Reiterated Wnt signaling during zebrafish neural crest development

While Wnt/beta-catenin signaling is known to be involved in the development of neural crest cells in zebrafish, it is unclear which Wnts are involved, and when they are required. To address these issues we employed a zebrafish line that was transgenic for an inducible inhibitor of Wnt/beta-catenin signaling, and inhibited endogenous Wnt/beta-catenin signaling at discrete times in development. Using this approach, we defined a critical period for Wnt signaling in the initial induction of neural crest, which is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interfered with neural crest formation without blocking development of dorsal spinal neurons. Transplantation experiments suggest that neural crest precursors must directly transduce a Wnt signal. With regard to identifying which endogenous Wnt is responsible for this initial critical period, we established that wnt8 is expressed in the appropriate time and place to participate in this process. Supporting a role for Wnt8, blocking its function with antisense morpholino oligonucleotides eliminates initial expression of neural crest markers. Taken together, these results demonstrate that Wnt signals are critical for the initial induction of zebrafish neural crest and suggest that this signaling pathway plays reiterated roles in its development.     

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

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

Cadherin-11 Mediates Contact Inhibition of Locomotion during Xenopus Neural Crest Cell Migration

Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration in vivo. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that Xenopus cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour in vivo is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC.     

Essential role of non-canonical Wnt signalling in neural crest migration

Migration of neural crest cells is an elaborate process that requires the delamination of cells from an epithelium and cell movement into an extracellular matrix. In this work, it is shown for the first time that the non-canonical Wnt signalling [planar cell polarity (PCP) or Wnt-Ca2+] pathway controls migration of neural crest cells. By using specific Dsh mutants, we show that the canonical Wnt signalling pathway is needed for neural crest induction, while the non-canonical Wnt pathway is required for neural crest migration. Grafts of neural crest tissue expressing non-canonical Dsh mutants, as well as neural crest cultured in vitro, indicate that the PCP pathway works in a cell-autonomous manner to control neural crest migration. Expression analysis of non-canonical Wnt ligands and their putative receptors show that Wnt11 is expressed in tissue adjacent to neural crest cells expressing the Wnt receptor Frizzled7 (Fz7). Furthermore, loss- and gain-of-function experiments reveal that Wnt11 plays an essential role in neural crest migration. Inhibition of neural crest migration by blocking Wnt11 activity can be rescued by intracellular activation of the non-canonical Wnt pathway. When Wnt11 is expressed opposite its normal site of expression, neural crest migration is blocked. Finally, time-lapse analysis of cell movement and cell protrusion in neural crest cultured in vitro shows that the PCP or Wnt-Ca2+ pathway directs the formation of lamellipodia and filopodia in the neural crest cells that are required for their delamination and/or migration.     

Frizzled7 mediates canonical Wnt signaling in neural crest induction

The neural crest is a multipotent cell population that migrates from the dorsal edge of the neural tube to various parts of the embryo where it differentiates into a remarkable variety of different cell types. Initial induction of neural crest is mediated by a combination of BMP, Wnt, FGF, Retinoic acid and Notch/Delta signaling. The two-signal model for neural crest induction suggests that BMP signaling induces the competence to become neural crest. The second signal involves Wnt acting through the canonical pathway and leads to expression of neural crest markers such as slug. Wnt signals from the neural plate, non-neural ectoderm and paraxial mesoderm have all been suggested to play a role in neural crest induction. We show that Xenopus frizzled7 (Xfz7) is expressed in the dorsal ectoderm including early neural crest progenitors and is a key mediator of the Wnt inductive signal. We demonstrate that Xfz7 expression is induced in response to a BMP antagonist, noggin, and that Xfz7 can induce neural crest specific genes in noggin-treated ectodermal explants (animal caps). Morpholino-mediated or dominant negative inhibition of Xfz7 inhibits Wnt induced Xslug expression in the animal cap assay and in the whole embryo leading to a loss of neural crest derived pigment cells. Full-length Xfz7 rescues the morpholino-induced phenotype, as does activated beta-catenin, suggesting that Xfz7 is signaling through the canonical pathway. We therefore demonstrate that Xfz7 is regulated by BMP antagonism and is required for neural crest induction by Wnt in the developing vertebrate embryo.     

Molecular analysis of neural crest migration

The neural crest (NC) cells have been called the 'explorers of the embryos' because they migrate all over the embryo where they differentiate into a variety of diverse kinds of cells. In this work, we analyse the role of different molecules controlling the migration of NC cells. First, we describe the strong similarity between the process of NC migration and metastasis in tumour cells. The epithelial-mesenchymal transition process that both kinds of cells undergo is controlled by the same molecular machinery, including cadherins, connexins, Snail and Twist genes and matrix metalloproteases. Second, we analysed the molecular signals that control the patterned migration of the cephalic and trunk NC cells. Most of the factors described so far, such as Eph/ephrins, semaphorins/neuropilins and Slit/Robo, are negative signals that prohibit the migration of NC cells into target areas of the embryo. Finally, we analyse how the direction of migration is controlled by regulation of cell polarity and how the planar cell polarity or non-canonical Wnt signalling is involved in this process.     

Activity of the RhoU/Wrch1 GTPase is critical for cranial neural crest cell migration

The neural crest (NC) is a stem cell-like population that arises at the border of neural and non-neural ectoderm. During development, NC undergoes an epithelio-mesenchymal transition (EMT), i.e. loss of epithelial junctions and acquisition of pro-migratory properties, invades the entire embryo and differentiates into a wide diversity of terminal tissues. We have studied the implication of Rho pathways in NC development and previously showed that RhoV is required for cranial neural crest (CNC) cell specification. We show here that the non-canonical Wnt response rhoU/wrch1 gene, closely related to rhoV, is also expressed in CNC cells but at later stages. Using both gain- and loss-of-function experiments, we demonstrate that the level of RhoU expression is critical for CNC cell migration and subsequent differentiation into craniofacial cartilages. In in vitro cultures, RhoU activates pathways that cooperate with PAK1 and Rac1 in epithelial adhesion, cell spreading and directional cell migration. These data support the conclusion that RhoU is an essential regulator of CNC cell migration.     

BMP signaling is essential for development of skeletogenic and neurogenic cranial neural crest

BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.     

Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns

The neural crest is a uniquely vertebrate cell type present in the most basal vertebrates, but not in cephalochordates. We have studied differences in regulation of the neural crest marker AP-2 across two evolutionary transitions: invertebrate to vertebrate, and agnathan to gnathostome. Isolation and comparison of amphioxus, lamprey and axolotl AP-2 reveals its extensive expansion in the vertebrate dorsal neural tube and pharyngeal arches, implying co-option of AP-2 genes by neural crest cells early in vertebrate evolution. Expression in non-neural ectoderm is a conserved feature in amphioxus and vertebrates, suggesting an ancient role for AP-2 genes in this tissue. There is also common expression in subsets of ventrolateral neurons in the anterior neural tube, consistent with a primitive role in brain development. Comparison of AP-2 expression in axolotl and lamprey suggests an elaboration of cranial neural crest patterning in gnathostomes. However, migration of AP-2-expressing neural crest cells medial to the pharyngeal arch mesoderm appears to be a primitive feature retained in all vertebrates. Because AP-2 has essential roles in cranial neural crest differentiation and proliferation, the co-option of AP-2 by neural crest cells in the vertebrate lineage was a potentially crucial event in vertebrate evolution.     

Wnt-frizzled signaling in the induction and differentiation of the neural crest

The neural crest is a transient population of multipotent progenitors arising at the lateral edge of the neural plate in vertebrate embryos. After delamination and migration from the neuroepithelium, these cells contribute to a diverse array of tissues including neurons, smooth muscle, craniofacial cartilage, bone cells, endocrine cells and pigment cells. Considerable progress in recent years has furthered our understanding at a molecular level of how this important group of cells is generated and how they are assigned to specific lineages. Here we review a number of recent studies supporting a role for Wnt signaling in neural crest induction, differentiation, and apoptosis. We also summarize the timing of expression of a number of Wnt ligands and receptors with respect to neural crest induction.     

A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration

Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.     

Dynamic alterations in gene expression after Wnt-mediated induction of avian neural crest

The Wnt signaling pathway is important in the formation of neural crest cells in many vertebrates, but the downstream targets of neural crest induction by Wnt are largely unknown. Here, we examined quantitative changes in gene expression regulated by Wnt-mediated neural crest induction using quantitative PCR (QPCR). Induction was recapitulated in vitro by adding soluble Wnt to intermediate neural plate tissue cultured in collagen, and induced versus control tissue were assayed using gene-specific primers at times corresponding to premigratory (18 and 24 h) or early (36 h) stages of crest migration. The results show that Wnt signaling up-regulates in a distinct temporal pattern the expression of several genes normally expressed in the dorsal neural tube (slug, Pax3, Msx1, FoxD3, cadherin 6B) at "premigratory" stages. While slug is maintained in early migrating crest cells, Pax3, FoxD3, Msx1 and cadherin 6B all are down-regulated by the start of migration. These results differ from the temporal profile of these genes in response to the addition of recombinant BMP4, where gene expression seems to be maintained. Interestingly, expression of rhoB is unchanged or even decreased in response to Wnt-mediated induction at all times examined, though it is up-regulated by BMP signals. The temporal QPCR profiles in our culture paradigm approximate in vivo expression patterns of these genes before neural crest migration, and are consistent with Wnt being an initial neural crest inducer with additional signals like BMP and other factors maintaining expression of these genes in vivo. Our results are the first to quantitatively describe changes in gene expression in response to a Wnt or BMP signal during transformation of a neural tube cell into a migratory neural crest cell.