SummaryDirectional collective migration is now a widely recognized mode of migration during embryogenesis and cancer. However, how a cluster of cells responds to chemoattractants is not fully understood. Neural crest cells are among the most motile cells in the embryo, and their behavior has been likened to malignant invasion. Here, we show that neural crest cells are collectively attracted toward the chemokine Sdf1. While not involved in initially polarizing cells, Sdf1 directionally stabilizes cell protrusions promoted by cell contact. At this cell contact, N-cadherin inhibits protrusion and Rac1 activity and in turn promotes protrusions and activation of Rac1 at the free edge. These results show a role for N-cadherin during contact inhibition of locomotion, and they reveal a mechanism of chemoattraction likely to function during both embryogenesis and cancer metastasis, whereby attractants such as Sdf1 amplify and stabilize contact-dependent cell polarity, resulting in directional collective migration.

Contact Inhibition of Locomotion was discovered by Abercrombie more than 50 years ago to describe the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction upon contact1,2. Its failure was suggested to contribute to malignant invasion3-6. However, the molecular basis of Contact Inhibition of Locomotion and whether it also occurs in vivo are still unknown. Here we show that neural crest cells, a highly migratory and multipotent embryonic cell population, whose behaviour has been likened to malignant invasion6-8, exhibit Contact Inhibition of Locomotion both in vivo and in vitro, and that this accounts for their directional migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display Contact Inhibition of Locomotion; instead, it invades the other tissue, like metastatic cancer cells3,5,9. We show that inhibition of non-canonical Wnt signalling abolishes both Contact Inhibition of Locomotion and the directionality of neural crest migration. Wnt signalling members localise at the site of cell contact, leading to activation of RhoA in this region. These results provide the first example of Contact Inhibition of Locomotion in vivo, present an explanation for coherent directional migration of group of cells and establish a novel role for non-canonical Wnt signalling.

Directed cell migration is crucial for development, but most of our current knowledge is derived from in vitro studies. We analyzed how neural crest (NC) cells migrate in the direction of their target during embryonic development. We show that the proteoglycan Syndecan-4 (Syn4) is expressed in the migrating neural crest of Xenopus and zebrafish embryos. Loss-of-function studies using an antisense morpholino against syn4 show that this molecule is required for NC migration, but not for NC induction. Inhibition of Syn4 does not affect the velocity of cell migration, but significantly reduces the directional migration of NC cells. Furthermore, we show that Syn4 and PCP signaling control the directional migration of NC cells by regulating the direction in which the cell protrusions are generated during migration. Finally, we perform FRET analysis of Cdc42, Rac and RhoA in vitro and in vivo after interfering with Syn4 and PCP signaling. This is the first time that FRET analysis of small GTPases has been performed in vivo. Our results show that Syn4 inhibits Rac activity, whereas PCP signaling promotes RhoA activity. In addition, we show that RhoA inhibits Rac in NC cells. We present a model in which Syn4 and PCP control directional NC migration by, at least in part, regulating membrane protrusions through the regulation of small GTPase activities.

The neural crest is induced by a combination of secreted signals. Although previous models of neural crest induction have proposed a step-wise activation of these signals, the actual spatial and temporal requirement has not been analysed. Through analysing the role of the mesoderm we show for the first time that specification of neural crest requires two temporally and chemically different steps: first, an induction at the gastrula stage dependent on signals arising from the dorsolateral mesoderm; and second, a maintenance step at the neurula stage dependent on signals from tissues adjacent to the neural crest. By performing tissue recombination experiments and using specific inhibitors of different inductive signals, we show that the first inductive step requires Wnt activation and BMP inhibition, whereas the later maintenance step requires activation of both pathways. This change in BMP necessity from BMP inhibition at gastrula to BMP activation at neurula stages is further supported by the dynamic expression of BMP4 and its antagonists, and is confirmed by direct measurements of BMP activity in the neural crest cells. The differential requirements of BMP activity allow us to propose an explanation for apparently discrepant results between chick and frog experiments. The demonstration that Wnt signals are required for neural crest induction by mesoderm solves an additional longstanding controversy. Finally, our results emphasise the importance of considering the order of exposure to signals during an inductive event.

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.

Wnt signalling is required for neural crest (NC) induction; however, the direct targets of the Wnt pathway during NC induction remain unknown. We show here that the homeobox gene Gbx2 is essential in this process and is directly activated by Wnt/β-catenin signalling. By ChIP and transgenesis analysis we show that the Gbx2 regulatory elements that drive expression in the NC respond directly to Wnt/β-catenin signalling. Gbx2 has previously been implicated in posteriorization of the neural plate. Here we unveil a new role for this gene in neural fold patterning. Loss-of-function experiments using antisense morpholinos against Gbx2 inhibit NC and expand the preplacodal domain, whereas Gbx2 overexpression leads to transformation of the preplacodal domain into NC cells. We show that the NC specifier activity of Gbx2 is dependent on the interaction with Zic1 and the inhibition of preplacodal genes such as Six1. In addition, we demonstrate that Gbx2 is upstream of the neural fold specifiers Pax3 and Msx1. Our results place Gbx2 as the earliest factor in the NC genetic cascade being directly regulated by the inductive molecules, and support the notion that posteriorization of the neural folds is an essential step in NC specification. We propose a new genetic cascade that operates in the distinction between anterior placodal and NC territories.

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.

During chick gastrulation, inhibition of BMP signaling is required for primitive streak formation and induction of Hensen's node. We have identified a unique secreted protein, Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family and is expressed in the primitive streak and Hensen's node. Grafts of cells expressing TSK in combination with the middle primitive streak induce an ectopic Hensen's node, while electroporation of TSK siRNA inhibits induction of the node. In Xenopus embryos, TSK can block BMP function and induce a secondary dorsal axis, while it can dorsalize ventral mesoderm and induce neural tissue in embryonic explants. Biochemical analysis shows that TSK binds directly to both BMP and chordin and forms a ternary complex with them. These observations indicate that TSK is an essential dorsalizing factor involved in the induction of Hensen's node.