Cranial Neural Crest Migration Research Articles

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca2+ channel CaV1.2 in nonexcitable cells. How abnormal Ca2+ influx through CaV1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established CaV1.2 roles. Here, we show that CaV1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca2+ influx through CaV1.2 regulates jaw development. Cranial neural crest migration was unaffected by CaV1.2 knockdown, suggesting a role for CaV1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca2+ signals through CaV1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca2+ channels in nonexcitable cells during development.

Commentary on the Differential Healing Capacity of Calvarial Bone

Commentary on the Differential Healing Capacity of Calvarial Bone

Tetraspanin18 is a FoxD3-responsive antagonist of cranial neural crest epithelial-to-mesenchymal transition that maintains cadherin-6B protein.

During epithelial-to-mesenchymal transition (EMT), tightly associated, polarized epithelial cells become individual mesenchymal cells capable of migrating. Here, we investigate the role of the transmembrane protein tetraspanin18 (Tspan18) in chick cranial neural crest EMT. Tspan18 mRNA is expressed in premigratory cranial neural crest cells, but is absent from actively migrating neural crest cells. Tspan18 knockdown leads to a concomitant loss of cadherin-6B (Cad6B) protein, whereas Cad6B protein persists when Tspan18 expression is extended. The temporal profile of Cad6B mRNA downregulation is unaffected in these embryos, which indicates that Tspan18 maintains Cad6B protein levels and reveals that Cad6B is regulated by post-translational mechanisms. Although downregulation of Tspan18 is necessary, it is not sufficient for neural crest migration: the timing of neural crest emigration, basal lamina breakdown and Cad7 upregulation proceed normally in Tspan18-deficient cells. This emphasizes the need for coordinated transcriptional and post-translational regulation of Cad6B during EMT and illustrates that Tspan18-antagonized remodeling of cell-cell adhesions is only one step in preparation for cranial neural crest migration. Unlike Cad6B, which is transcriptionally repressed by Snail2, Tspan18 expression is downstream of the winged-helix transcription factor FoxD3, providing a new transcriptional input into cranial neural crest EMT. Together, our data reveal post-translational regulation of Cad6B protein levels by Tspan18 that must be relieved by a FoxD3-dependent mechanism in order for cranial neural crest cells to migrate. These results offer new insight into the molecular mechanisms of cranial neural crest EMT and expand our understanding of tetraspanin function relevant to metastasis.

Elk3 is essential for the progression from progenitor to definitive neural crest cell

Elk3/Net/Sap2 (here referred to as Elk3) is an Ets ternary complex transcriptional repressor known for its involvement in angiogenesis during embryonic development. Although Elk3 is expressed in various tissues, additional roles for the protein outside of vasculature development have yet to be reported. Here, we characterize the early spatiotemporal expression pattern of Elk3 in the avian embryo using whole mount in situ hybridization and quantitative RT-PCR and examine the effects of its loss of function on neural crest development. At early stages, Elk3 is expressed in the head folds, head mesenchyme, intersomitic vessels, and migratory cranial neural crest (NC) cells. Loss of the Elk3 protein results in the retention of Pax7+ precursors in the dorsal neural tube that fail to upregulate neural crest specifier genes, FoxD3, Sox10 and Snail2, resulting in embryos with severe migration defects. The results putatively place Elk3 downstream of neural plate border genes, but upstream of neural crest specifier genes in the neural crest gene regulatory network (NC-GRN), suggesting that it is critical for the progression from progenitor to definitive neural crest cell.

Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development

Craniofacial and ocular morphogenesis require proper regulation of cranial neural crest migration, proliferation, survival and differentiation. Although alterations in maternal thyroid hormone (TH) are associated with congenital craniofacial anomalies, the role of TH on the neural crest has not been previously described. Using zebrafish, we demonstrate that pharmacologic and genetic alterations in TH signaling disrupt cranial neural crest migration, proliferation, and survival, leading to craniofacial, extraocular muscle, and ocular developmental abnormalities. In the rostral cranial neural crest that gives rise to the periocular mesenchyme and the frontonasal process, retinoic acid (RA) rescued migratory defects induced by decreased TH signaling. In the caudal cranial neural crest, TH and RA had reciprocal effects on anterior and posterior pharyngeal arch development. The interactions between TH and RA signaling were partially mediated by the retinoid X receptor. We conclude that TH regulates both rostral and caudal cranial neural crest. Further, coordinated interactions of TH and RA are required for proper craniofacial and ocular development.

Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis

Development of the palate in vertebrates involves cranial neural crest migration, convergence of facial prominences and extension of the cartilaginous framework. Dysregulation of palatogenesis results in orofacial clefts, which represent the most common structural birth defects. Detailed analysis of zebrafish palatogenesis revealed distinct mechanisms of palatal morphogenesis: extension, proliferation and integration. We show that wnt9a is required for palatal extension, wherein the chondrocytes form a proliferative front, undergo morphological change and intercalate to form the ethmoid plate. Meanwhile, irf6 is required specifically for integration of facial prominences along a V-shaped seam. This work presents a mechanistic analysis of palate morphogenesis in a clinically relevant context.

Defective neural crest migration revealed by a Zebrafish model of Alx1-related frontonasal dysplasia

Frontonasal dysplasia (FND) refers to a class of midline facial malformations caused by abnormal development of the facial primordia. The term encompasses a spectrum of severities but characteristic features include combinations of ocular hypertelorism, malformations of the nose and forehead and clefting of the facial midline. Several recent studies have drawn attention to the importance of Alx homeobox transcription factors during craniofacial development. Most notably, loss of Alx1 has devastating consequences resulting in severe orofacial clefting and extreme microphthalmia. In contrast, mutations of Alx3 or Alx4 cause milder forms of FND. Whilst Alx1, Alx3 and Alx4 are all known to be expressed in the facial mesenchyme of vertebrate embryos, little is known about the function of these proteins during development. Here, we report the establishment of a zebrafish model of Alx-related FND. Morpholino knock-down of zebrafish alx1 expression causes a profound craniofacial phenotype including loss of the facial cartilages and defective ocular development. We demonstrate for the first time that Alx1 plays a crucial role in regulating the migration of cranial neural crest (CNC) cells into the frontonasal primordia. Abnormal neural crest migration is coincident with aberrant expression of foxd3 and sox10, two genes previously suggested to play key roles during neural crest development, including migration, differentiation and the maintenance of progenitor cells. This novel function is specific to Alx1, and likely explains the marked clinical severity of Alx1 mutation within the spectrum of Alx-related FND.

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

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

Williams Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis

Williams Syndrome Transcription Factor (WSTF) is one of ∼25 haplodeficient genes in patients with the complex developmental disorder Williams Syndrome (WS). WS results in visual/spatial processing defects, cognitive impairment, unique behavioral phenotypes, characteristic “elfin” facial features, low muscle tone and heart defects. WSTF exists in several chromatin remodeling complexes and has roles in transcription, replication, and repair. Chromatin remodeling is essential during embryogenesis, but WSTF’s role in vertebrate development is poorly characterized. To investigate the developmental role of WSTF, we knocked down WSTF in Xenopus laevis embryos using a morpholino that targets WSTF mRNA. BMP4 shows markedly increased and spatially aberrant expression in WSTF-deficient embryos, while SHH, MRF4, PAX2, EPHA4 and SOX2 expression are severely reduced, coupled with defects in a number of developing embryonic structures and organs. WSTF-deficient embryos display defects in anterior neural development. Induction of the neural crest, measured by expression of the neural crest-specific genes SNAIL and SLUG, is unaffected by WSTF depletion. However, at subsequent stages WSTF knockdown results in a severe defect in neural crest migration and/or maintenance. Consistent with a maintenance defect, WSTF knockdowns display a specific pattern of increased apoptosis at the tailbud stage in regions corresponding to the path of cranial neural crest migration. Our work is the first to describe a role for WSTF in proper neural crest function, and suggests that neural crest defects resulting from WSTF haploinsufficiency may be a major contributor to the pathoembryology of WS.

ADAM13 function is required in the 3 dimensional context of the embryo during cranial neural crest cell migration in Xenopus laevis

The cranial neural crest (CNC) is a population of cells that arises from the lateral part of the developing brain, migrates ventrally and coordinates the entire craniofacial development of vertebrates. Many molecules are involved in CNC migration including the transmembrane metalloproteases ADAM13 and 19. We have previously shown that these ADAMs cleave a number of extracellular proteins and modify the transcription of a number of genes, and that both of these activities are important for cell migration. Here we show that the knock down of ADAM13 inhibits CNC migration in vivo but not in vitro, indicating that ADAM13 function is required in the 3-dimentional context of the embryo. We further show that the migration of CNC that do not express ADAM13 and ADAM19 can be rescued in vivo by co-grafting wild type CNC. Furthermore, the migration of CNC lacking ADAM13 can be rescued by mechanically separating the CNC from the surrounding ectoderm and mesoderm. Finally, we show that ADAM13 function is autonomous to CNC tissue, as the migration of morphant CNC can only be rescued by ADAM13 expression in the CNC and not the surrounding tissues. Together our results suggest that ADAM13 changes CNC interaction with the extracellular environment and that this change is necessary for their migration in vivo.

Signaling and transcriptional regulation in neural crest specification and migration: lessons from xenopus embryos

The neural crest is a population of highly migratory and multipotent cells, which arises from the border of the neural plate in vertebrate embryos. In the last few years, the molecular actors of neural crest early development have been intensively studied, notably by using the frog embryo, as a prime model for the analysis of the earliest embryonic inductions. In addition, tremendous progress has been made in understanding the molecular and cellular basis of Xenopus cranial neural crest migration, by combining in vitro and in vivo analysis. In this review, we examine how the action of previously known neural crest-inducing signals [bone morphogenetic protein (BMP), wingless-int (Wnt), fibroblast growth factor (FGF)] is controlled by newly discovered modulators during early neural plate border patterning and neural crest specification. This regulation controls the induction of key transcription factors that cooperate to pattern the premigratory neural crest progenitors. These data are discussed in the perspective of the gene regulatory network that controls neural and neural crest patterning. We then address recent findings on noncanonical Wnt signaling regulation, cell polarization, and collective cell migration which highlight how cranial neural crest cells populate their target tissue, the branchial arches, in vivo. More than ever, the neural crest stands as a powerful and attractive model to decipher complex vertebrate regulatory circuits in vivo.

The p21-activated kinase Pak1 regulates induction and migration of the neural crest in Xenopus

Pak1 is a member of the PAK family of serine/threonine kinases that are downstream effectors of Rac1 and Cdc42 small GTPases and are implicated in cytoskeleton reorganization. Early expression of Pak1 in Xenopus embryos is tissue restricted, suggesting a role in organogenesis and in cranial neural crest (CNC) cell migration. By observing CNC in vivo and after transplantation, we show that a dominant-negative (DN) Pak1 inhibits its migration. DN-Pak1 also specifically modified the expression of several NC markers. Twist expression was decreased and Snail1 expression posteriorized, but Snail2 (Slug), Sox9 and AP2 were unaffected. DN-Pak1 inhibition of CNC migration could be rescued with Snail1 but not with Twist, which, in fact, cooperated with DN-Pak1 in inhibiting migration. The data confirm that neither Snail1 nor Snail2 expression alone is sufficient for Xenopus CNC migration. Furthermore, they show that, in this tissue, Snail1 and Snail2 expression is not interdependent, nor are these factors subject to obligatory co-regulation, and that their expression depends on signal transduction. Our results also represent the first evidence that Pak1 links extracellular signals to the genetic cascade of transcription factors necessary for CNC specification.

Histone deacetylase-4 is required during early cranial neural crest development for generation of the zebrafish palatal skeleton

BackgroundHistone deacetylase-4 (Hdac4) is a class II histone deacetylase that inhibits the activity of transcription factors. In humans, HDAC4 deficiency is associated with non-syndromic oral clefts and brachydactyly mental retardation syndrome (BDMR) with craniofacial abnormalities.ResultsWe identify hdac4 in zebrafish and characterize its function in craniofacial morphogenesis. The gene is present as a single copy, and the deduced Hdac4 protein sequence shares all known functional domains with human HDAC4. The zebrafish hdac4 transcript is widely present in migratory cranial neural crest (CNC) cells of the embryo, including populations migrating around the eye, which previously have been shown to contribute to the formation of the palatal skeleton of the early larva. Embryos injected with hdac4 morpholinos (MO) have reduced or absent CNC populations that normally migrate medial to the eye. CNC-derived palatal precursor cells do not recover at the post-migratory stage, and subsequently we found that defects in the developing cartilaginous palatal skeleton correlate with reduction or absence of early CNC cells. Palatal skeletal defects prominently include a shortened, clefted, or missing ethmoid plate, and are associated with a shortening of the face of young larvae.ConclusionsOur results demonstrate that Hdac4 is a regulator of CNC-derived palatal skeletal precursors during early embryogenesis. Cleft palate resulting from HDAC4 mutations in human patients may result from defects in a homologous CNC progenitor cell population.

Caldesmon regulates actin dynamics to influence cranial neural crest migration inXenopus

Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.

Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

The eye as an organizer of craniofacial development

The formation and invagination of the optic stalk coincides with the migration of cranial neural crest (CNC) cells, and a growing body of data reveals that the optic stalk and CNC cells communicate to lay the foundations for periocular and craniofacial development. Following migration, the interaction between the developing eye and surrounding periocular mesenchyme (POM) continues, leading to induction of transcriptional regulatory cascades that regulate craniofacial morphogenesis. Studies in chick, mice, and zebrafish have revealed a remarkable level of genetic and mechanistic conservation, affirming the power of each animal model to shed light on the broader morphogenic process. This review will focus on the role of the developing eye in orchestrating craniofacial morphogenesis, utilizing morphogenic gradients, paracrine signaling, and transcriptional regulatory cascades to establish an evolutionarily-conserved facial architecture. We propose that in addition to the forebrain, the eye functions during early craniofacial morphogenesis as a key organizer of facial development, independent of its role in vision.

An additional patient with mycophenolate mofetil embryopathy: Cardiac and facial analyses

We describe an infant male of Cambodian background who has typical craniofacial features of mycophenolate mofetil (MMF) embryopathy and a complex congenital heart defect (CHD) (double outlet right ventricle, mitral atresia, pulmonic stenosis, and total anomalous pulmonary venous return). Together with four case reports and the 20 patients included in two recent reviews, we report 24 (19 affected, five normal) patients with this pattern of anomalies. Eight (33%) have a CHD, most commonly, conotruncal or aortic arch defects (6/8, 75%). This would support the hypothesis that disturbance of cranial neural crest migration occurs in exposed infants, and may predict which additional anomalies will be observed in the future. We also attempted to score the severity of the facial anomalies in each MMF patient using a system created by plastic surgeons for patients with hemifacial microsomia. This classification had modest utility in comparing severity and correlating facial to extracranial defects. The findings are viewed with caution because of the preliminary methodology. Finally, since several exposed infants have been reported to be minimally affected, we remind clinicians to be sensitive to the potential mild expression of the effects of this teratogen. This awareness may influence clinical management of apparently normal MMF-exposed individuals.

Translocation of the Cytoplasmic Domain of ADAM13 to the Nucleus Is Essential for Calpain8-a Expression and Cranial Neural Crest Cell Migration

ADAMs are transmembrane metalloproteases that control cell behavior by cleaving both cell adhesion and signaling molecules. The cytoplasmic domain of ADAMs can regulate the proteolytic activity by controlling the subcellular localization and/or the activation of the protease domain. Here, we show that the cytoplasmic domain of ADAM13 is cleaved and translocates into the nucleus. Preventing this translocation renders the protein incapable of promoting cranial neural crest (CNC) cell migration invivo, without affecting its proteolytic activity. In addition, the cytoplasmic domain of ADAM13 regulates the expression of multiple genes in CNC, including the protease Calpain8-a. Restoring the expression of Calpain8-a is sufficient to rescue CNC migration in the absence of the ADAM13 cytoplasmic domain. This study shows that the cytoplasmic domain of ADAM metalloproteases can perform essential functions in the nucleus of cells and may contribute substantially to the overall function of the protein.

Zebrafish wnt9a is expressed in pharyngeal ectoderm and is required for palate and lower jaw development

Wnt activity is critical in craniofacial morphogenesis. Dysregulation of Wnt/β-catenin signaling results in significant alterations in the facial form, and has been implicated in cleft palate phenotypes in mouse and man. In zebrafish, we show that wnt9a is expressed in the pharyngeal arch, oropharyngeal epithelium that circumscribes the ethmoid plate, and ectodermal cells superficial to the lower jaw structures. Alcian blue staining of morpholino-mediated knockdown of wnt9a results in loss of the ethmoid plate, absence of lateral and posterior parachordals, and significant abrogation of the lower jaw structures. Analysis of cranial neural crest cells in the sox10:eGFP transgenic demonstrates that the wnt9a is required early during pharyngeal development, and confirms that the absence of Alcian blue staining is due to absence of neural crest derived chondrocytes. Molecular analysis of genes regulating cranial neural crest migration and chondrogenic differentiation suggest that wnt9a is dispensable for early cranial neural crest migration, but is required for chondrogenic development of major craniofacial structures. Taken together, these data corroborate the central role for Wnt signaling in vertebrate craniofacial development, and reveal that wnt9a provides the signal from the pharyngeal epithelium to support craniofacial chondrogenic morphogenesis in zebrafish.