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Patterns and Control of Cell Motility in the Xenopus Gastrula

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Patterns and Control of Cell Motility in the Xenopus Gastrula Development 125, 1931-1942 (1998) 1931 Printed in Great Britain © The Company of Biologists Limited 1998 DEV6326 Patterns and control of cell motility in the Xenopus gastrula Stephan Wacker1, Anja Brodbeck1, Patrick Lemaire2, Christof Niehrs3 and Rudolf Winklbauer1,* 1Universität zu Köln, Zoologisches Institut, Weyertal 119, 50931 Köln, Germany 2Laboratoire de Génétique et Physiologie de Développement, CNRS-Université de la Méditerranée, Institut de Biologie du Développement de Marseille, Case 907, Campus de Luminy, F-13288 Marseille Cedex 09, France 3Division of Molecular Embryology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany *Author for correspondence (e-mail: [email protected]) Accepted 3 March; published on WWW 22 April 1998 SUMMARY By comparing cells with respect to several motility-related another immediate early gene, is without effect when properties and the ability to migrate on fibronectin, three expressed alone in animal cap cells, but it acts cell types can be distinguished in the Xenopus gastrula. synergistically with Mix.1 in the control of adhesion, and These occur in a distinct spatial pattern, thus defining three antagonistically in the polarization of protrusive activity. motility domains which do not correspond to the bFGF also induces migration, lamellipodia formation and prospective germ layers. Migratory behavior is confined to polarization in animal cap cells, but has no effect on a region encompassing the anterior mesoderm and adhesion. By the various treatments of animal cap cells, endoderm. When stationary animal cap cells are induced new combinations of motile properties can be generated, to migrate by treatment with activin, cells become adhesive yielding cell types which are not found in the embryo. at low concentrations of fibronectin, show polarized protrusive activity, and form lamellipodia. Adhesion and polarization, but not lamellipodia formation, are mimicked Key words: Xenopus, Mesoderm induction, Cell migration, Motility, by the immediate early response gene Mix.1. Goosecoid, goosecoid, Mix.1, Activin, FGF, Adhesion, Cell polarity, Gastrula INTRODUCTION followed by prospective axial and paraxial mesoderm, until all of the mesoderm has become internalized. The vegetal cell During amphibian gastrulation, several morphogenetic mass is held to be moved passively to the interior. The processes cooperate to transform a simple vesicle-like blastula prospective ectoderm remains on the outside and spreads to into a multilayered structure representing the basic body plan cover the whole embryo in the process of epiboly (Keller, 1986; of the organism. These region-specific morphogenetic Keller and Winklbauer, 1992, for review). movements are thought to be driven by defined, spatially Once inside the embryo, the mesoderm attaches to the BCR differentiated cell behaviors, such as, active cell shape changes, and moves toward the animal pole. Two processes are known cell intercalation, or migration (Keller, 1986; Gerhart and to be associated with mesoderm translocation. First, cell Keller, 1986). While at the descriptive level, knowledge on cell intercalation leads to substrate-independent narrowing and behavior in different regions of the amphibian gastrula has lengthening of the axial/paraxial mesoderm, i.e. to dorsal accumulated (for review see Keller and Winklbauer, 1992), not convergence and extension (Keller et al., 1992). Second, much is known yet about the molecular pathways that control mesoderm cells which contact the BCR show migratory these cellular activities. behavior (Nakatsuji and Johnson, 1982; Winklbauer and In the Xenopus blastula, the wall enclosing the blastocoel Nagel, 1991). This latter process will be examined in the cavity is formed by a thin blastocoel roof (BCR) and a massive present paper. blastocoel floor. The BCR consists of small animal In Xenopus, fibronectin (FN) which forms a fibril network blastomeres, and the blastocoel floor of large, yolk-rich vegetal on the BCR is essential for migration. When interaction with blastomeres. The transition zone between the two regions, the FN is inhibited, mesoderm cells adhere to the BCR, but cease marginal zone, is fated to become mainly mesoderm, whereas to form locomotory protrusions and to migrate (Winklbauer the BCR above it represents prospective ectoderm, and the and Keller, 1996). Mesoderm cell migration can also be studied vegetal base will contribute to the endoderm (for review see on FN in vitro. As in the embryo, cells employ lamellipodia Keller, 1986). for translocation, which are induced by contact to FN. Isolated At the beginning of gastrulation, a blastopore invaginates at cells are typically spindle-shaped and move in an intermittent the vegetal boundary of the mesoderm mantle. The mesoderm and non-persistent fashion (Nakatsuji and Johnson, 1982; above it begins to involute by rolling over the blastopore lip. Winklbauer and Selchow, 1992; Winklbauer and Keller, 1996). Dorsally, prospective head mesoderm is first to involute, The Xenopus embryo offers an opportunity to study the 1932 S. Wacker and others control of cell migration at the molecular level. Stationary BCR Adhesion assay cells can be induced to form mesoderm by treatment with A field in a Greiner 35 mm Petri dish (TC quality) was coated with growth factors, and induced cells migrate on FN in vitro (Smith 20 µg/ml of bovine plasma fibronectin (30 minutes) and saturated with et al., 1990a; Smith and Howard, 1992; Howard and Smith, 5% BSA. Stage 10+ animal cap or head mesoderm explants were 1993; Ramos and DeSimone, 1996). Mesoderm inducing dissociated. Cells were seeded onto the FN-coated area. After 45 factors include activins (Asashima et al., 1990; Smith et al., minutes in MBS, cells were counted, non-adherent cells were removed 1990b) and fibroblast growth factors (Slack et al., 1987; by inverting the dish in a tank containing MBS, and remaining cells Kimelman and Kirschner, 1987). The inductive signals become were counted again. effective at the onset of zygotic transcription in the middle Migration assay blastula, when they direct a first wave of mesodermal gene Petri dishes were coated with 100-200 µg/ml of FN and saturated with expression which is independent of protein synthesis and 5% BSA. After spreading on this substrate, cells were recorded for at therefore qualifies as an immediate early response to induction. least 1 hour using a Zeiss IM 35 microscope, and a Panasonic CL- Many of these early expressed genes code for transcription 700 CCD-camera and AG-6720 video recorder in the time lapse mode factors which are assumed to control target genes responsible (40 fold). For each cell, positions were determined at 10 minute for eventual mesoderm differentiation. Among the early genes intervals over 1 hour. From the length of its path, the average velocity are the activin-induced, paired-class homeodomain containing of a cell was calculated. genes goosecoid (gsc) (Cho et al., 1991) and Mix.1 (Rosa, Cell polarity assay 1989). Cells were seeded into dishes rendered non-adhesive by blocking with Here we further dissect the control mechanisms that 5% BSA. Protrusive activity in MBS was visualized by phase contrast determine the motile behavior of mesodermal cells. We microscopy and recorded in the time lapse mode (8 fold) for at least distinguish, on the basis of a small set of elementary, motility- 2 hours. Angular distances between successively appearing filopodia related features, three types of cells in the gastrula, one were measured. migratory and two stationary. We investigate how specific features are changed when BCR cells are treated with mesoderm inducers or downstream effectors of induction and we show that some of the effects of mesoderm induction on RESULTS cell motility appear to be mediated by gsc and Mix.1. Anterior mesoderm and endoderm cells are migratory at gastrulation Cells from all regions of the early gastrula adhere at high FN MATERIALS AND METHODS density and can be tested for migration on this substrate (Fig. 1). Animal cap cells are stationary on FN: they are motile and Embryos and explants move incessantly back and forth, which yields velocities above Xenopus laevis embryos obtained from induced spawnings were zero, but they do not translocate (Fig. 1A). Prospective staged according to Nieuwkoop and Faber (1967). Operation neuroectodermal cells from the margin of the BCR show the techniques and buffers (modified Barth’s solution, MBS; dissociation same behavior (Fig. 1B). buffer) have been described by Winklbauer (1990). Adjacent to the BCR, prospective axial/paraxial mesoderm Mesoderm induction in BCR cells forms the dorsal blastopore lip of the early gastrula. These cells (a) 200 nl of MBS containing 200 units/ml of human recombinant show a broad distribution of velocities. Below 1 µm/minute, it activin A were injected into the blastocoel of stage 9 embryos. At overlaps with that of the stationary BCR cells, but a large stage 10+, animal caps were explanted and dissociated. (b) Stage 9.5 fraction of cells is migratory and translocates at rates up to 3 animal cap was explanted and dissociated for 45 minutes. Cells were µm/minute (Fig. 1C). The more anterior dorsal mesoderm, i.e. seeded onto FN in MBS containing 20 units/ml of activin. (c) Animal prospective head mesoderm, has already involuted. All cells caps were explanted at stage 8, incubated for 2 hours in 50 ng/ml of from this region migrate, and the distribution of velocities does human recombinant bFGF in MBS (TEBU, Frankfurt,
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