Cornelis J. Weijer

Cell Movement Patterns during Gastrulation in the Chick Are Controlled by Positive and Negative Chemotaxis Mediated by FGF4 and FGF8

During gastrulation in amniotes, epiblast cells ingress through the primitive streak and migrate away to form endodermal, mesodermal, and extraembryonic structures. Here we analyze the detailed movement trajectories of cells emerging at different anterior-posterior positions from the primitive streak, using in vivo imaging of the movement of GFP-tagged streak cells. Cells emerging at different anterior-posterior positions from the streak show characteristic cell migration patterns, in response to guidance signals from neighboring tissues. Streak cells are attracted by sources of FGF4 and repelled by sources of FGF8. The observed movement patterns of anterior streak cells can be explained by an FGF8-mediated chemorepulsion of cells away from the streak followed by chemoattraction toward an FGF4 signal produced by the forming notochord.

Collective cell migration in development

Collective cell migration is a key process during the development of most organisms. It can involve either the migration of closely packed mesenchymal cells that make dynamic contacts with frequently changing neighbour cells, or the migration of epithelial sheets that typically display more stable cell-cell interactions and less frequent changes in neighbours. These collective movements can be controlled by short- or long-range dynamic gradients of extracellular signalling molecules, depending on the number of cells involved and their distance of migration. These gradients are sensed by some or all of the migrating cells and translated into directed migration, which in many settings is further modulated by cell-contact-mediated attractive or repulsive interactions that result in contact-following or contact-inhibition of locomotion, respectively. Studies of collective migration of groups of epithelial cells during development indicate that, in some cases, only leader cells sense and migrate up an external signal gradient, and that adjacent cells follow through strong cell-cell contacts. In this Commentary, I review studies of collective cell migration of differently sized cell populations during the development of several model organisms, and discuss our current understanding of the molecular mechanisms that coordinate this migration.

PtdIns(3,4,5)P3-Dependent and -Independent Roles for PTEN in the Control of Cell Migration

Summary Background Phosphatase and tensin homolog (PTEN) mediates many of its effects on proliferation, growth, survival, and migration through its PtdIns(3,4,5)P3 lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways. PTEN also possesses a protein phosphatase activity, the role of which is less well characterized. Results We have investigated the role of PTEN in the control of cell migration of mesoderm cells ingressing through the primitive streak in the chick embryo. Overexpression of PTEN strongly inhibits the epithelial-to-mesenchymal transition (EMT) of mesoderm cells ingressing through the anterior and middle primitive streak, but it does not affect EMT of cells located in the posterior streak. The inhibitory activity on EMT is completely dependent on targeting PTEN through its C-terminal PDZ binding site, but can be achieved by a PTEN mutant (PTEN G129E) with only protein phosphatase activity. Expression either of PTEN lacking the PDZ binding site or of the PTEN C2 domain, or inhibition of PI3K through specific inhibitors, does not inhibit EMT, but results in a loss of both cell polarity and directional migration of mesoderm cells. The PTEN-related protein TPTE, which normally lacks any detectable lipid and protein phosphatase activity, can be reactivated through mutation, and only this reactivated mutant leads to nondirectional migration of these cells in vivo. Conclusions PTEN modulates cell migration of mesoderm cells in the chick embryo through at least two distinct mechanisms: controlling EMT, which involves its protein phosphatase activity; and controlling the directional motility of mesoderm cells, through its lipid phosphatase activity.

In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium phagocytosis and chemotaxis

Phagocytosis and chemotaxis are receptor-mediated processes that require extensive rearrangements of the actin cytoskeleton, and are controlled by lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We used a panel of pleckstrin homology (PH) domains with distinct binding specificities for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 to study the spatiotemporal dynamics of these phosphoinositides in vivo. During phagocytosis and macropinocytosis PtdIns(3,4,5)P3 levels transiently increased at sites of engulfment, followed by a rapid PtdIns(3,4)P2 production round the phagosome/macropinosome upon its internalisation, suggesting that PtdIns(3,4,5)P3 is degraded to PtdIns(3,4)P2. PTEN null mutants, which are defective in phagocytosis, showed normal rates of PtdIns(3,4,5)P3degradation, but unexpectedly an accelerated PtdIns(3,4)P2 degradation. During chemotaxis to cAMP only PtdIns(3,4,5)P3 was formed in the plasma membrane, and no PtdIns(3,4)P2 was detectable, showing that all PtdIns(3,4,5)P3 was degraded by PTEN to PtdIns(4,5)P2. Furthermore, we showed that different PtdIns(3,4,5)P3 binding PH domains gave distinct spatial and temporal readouts of the same underlying PtdIns(3,4,5)P3 signal, enabling distinct biological responses to one signal.

Imaging of cell migration

Cell migration is an essential process during many phases of development and adult life. Cells can either migrate as individuals or move in the context of tissues. Movement is controlled by internal and external signals, which activate complex signal transduction cascades resulting in highly dynamic and localised remodelling of the cytoskeleton, cell–cell and cell–substrate interactions. To understand these processes, it will be necessary to identify the critical structural cytoskeletal components, their spatio‐temporal dynamics as well as those of the signalling pathways that control them. Imaging plays an increasingly important and powerful role in the analysis of these spatio‐temporal dynamics. We will highlight a variety of imaging techniques and their use in the investigation of various aspects of cell motility, and illustrate their role in the characterisation of chemotaxis in Dictyostelium and cell movement during gastrulation in chick embryos in more detail.

Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN

PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTENs lipid phosphatase activity in regulating the PI3K signalling pathway is recognized, the significance of PTENs other mechanisms of action is currently unclear. In this study, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant, we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells, the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP3 levels or the downstream protein kinase Akt/PKB. Finally, in three-dimensional Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provide a novel tool to address the significance of PTENs separable lipid and protein phosphatase activities and suggest that both activities suppress proliferation and together suppress invasion.

Visualizing PI3 Kinase-Mediated Cell-Cell Signaling during Dictyostelium Development

BACKGROUND Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signaling. The spatiotemporal dynamics of PI3 kinase signaling can be followed in individual cells via the cAMP-induced membrane recruitment of a GFP-tagged PH domain-containing protein, CRAC, which is required for the activation of adenylylcyclase. RESULTS We show that polarized periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows rapid adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact, it takes 10 s for the signal to propagate from one cell to the next. In slugs, all cells show a permanent polarized PI3 kinase signaling in their leading edge, which is dependent on cell-cell contact. CONCLUSIONS Measuring the redistribution of GFP-tagged CRAC has enabled us to study the dynamics of PI3 kinase-mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signaling, cell polarization, and movement in the development of other organisms.

Wnt3a-mediated chemorepulsion controls movement patterns of cardiac progenitors and requires RhoA function

The heart is the first organ to function during vertebrate development and cardiac progenitors are among the first cell lineages to be established. In the chick, cardiac progenitors have been mapped in the epiblast of pre-streak embryos, and in the early gastrula they are located in the mid-primitive streak, from which they enter the mesoderm bilaterally. Signals controlling the specification of cardiac cells have been well documented; however, migration routes of cardiac progenitors have not been directly observed within the embryo and the factor(s) controlling their movement are not known. In addition, it is not clear how cell movement is coordinated with cell specification in the early embryo. Here we use live imaging to show that cardiac progenitors migrate in highly directed trajectories, which can be controlled by Wnt3a. Ectopic Wnt3a altered movement trajectories and caused cardia bifida. This was rescued by electroporation of dominant-negative DN-Wnt3a into prospective cardiac cells. Explant essays and mutant analysis showed that cellular guidance involved repulsion in response to Wnt3a and required RhoA function. It has been shown that Wnt3a inhibits cardiogenic cell specification through a β-catenin-dependent pathway. On the basis of our results, we propose that Wnt3a concomitantly guides the movement of cardiac progenitors by a novel mechanism involving RhoA-dependent chemorepulsion.

Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation

Primitive streak formation in the chick embryo involves large-scale highly coordinated flows of more than 100,000 cells in the epiblast. These large-scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combination of light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical–basal axis followed by ingression, and asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localization and direction of which correlate strongly with the appearance of active myosin II cables in aligned apical junctions in neighbouring cells. Use of class specific myosin inhibitors and gene-specific knockdown shows that apical contraction and intercalation are myosin II dependent and also reveal critical roles for myosin I and myosin V family members in the assembly of junctional myosin II cables.

Paxillin is required for cell-substrate adhesion, cell sorting and slug migration during Dictyostelium development

Paxillin is a key regulatory component of focal adhesion sites, implicated in controlling cell-substrate interactions and cell movement. We analyse the function of aDictyostelium discoideumpaxillin homologue, PaxB, which contains four highly conserved LD and four LIM domains, but lacks two characteristic tyrosine residues, that form the core of vertebrate SH2-binding domains. PaxB is expressed during growth and all stages of development, but expression peaks during slug formation. Using apaxB-gfpknockin strain we show the existence of focal adhesions and characterise their dynamics. During multicellular development PaxB is not only found in focal adhesions at the cell-substrate interface, but also in the tips of filopodial structures predominantly located at the trailing ends of cells.paxB–strains are less adhesive to the substrate, they can aggregate but multicellular development from the mound stage onwards is severely impeded.paxB– strains are defective in proper cell type proportioning, cell sorting, slug migration and form-defective fruiting bodies. Mutation of a conserved JNK phosphorylation site, implicated in the control of cell migration, does not have any major effects on cell sorting, slug migration or morphogenesis inDictyostelium. PaxB does not appear to function redundantly with its closest relative Lim2 (paxA), which when deleted also results in a mound arrest phenotype. However, analysis ofpaxA–andpaxB–single and double null mutants suggest that PaxB may act upstream of Lim2.