Summary3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype.

Tie2 belongs to the receptor tyrosine kinase family and functions as a receptor for Angiopoietin-1 (Ang1). Gene-targeting analyses of either Ang1 or Tie2 in mice reveal a critical role of Ang1-Tie2 signalling in developmental vascular formation. It remains elusive how the Tie2 signalling pathway plays distinct roles in both vascular quiescence and angiogenesis. We demonstrate here that Ang1 bridges Tie2 at cell-cell contacts, resulting in trans-association of Tie2 in the presence of cell-cell contacts. In clear contrast, in isolated cells, extracellular matrix-bound Ang1 locates Tie2 at cell-substratum contacts. Furthermore, Tie2 activated at cell-cell or cell-substratum contacts leads to preferential activation of Akt and Erk, respectively. Microarray analyses and real-time PCR validation clearly show the differential gene expression profile in vascular endothelial cells upon Ang1 stimulation in the presence or absence of cell-cell contacts, implying downstream signalling is dependent upon the spatial localization of Tie2.

Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherindependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with ␤-catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca 2؉ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca 2؉ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion. INTRODUCTIONIntercellular adhesion of vascular endothelial cells is essential for connecting neighboring endothelial cells to develop a vascular tree and to function as a barrier separating blood and tissues. Vascular endothelial cell adhesion is characterized by the overlapping of adherens junctions (AJs) and tight junctions (TJs). AJs are constituted by vascular endothelial cadherin (VE-cadherin) in close cooperation with platelet and endothelial adhesion molecule-1 (PECAM-1) and nectin. VE-cadherin-mediated cell adhesion depends on extracellular Ca 2ϩ , but not those mediated by PECAM-1 and nectin. TJs are made up of junctional adhesion molecule (JAM) family members, occuludin, claudin-5, and nectin (reviewed in Dejana, 2004).VE-cadherin has an extracellular domain constituted by five cadherin domains, a transmembrane domain, and a cytoplasmic domain connected to p120 catenin and ␤-catenin (Iyer et al., 2004). Through ␤-catenin, VE-cadherin is linked to ␣-catenin that is associated with the actin cytoskeleton, which results in the maintenance of cell-cell adhesion in conjunction with cytoskeleton (Herren et al., 1998;Navarro et al., 1998;Kobielak and Fuchs, 2004). Tyrosine-phosphorylated VE-cadherin in its cytoplasmic domain provides docking sites for signal-transmitting molecules (Esser et al., 1998;Zanetti et al., 2002;Hudry-Clergeon et al., 2005). Conversely, cytoplasmic domain modified by phosphorylation or associated with signaling molecules triggers the insideout signal that regulates the VE-cadherin-mediated cell adhesion (Nwariaku et al., 2004). ␤-catenin binds to other signaling molecules including PI3-K and MAGUK with inverted domain structure-1 (MAGI-1) as well as ␣-catenin (Kotelevets et al., 2005).MAGI-1 consists of six PSD95/DiscLarge/ZO-1 (PDZ) domains, a guanylate ...

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group previously showed that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts. We further demonstrated that the former up-regulates Notch ligand delta-like 4 (Dll4) only in the presence of cell-cell contacts. Because Dll4/Notch signal restricts sprouting angiogenesis and promotes vascular stabilization, we investigated the mechanism of how the Ang1/Tie2 signal induces Dll4 expression to clarify the role of the Dll4/Notch signal in Ang1/Tie2 signal-mediated vascular quiescence. Under confluent endothelial cells, the basal Notch signal was observed. Ang1, moreover, induced Dll4 expression and production of the Notch intracellular domain (NICD). Ang1 stimulated transcriptional activity of ␤-catenin through phosphoinositide 3-kinase (PI3K)/AKTmediated phosphorylation of glycogen synthase kinase 3␤ (GSK3␤). Correspondingly, the GSK3␤ inhibitor up-regulated Dll4, whereas depletion of ␤-catenin by siRNA blocked Ang1-induced Dll4 expression, indicating the indispensability of ␤-catenin in Ang1-mediated up-regulation of Dll4. In addition, Dll4 expression by the GSK3␤ inhibitor was only observed in confluent cells, and was impeded by DAPT, a ␥-secretase inhibitor, implying requirement of the Notch signal in ␤-catenin-dependent Dll4 expression. Consistently, we found that either Ang1 or NICD up-regulates Dll4 through the RBP-J binding site within intron 3 of the DLL4 gene and that ␤-catenin forms a complex with NICD/RBP-J to enhance Dll4 expression. Ang1 induced the deposition of extracellular matrix that is preferable for basement membrane formation through Dll4/Notch signaling. Collectively, the Ang1/Tie2 signal potentiates basal Notch signal controlling vascular quiescence by up-regulating Dll4 through AKT-mediated activation of ␤-catenin.

During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation.