknown. To this end, we used a combination of in vivo and ex vivo experiments to assess gap junction formation, and Cx43 function, in chick cranial neural crest cells. Our results demonstrate that gap junctions exist between premigratory and migratory cranial neural crest cells and depend on Cx43 for their function. In the embryo, Cx43 knockdown just prior to EMT delays the emergence of Cx43-depleted neural crest cells from the neural tube, but these cells eventually successfully emigrate and join the migratory stream. This delay can be rescued by introduction of full-length Cx43 into Cx43-depleted cells. Furthermore, Cx43 depletion reduces the size of the premigratory neural crest cell domain through an early effect on neural crest cell specification. Collectively, these data identify new roles for Cx43 in chick cranial neural crest cell development.

A new transgenic reporter line reveals Wnt-dependent Snai2 re-expression and cranial neural crest differentiation in Xenopus

During vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and differentiates into many types of cells. The transcription factor Snai2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snai2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear. We generated a transgenic X. tropicalis line that expresses enhanced green fluorescent protein (eGFP) driven by the snai2 promoter/enhancer, and observed eGFP expression not only in the pre-migratory and migrating CNC, but also the differentiating CNC. This transgenic line can be used directly to detect deficiencies in CNC development at various stages, including subtle perturbation of CNC differentiation. In situ hybridization and immunohistochemistry confirm that Snai2 is re-expressed in the differentiating CNC. Using a separate transgenic Wnt reporter line, we show that canonical Wnt signaling is also active in the differentiating CNC. Blocking Wnt signaling shortly after CNC migration causes reduced snai2 expression and impaired differentiation of CNC-derived head cartilage structures. These results suggest that Wnt signaling is required for snai2 re-expression and CNC differentiation.

Sema3E is required for migration of cranial neural crest cells in zebrafish: Implications for the pathogenesis of CHARGE syndrome.

CHARGE syndrome is a congenital disorder with multiple malformations in the craniofacial structures, and cardiovascular and genital systems, which are mainly affected by neural crest defects caused by loss-of-function mutations within chromodomain helicase DNA-binding protein 7 (CHD7). However, many patients with CHARGE syndrome test negative for CHD7. Semaphorin 3E (sema3E) is a gene reported to be mutated in patients with CHARGE syndrome. However, its role in the pathogenesis of CHARGE syndrome has not been verified experimentally. Here, we report that the knockdown of sema3E results in severe craniofacial malformations, including small eyes, defective cartilage and an abnormal number of otoliths in zebrafish embryos, which resemble the major features of CHARGE syndrome. Further analysis reveals that the migratory cranial neural crest cells are scattered in the region of the hindbrain, and the postmigratory neural crest cells are reduced in the pharyngeal arches upon sema3E knockdown. Notably, immunostaining and time-lapse imaging analyses of a neural crest cell-labelled transgenic fish line, sox10:EGFP, show that the migration of cranial neural crest cells is severely impaired, and many of these cells are misrouted upon sema3E knockdown. Furthermore, the sox10-expressing cranial neural crest cells are scattered in chd7 homozygous mutants, which phenocopied the phenotype in sema3E morphants. Overexpression of sema3E rescues the phenotype of scattered cranial neural crest cells in chd7 homozygotes, indicating that chd7 may control the expression of sema3E to regulate cranial neural crest cell migration. Collectively, our data demonstrate that sema3E is involved in the pathogenesis of CHARGE syndrome by modulating cranial neural crest cell migration.

Impact of prenatal arsenate exposure on gene expression in a pure population of migratory cranial neural crest cells

Prenatal exposure to arsenic, a naturally occurring toxic element, causes neural tube defects (NTDs) and, in animal models, orofacial anomalies. Since aberrant development or migration of cranial neural crest cells (CNCCs) can also cause similar anomalies within developing embryos, we examined the effects of in utero exposure to sodium arsenate on gene expression patterns in pure populations of CNCCs, isolated by fluorescence activated cell sorting (FACS), from Cre/LoxP reporter mice. Changes in gene expression were analyzed using Affymetrix GeneChip® microarrays and expression of selected genes was verified by TaqMan quantitative real-time PCR. We report, for the first time, arsenate-induced alterations in the expression of a number of novel candidate genes and canonical cascades that may contribute to the pathogenesis of orofacial defects. Ingenuity Pathway and NIH-DAVID analyses revealed cellular response pathways, biological themes, and potential upstream regulators, that may underlie altered fetal programming of arsenate exposed CNCCs.

Prickle1 is required for EMT and migration of zebrafish cranial neural crest

The neural crest—a key innovation of the vertebrates—gives rise to diverse cell types including melanocytes, neurons and glia of the peripheral nervous system, and chondrocytes of the jaw and skull. Proper development of the cephalic region is dependent on the tightly-regulated specification and migration of cranial neural crest cells (NCCs). The core PCP proteins Frizzled and Disheveled have previously been implicated in NCC migration. Here we investigate the functions of the core PCP proteins Prickle1a and Prickle1b in zebrafish cranial NCC development. Using analysis of pk1a and pk1b mutant embryos, we uncover similar roles for both genes in facilitating cranial NCC migration. Disruption of either gene causes pre-migratory NCCs to cluster together at the dorsal aspect of the neural tube, where they adopt aberrant polarity and movement. Critically, in investigating Pk1-deficient cells that fail to migrate ventrolaterally, we have also uncovered roles for pk1a and pk1b in the epithelial-to-mesenchymal transition (EMT) of pre-migratory NCCs that precedes their collective migration to the periphery. Normally, during EMT, pre-migratory NCCs transition from a neuroepithelial to a bleb-based and subsequently, mesenchymal morphology capable of directed migration. When either Pk1a or Pk1b is disrupted, NCCs continue to perform blebbing behaviors characteristic of pre-migratory cells over extended time periods, indicating a block in a key transition during EMT. Although some Pk1-deficient NCCs transition successfully to mesenchymal, migratory morphologies, they fail to separate from neighboring NCCs. Additionally, Pk1b-deficient NCCs show elevated levels of E-Cadherin and reduced levels of N-Cadherin, suggesting that Prickle1 molecules regulate Cadherin levels to ensure the completion of EMT and the commencement of cranial NCC migration. We conclude that Pk1 plays crucial roles in cranial NCCs both during EMT and migration. These roles are dependent on the regulation of E-Cad and N-Cad.

Family-based exome sequencing and case-control analysis implicate CEP41 as an ASD gene

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with a strong genetic component. Although next-generation sequencing (NGS) technologies have been successfully applied to gene identification in de novo ASD, the genetic architecture of familial ASD remains largely unexplored. Our approach, which leverages the high specificity and sensitivity of NGS technology, has focused on rare variants in familial autism. We used NGS exome sequencing in 26 families with distantly related affected individuals to identify genes with private gene disrupting and missense variants of interest (VOI). We found that the genes carrying VOIs were enriched for biological processes related to cell projection organization and neuron development, which is consistent with the neurodevelopmental hypothesis of ASD. For a subset of genes carrying VOIs, we then used targeted NGS sequencing and gene-based variant burden case-control analysis to test for association with ASD. Missense variants in one gene, CEP41, associated significantly with ASD (p = 6.185e−05). Homozygous gene-disrupting variants in CEP41 were initially found to be responsible for recessive Joubert syndrome. Using a zebrafish model, we evaluated the mechanism by which the CEP41 variants might contribute to ASD. We found that CEP41 missense variants affect development of the axonal tract, cranial neural crest migration and social behavior phenotype. Our work demonstrates the involvement of CEP41 heterozygous missense variants in ASD and that biological processes involved in cell projection organization and neuron development are enriched in ASD families we have studied.

Cadherin-7 mediates proper neural crest cell-placodal neuron interactions during trigeminal ganglion assembly.

The cranial trigeminal ganglia play a vital role in the peripheral nervous system through their relay of sensory information from the vertebrate head to the brain. These ganglia are generated from the intermixing and coalescence of two distinct cell populations: cranial neural crest cells and placodal neurons. Trigeminal ganglion assembly requires the formation of cadherin-based adherens junctions within the neural crest cell and placodal neuron populations; however, the molecular composition of these adherens junctions is still unknown. Herein, we aimed to define the spatio-temporal expression pattern and function of Cadherin-7 during early chick trigeminal ganglion formation. Our data reveal that Cadherin-7 is expressed exclusively in migratory cranial neural crest cells and is absent from trigeminal neurons. Using molecular perturbation experiments, we demonstrate that modulation of Cadherin-7 in neural crest cells influences trigeminal ganglion assembly, including the organization of neural crest cells and placodal neurons within the ganglionic anlage. Moreover, alterations in Cadherin-7 levels lead to changes in the morphology of trigeminal neurons. Taken together, these findings provide additional insight into the role of cadherin-based adhesion in trigeminal ganglion formation, and, more broadly, the molecular mechanisms that orchestrate the cellular interactions essential for cranial gangliogenesis.

TBC1d24-ephrinB2 interaction regulates contact inhibition of locomotion in neural crest cell migration

Although Eph-ephrin signalling has been implicated in the migration of cranial neural crest (CNC) cells, it is still unclear how ephrinB transduces signals regulating this event. We provide evidence that TBC1d24, a putative Rab35-GTPase activating protein (Rab35 GAP), complexes with ephrinB2 via the scaffold Dishevelled (Dsh) and mediates a signal affecting contact inhibition of locomotion (CIL) in CNC cells. Moreover, we found that, in migrating CNC, the interaction between ephrinB2 and TBC1d24 negatively regulates E-cadherin recycling in these cells via Rab35. Upon engagement of the cognate Eph receptor, ephrinB2 is tyrosine phosphorylated, which disrupts the ephrinB2/Dsh/TBC1d24 complex. The dissolution of this complex leads to increasing E-cadherin levels at the plasma membrane, resulting in loss of CIL and disrupted CNC migration. Our results indicate that TBC1d24 is a critical player in ephrinB2 control of CNC cell migration via CIL.

Ric-8A, a GEF for heterotrimeric G-proteins, controls cranial neural crest cell polarity during migration

The neural crest (NC) is a transient embryonic cell population that migrates extensively during development. Ric-8A, a guanine nucleotide exchange factor (GEF) for different Gα subunits regulates cranial NC (CNC) cell migration in Xenopus through a mechanism that still remains to be elucidated. To properly migrate, CNC cells establish an axis of polarization and undergo morphological changes to generate protrusions at the leading edge and retraction of the cell rear. Here, we aim to study the role of Ric-8A in cell polarity during CNC cell migration by examining whether its signaling affects the localization of GTPase activity in Xenopus CNC using GTPase-based probes in live cells and aPKC and Par3 as polarity markers. We show that the levels of Ric-8A are critical during migration and affect the localization of polarity markers and the subcellular localization of GTPase activity, suggesting that Ric-8A, probably through heterotrimeric G-protein signaling, regulates cell polarity during CNC migration.