p53 coordinates cranial neural crest cell growth and epithelial-mesenchymal transition/delamination processes

Neural crest development involves epithelial-mesenchymal transition (EMT), during which epithelial cells are converted into individual migratory cells. Notably, the same signaling pathways regulate EMT function during both development and tumor metastasis. p53 plays multiple roles in the prevention of tumor development; however, its precise roles during embryogenesis are less clear. We have investigated the role of p53 in early cranial neural crest (CNC) development in chick and mouse embryos. In the mouse, p53 knockout embryos displayed broad craniofacial defects in skeletal, neuronal and muscle tissues. In the chick, p53 is expressed in CNC progenitors and its expression decreases with their delamination from the neural tube. Stabilization of p53 protein using a pharmacological inhibitor of its negative regulator, MDM2, resulted in reduced SNAIL2 (SLUG) and ETS1 expression, fewer migrating CNC cells and in craniofacial defects. By contrast, electroporation of a dominant-negative p53 construct increased PAX7(+) SOX9(+) CNC progenitors and EMT/delamination of CNC from the neural tube, although the migration of these cells to the periphery was impaired. Investigating the underlying molecular mechanisms revealed that p53 coordinates CNC cell growth and EMT/delamination processes by affecting cell cycle gene expression and proliferation at discrete developmental stages; disruption of these processes can lead to craniofacial defects.     

Protocadherin PAPC is expressed in the CNC and can compensate for the loss of PCNS

Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration. genesis 52:120–126. © 2013 Wiley Periodicals, Inc.     

Cell lineage analysis in neural crest ontogeny

The neural crest is a transitory and pluripotent structure of the vertebrate embryo composed of cells endowed with developmentally regulated migratory properties. We review here a series of studies carried out both in vivo and in vitro on the ontogeny of the neural crest in the avian embryo. Through in vivo studies we established the fate map of the neural crest along the neuraxis prior to the onset of the migration and we demonstrated the crucial role played by the tissue environment in which the crest cells migrate in determining their fate. Moreover, the pathways of neural crest cell migration could also be traced by the quail-chick marker system and the use of the HNK1/NC1 monoclonal antibody (Mab). A large series of clonal cultures of isolated neural crest cells showed that, at migration time, most crest cells are pluripotent. Some, however, are already committed to a particular pathway of differentiation. The differentiation capacities of the pluripotent progenitors are highly variable from one to the other cell. Rare totipotent progenitors able to give rise to representatives of all the phenotypes (neuronal, glial, melanocytic, and mesectodermal) encountered in neural crest derivatives were also found. As a whole we propose a model according to which totipotent neural crest cells become progressively restricted (according to a stochastic rather than a sequentially ordered mechanism) in their potentialities, while they actively divide during the migration process. At the sites of gangliogenesis, selective forces allow only certain crest cells potentialities to be expressed in each type of peripheral nervous system (PNS) ganglia. © 1993 John Wiley & Sons, Inc.     

Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development

Neural crest cells escape the neural tube by undergoing an epithelial to mesenchymal transition (EMT). This is followed by extensive migration along specific pathways that are lined with extracellular matrix (ECM). In this study, we have examined the roles of matrix receptors containing beta1 integrin subunits in neural crest cell morphogenesis using antisense morpholino oligos electroporated in ovo into avian neural crest cell precursors. Our results show that reduced levels of expression of beta1 integrin subunits in the dorsal neural tube results in an abnormal epithelial to mesenchymal transition. In approximately half of the experimental embryos, however, some neural crest cells filled with beta1 antisense are able to escape the neural tube and migrate ventrally, indicating that grossly normal migration of trunk neural crest cells can take place after beta1 integrin expression is reduced. This study shows the potential of this novel method for investigating the roles of genes that are required for the survival of early mouse embryos in later development events.     

Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/Wrch‐1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N‐ and C‐terminal domains. The N‐terminal extension promotes binding to SH3 (Src homology 3)‐domain‐containing adaptors, whereas the C‐terminal extension mediates membrane targeting through palmitoylation of its non‐conventional CAAX box. RhoU/Wrch‐1 possesses transforming activity, which is negatively regulated by its N‐terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c‐Src‐expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N‐terminal extension and the palmitoylation site were dispensable, whereas the C‐terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked‐down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C‐terminal domains are critical for these functions.     

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

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

Fascin1-Dependent Filopodia are Required for Directional Migration of a Subset of Neural Crest Cells

Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development.     

Ultrastructural and tissue-culture studies on the role of fibronectin,collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo

Summary The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15–40 nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin ( 3 nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker ( 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen.Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates. However, partially desulphated chondroitin sulphate (5mg/ml) strongly retarded the migration of NC cells.The in vivo and in vitro studies suggest that fibronectin may dictate the pathways of NC cell migration by acting as a highly preferred physical substrate. However, the utilization of these pathways may be reduced by the presence of proteoglycans bearing undersulphated chondroitin sulphate.Abbreviations NC neural crest - ECM extracellular material - GAG glycosaminoglycan - FN fibronectin - CIG cold insoluble globulin - TEM transmission electron microscopy - SEM scanning electron microscopy - DMEM-H HEPES buffered Dulbecco's modified Eagle's medium - FCS foetal calf serum - CEE chick embryo extract - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

Control of the onset of migration of neural crest cells in avian embryos

Summary To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of Ca^{+ +} and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3–8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca^{+ +}- and Mg^{+ +}-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca^{+ +}-free medium duplicated the effects of CMF, but neither Mg^{+ +}-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane. Ca^{+ +}-channel antagonists D600 and Nifedipine, without any exernal Ca^{+ +}-depletion.The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca^{+ +}-depletion inactivates Ca^{+ +}-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca^{+ +}-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca^{+ +}-channels suggest that Ca^{+ +} levels may be involved in regulating this system.     

Smad4 is required to regulate the fate of cranial neural crest cells

Smad4 is the central mediator for TGF-β/BMP signals, which are involved in regulating cranial neural crest (CNC) cell formation, migration, proliferation and fate determination. It is unclear whether TGF-β/BMP signals utilize Smad-dependent or -independent pathways to control the development of CNC cells. To investigate the functional significance of Smad4 in regulating CNC cells, we generated mice with neural crest specific inactivation of the Smad4 gene. Our study shows that Smad4 is not required for the migration of CNC cells, but is required in neural crest cells for the development of the cardiac outflow tract. Smad4 is essential in mediating BMP signaling in the CNC-derived ectomesenchyme during early stages of tooth development because conditional inactivation of Smad4 in neural crest derived cells results in incisor and molar development arrested at the dental lamina stage. Furthermore, Smad-mediated TGF-β/BMP signaling controls the homeobox gene patterning of oral/aboral and proximal/distal domains within the first branchial arch. At the cellular level, a Smad4-mediated downstream target gene(s) is required for the survival of CNC cells in the proximal domain of the first branchial arch. Smad4 mutant mice show underdevelopment of the first branchial arch and midline fusion defects. Taken together, our data show that TGF-β/BMP signals rely on Smad-dependent pathways in the ectomesenchyme to mediate epithelial-mesenchymal interactions that control craniofacial organogenesis.     

Mechanisms of neural crest cell migration

Neural crest cells are remarkable in their extensive and stereotypic patterns of migration. The pathways of neural crest migration have been documented by cell marking techniques, including interspecific neural tube grafts, immunocytochemistry and Dil-labelling. In the trunk, neural crest cells migrate dorsally under the skin or ventrally through the somites, where they move in a segmental fashion through the rostral half of each sclerotome. The segmental migration of neural crest cells appears to be prescribed by the somites, perhaps by an inhibitory cue from the caudal half. Within the rostral sclerotome, neural crest cells fill the available space except for a region around the notochord, suggesting the notochord may inhibit neural crest cells in its vicinity. In the cranial region, antibody perturbation experiments suggest that multiple cell-matrix interactions are required for proper in vivo migration of neural crest cells. Neural crest cells utilize integrin receptors to bind to a number of extracellular matrix molecules. Substrate selective inhibition of neural crest cell attachment in vitro by integrin antibodies and antisense oligonucleotides has demonstrated that they possess at least three integrins, one being an α₁β₁ integrin which functions in the absence of divalent cations. Thus, neural crest cells utilize complex sets of interactions which may differ at different axial levels.     

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.     

Fibronectin and integrin alpha 5 play essential roles in the development of the cardiac neural crest

Cardiac neural crest (CNC) plays a requisite role during cardiovascular development and defects in the formation of CNC-derived structures underlie several common forms of human congenital birth defects. Migration of the CNC cells to their destinations as well as expansion and maintenance of these cells are important for the normal development of the cardiac outflow tract and aortic arch arteries; however, molecular mechanisms regulating these processes are not well-understood. Fibronectin (FN) protein is present along neural crest migration paths and neural crest cells migrate when plated on FN in vitro; therefore, we tested the role of FN during the development of the CNC in vivo. Our analysis of the fate of the neural crest shows that CNC cells reach their destinations in the branchial arches and the cardiac outflow tract in the absence of FN or its cellular receptor integrin α5β1. However, we found that FN and integrin α5 modulate CNC proliferation and survival, and are required for the presence of normal numbers of CNC cells at their destinations.     

A Novel Role for VICKZ Proteins in Maintaining Epithelial Integrity during Embryogenesis

Background

VICKZ (IGF2BP1,2,3/ZBP1/Vg1RBP/IMP1,2,3) proteins bind RNA and help regulate many RNA-mediated processes. In the midbrain region of early chick embryos, VICKZ is expressed in the neural folds and along the basal surface of the neural epithelium, but, upon neural tube closure, is down-regulated in prospective cranial neural crest (CNC) cells, concomitant with their emigration and epithelial-to-mesenchymal transition (EMT). Electroporation of constructs that modulate cVICKZ expression demonstrates that this down-regulation is both necessary and sufficient for CNC EMT. These results suggest that VICKZ down-regulation in CNC cell-autonomously promotes EMT and migration. Reduction of VICKZ throughout the embryo, however, inhibits CNC migration non-cell-autonomously, as judged by transplantation experiments in Xenopus embryos.

Results and Conclusions

Given the positive role reported for VICKZ proteins in promoting cell migration of chick embryo fibroblasts and many types of cancer cells, we have begun to look for specific mRNAs that could mediate context-specific differences. We report here that the laminin receptor, integrin alpha 6, is down-regulated in the dorsal neural tube when CNC cells emigrate, this process is mediated by cVICKZ, and integrin alpha 6 mRNA is found in VICKZ ribonucleoprotein complexes. Significantly, prolonged inhibition of cVICKZ in either the neural tube or the nascent dermomyotome sheet, which also dynamically expresses cVICKZ, induces disruption of these epithelia. These data point to a previously unreported role for VICKZ in maintaining epithelial integrity.     

Draxin,an axon guidance protein,affects chick trunk neural crest migration

The neural crest is a multipotent population of migratory cells that arises in the central nervous system and subsequently migrates along defined stereotypic pathways. In the present work, we analyzed the role of a repulsive axon guidance protein, draxin, in the migration of neural crest cells. Draxin is expressed in the roof plate of the chick trunk spinal cord and around the early migration pathway of neural crest cells. Draxin modulates chick neural crest cell migration in vitro by reducing the polarization of these cells. When exposed to draxin, the velocity of migrating neural crest cells was reduced, and the cells changed direction so frequently that the net migration distance was also reduced. Overexpression of draxin also caused some early migrating neural crest cells to change direction to the dorsolateral pathway in the chick trunk region, presumably due to draxin’s inhibitory activity. These results demonstrate that draxin, an axon guidance protein, can also affect trunk neural crest migration in the chick embryo.     

Alcama mediates Edn1 signaling during zebrafish cartilage morphogenesis

The zebrafish pharyngeal cartilage is derived from the pharyngeal apparatus, a vertebrate-specific structure derived from all three germ layers. Developmental aberrations of the pharyngeal apparatus lead to birth defects such as Treacher-Collins and DiGeorge syndromes. While interactions between endoderm and neural crest (NC) are known to be important for cartilage formation, the full complement of molecular players involved and their roles remain to be elucidated. Activated leukocyte cell adhesion molecule a (alcama), a member of the immunoglobulin (Ig) superfamily, is among the prominent markers of pharyngeal pouch endoderm, but to date no role has been assigned to this adhesion molecule in the development of the pharyngeal apparatus. Here we show that alcama plays a crucial, non-autonomous role in pharyngeal endoderm during zebrafish cartilage morphogenesis. alcama knockdown leads to defects in NC differentiation, without affecting NC specification or migration. These defects are reminiscent of the phenotypes observed when Endothelin 1 (Edn1) signaling, a key regulator of cartilage development is disrupted. Using gene expression analysis and rescue experiments we show that Alcama functions downstream of Edn1 signaling to regulate NC differentiation and cartilage morphogenesis. In addition, we also identify a role for neural adhesion molecule 1.1 (nadl1.1), a known interacting partner of Alcama expressed in neural crest, in NC differentiation. Our data shows that nadl1.1 is required for alcama rescue of NC differentiation in edn1^−/− mutants and that Alcama interacts with Nadl1.1 during chondrogenesis. Collectively our results support a model by which Alcama on the endoderm interacts with Nadl1.1 on NC to mediate Edn1 signaling and NC differentiation during chondrogenesis.     

The role of TGF-beta signaling in regulating chondrogenesis and osteogenesis during mandibular development

During craniofacial development, Meckel's cartilage and the mandible bone derive from the first branchial arch, and their development depends upon the contribution of cranial neural crest (CNC) cells. We previously demonstrated that conditional inactivation of Tgfbr2 in the neural crest of mice (Tgfbr2^fl/fl;Wnt1-Cre) results in severe defects in mandibular development, although the specific cellular and molecular mechanisms by which TGF-β signaling regulates the fate of CNC cells during mandibular development remain unknown. We show here that loss of Tgfbr2 does not affect the migration of CNC cells during mandibular development. TGF-β signaling is specifically required for cell proliferation in Meckel's cartilage and the mandibular anlagen and for the formation of the coronoid, condyle and angular processes. TGF-β-mediated connective tissue growth factor (CTGF) signaling is critical for CNC cell proliferation. Exogenous CTGF rescues the cell proliferation defect in Meckel's cartilage of Tgfbr2^fl/fl;Wnt1-Cre mutants, demonstrating the biological significance of this signaling cascade in chondrogenesis during mandibular development. Furthermore, TGF-β signaling controls Msx1 expression to regulate mandibular osteogenesis as Msx1 expression is significantly reduced in Tgfbr2^fl/fl;Wnt1-Cre mutants. Collectively, our data suggest that there are differential signal cascades in response to TGF-β to control chondrogenesis and osteogenesis during mandibular development.