Patterns and control of cell motility in the Xenopus gastrula

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1 Development 125, (1998) Printed in Great Britain The Company of Biologists Limited 1998 DEV Patterns and control of cell motility in the Xenopus gastrula Stephan Wacker 1, Anja Brodbeck 1, Patrick Lemaire 2, Christof Niehrs 3 and Rudolf Winklbauer 1, * 1 Universität zu Köln, Zoologisches Institut, Weyertal 119, 931 Köln, Germany 2 Laboratoire de Génétique et Physiologie de Développement, CNRS-Université de la Méditerranée, Institut de Biologie du Développement de Marseille, Case 97, Campus de Luminy, F Marseille Cedex 9, France 3 Division of Molecular Embryology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 28, 6912 Heidelberg, Germany *Author for correspondence ( rwinkl@novell.biolan.uni-koeln.de) Accepted 3 March; published on WWW 22 April 1998 SUMMARY By comparing cells with respect to several motility-related properties and the ability to migrate on fibronectin, three cell types can be distinguished in the Xenopus gastrula. These occur in a distinct spatial pattern, thus defining three motility domains which do not correspond to the prospective germ layers. Migratory behavior is confined to a region encompassing the anterior mesoderm and endoderm. When stationary animal cap cells are induced to migrate by treatment with activin, cells become adhesive at low concentrations of fibronectin, show polarized protrusive activity, and form lamellipodia. Adhesion and polarization, but not lamellipodia formation, are mimicked by the immediate early response gene Mix.1. Goosecoid, another immediate early gene, is without effect when expressed alone in animal cap cells, but it acts synergistically with Mix.1 in the control of adhesion, and antagonistically in the polarization of protrusive activity. bfgf also induces migration, lamellipodia formation and polarization in animal cap cells, but has no effect on adhesion. By the various treatments of animal cap cells, new combinations of motile properties can be generated, yielding cell types which are not found in the embryo. Key words: Xenopus, Mesoderm induction, Cell migration, Motility, goosecoid, Mix.1, Activin, FGF, Adhesion, Cell polarity, Gastrula INTRODUCTION During amphibian gastrulation, several morphogenetic processes cooperate to transform a simple vesicle-like blastula into a multilayered structure representing the basic body plan of the organism. These region-specific morphogenetic movements are thought to be driven by defined, spatially differentiated cell behaviors, such as, active cell shape changes, cell intercalation, or migration (Keller, 1986; Gerhart and Keller, 1986). While at the descriptive level, knowledge on cell behavior in different regions of the amphibian gastrula has accumulated (for review see Keller and Winklbauer, 1992), not much is known yet about the molecular pathways that control these cellular activities. In the Xenopus blastula, the wall enclosing the blastocoel cavity is formed by a thin blastocoel roof (BCR) and a massive blastocoel floor. The BCR consists of small animal blastomeres, and the blastocoel floor of large, yolk-rich vegetal blastomeres. The transition zone between the two regions, the marginal zone, is fated to become mainly mesoderm, whereas the BCR above it represents prospective ectoderm, and the vegetal base will contribute to the endoderm (for review see Keller, 1986). At the beginning of gastrulation, a blastopore invaginates at the vegetal boundary of the mesoderm mantle. The mesoderm above it begins to involute by rolling over the blastopore lip. Dorsally, prospective head mesoderm is first to involute, followed by prospective axial and paraxial mesoderm, until all of the mesoderm has become internalized. The vegetal cell mass is held to be moved passively to the interior. The prospective ectoderm remains on the outside and spreads to cover the whole embryo in the process of epiboly (Keller, 1986; Keller and Winklbauer, 1992, for review). Once inside the embryo, the mesoderm attaches to the BCR and moves toward the animal pole. Two processes are known to be associated with mesoderm translocation. First, cell intercalation leads to substrate-independent narrowing and lengthening of the axial/paraxial mesoderm, i.e. to dorsal convergence and extension (Keller et al., 1992). Second, mesoderm cells which contact the BCR show migratory behavior (Nakatsuji and Johnson, 1982; Winklbauer and Nagel, 1991). This latter process will be examined in the present paper. In Xenopus, fibronectin (FN) which forms a fibril network on the BCR is essential for migration. When interaction with FN is inhibited, mesoderm cells adhere to the BCR, but cease to form locomotory protrusions and to migrate (Winklbauer and Keller, 1996). Mesoderm cell migration can also be studied on FN in vitro. As in the embryo, cells employ lamellipodia for translocation, which are induced by contact to FN. Isolated cells are typically spindle-shaped and move in an intermittent and non-persistent fashion (Nakatsuji and Johnson, 1982; Winklbauer and Selchow, 1992; Winklbauer and Keller, 1996). The Xenopus embryo offers an opportunity to study the

2 1932 S. Wacker and others control of cell migration at the molecular level. Stationary BCR cells can be induced to form mesoderm by treatment with growth factors, and induced cells migrate on FN in vitro (Smith et al., 199a; Smith and Howard, 1992; Howard and Smith, 1993; Ramos and DeSimone, 1996). Mesoderm inducing factors include activins (Asashima et al., 199; Smith et al., 199b) and fibroblast growth factors (Slack et al., 1987; Kimelman and Kirschner, 1987). The inductive signals become effective at the onset of zygotic transcription in the middle blastula, when they direct a first wave of mesodermal gene expression which is independent of protein synthesis and therefore qualifies as an immediate early response to induction. Many of these early expressed genes code for transcription factors which are assumed to control target genes responsible for eventual mesoderm differentiation. Among the early genes are the activin-induced, paired-class homeodomain containing genes goosecoid (gsc) (Cho et al., 1991) and Mix.1 (Rosa, 1989). Here we further dissect the control mechanisms that determine the motile behavior of mesodermal cells. We distinguish, on the basis of a small set of elementary, motilityrelated features, three types of cells in the gastrula, one 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 cell motility appear to be mediated by gsc and Mix.1. MATERIALS AND METHODS Embryos and explants Xenopus laevis embryos obtained from induced spawnings were staged according to Nieuwkoop and Faber (1967). Operation techniques and buffers (modified Barth s solution, MBS; dissociation buffer) have been described by Winklbauer (199). Mesoderm induction in BCR cells (a) 2 nl of MBS containing 2 units/ml of human recombinant activin A were injected into the blastocoel of stage 9 embryos. At stage +, animal caps were explanted and dissociated. (b) Stage 9.5 animal cap was explanted and dissociated for 45 minutes. Cells were seeded onto FN in MBS containing 2 units/ml of activin. (c) Animal caps were explanted at stage 8, incubated for 2 hours in ng/ml of human recombinant bfgf in MBS (TEBU, Frankfurt, Germany), cultured in MBS as required, and dissociated. mrna synthesis and injection Full length goosecoid mrna was synthesized from EcoRI linearized pspgsc (Niehrs et al., 1994) by transcription with SP6 RNA polymerase, Mix.1 mrna from Sfi1 linearized pbsrn3mix.1 (Lemaire et al., unpublished) by transcription with T3 RNA polymerase. Dominant negative (XFD) and nonfunctional (d) FGF receptor clones constructed by Enrique Amaya and Marc Kirschner (Amaya et al., 1991) were used. Linearized plasmids were transcribed with SP6 RNA polymerase. At the 8-cell stage, 8 nl of RNA solution was injected in each of the 4 animal blastomeres. TRITC-phalloidin staining Cells on FN were fixed in 4 formaldehyde in MBS for 5 minutes, permeabilized by the addition of.1 Triton X- for another minutes, blocked with.1 BSA, and stained with 1 µg/ml of TRITC-phalloidin for 2 minutes. Adhesion assay A field in a Greiner 35 mm Petri dish (TC quality) was coated with 2 µg/ml of bovine plasma fibronectin ( minutes) and saturated with 5 BSA. Stage + animal cap or head mesoderm explants were dissociated. Cells were seeded onto the FN-coated area. After 45 minutes in MBS, cells were counted, non-adherent cells were removed by inverting the dish in a tank containing MBS, and remaining cells were counted again. Migration assay Petri dishes were coated with -2 µg/ml of FN and saturated with 5 BSA. After spreading on this substrate, cells were recorded for at least 1 hour using a Zeiss IM 35 microscope, and a Panasonic CL- 7 CCD-camera and AG-672 video recorder in the time lapse mode (4 fold). For each cell, positions were determined at minute intervals over 1 hour. From the length of its path, the average velocity of a cell was calculated. Cell polarity assay Cells were seeded into dishes rendered non-adhesive by blocking with 5 BSA. Protrusive activity in MBS was visualized by phase contrast microscopy and recorded in the time lapse mode (8 fold) for at least 2 hours. Angular distances between successively appearing filopodia were measured. RESULTS Anterior mesoderm and endoderm cells are migratory at gastrulation Cells from all regions of the early gastrula adhere at high FN density and can be tested for migration on this substrate (Fig. 1). Animal cap cells are stationary on FN: they are motile and move incessantly back and forth, which yields velocities above zero, but they do not translocate (Fig. 1A). Prospective neuroectodermal cells from the margin of the BCR show the same behavior (Fig. 1B). Adjacent to the BCR, prospective axial/paraxial mesoderm forms the dorsal blastopore lip of the early gastrula. These cells show a broad distribution of velocities. Below 1 ute, it overlaps with that of the stationary BCR cells, but a large fraction of cells is migratory and translocates at rates up to 3 ute (Fig. 1C). The more anterior dorsal mesoderm, i.e. prospective head mesoderm, has already involuted. All cells from this region migrate, and the distribution of velocities does not overlap with that of BCR cells (Fig. 1D). Occasionally, head mesoderm preparations are contaminated by large cells from the adjacent endoderm. These are also migratory (not shown). In contrast, cells from the vegetal base endoderm are mostly stationary (Fig. 1E). In the late gastrula, the most anterior part of the cell mass advancing on the BCR consists of large endoderm cells which are separated from the BCR by a thin layer of small cells extending up to the leading edge (e.g. Hausen and Riebesell, 1991), presumably prospective head mesoderm. Although not in contact with the BCR substrate in the embryo, the dorsalanterior prospective endoderm cells migrate well on FN in vitro (Fig. 1F). Also, head mesoderm cells migrate vigorously (not shown), as before (see Fig. 1D). By the late gastrula stage, most of the axial/paraxial mesoderm has left the blastopore lip and become apposed to the BCR. Anteriorly, these cells are mainly migratory (Fig.

3 Cell motility in the Xenopus gastrula G), but posteriorly, cells are predominantly stationary (Fig. 1H). Super-imposing the velocity distributions of anterior and posterior axial/paraxial mesoderm would yield a distribution similar to that of the early gastrula blastopore lip. This suggests that involution of the axial/paraxial mesoderm is not accompanied by a transition from stationary to migratory behavior. In summary, we conclude that the cells of the anterior mesoderm, including the head mesoderm and the anterior axial/paraxial mesoderm, and the adjacent dorsalanterior endoderm cells are migratory on FN. In contrast, most of the posterior mesoderm cells, and all cells of the BCR, are stationary. Also, most vegetal cells do not translocate on FN. Protrusive activity differs between migratory mesoderm cells, BCR cells, and vegetal cells On non-adhesive substrate, all gastrula cells extend filopodia, but the pattern differs between cell types. In head mesoderm cells, most filopodia form within a narrow zone, giving cells a polarized appearance (Fig. 2A; Winklbauer and Selchow, 1992). Consequently, angles between successively appearing filopodia are typically small (Fig. 2C). In contrast, cells from the BCR are non-polar. Filopodia extend in all directions (Fig. 2B), leading to an even distribution of angles (Fig. 2D,E). The same non-polarized distribution is observed for vegetal cells (Fig. 2F). Gastrula cells differ also in the A B C D E F AC NIMZ BPL HM VG DAE S.25 S.25 S.25 S.5 S.25 S12 Fig. 1. FN-dependent migration of gastrula cells. For defined regions (boxed region in right panel), average velocities () of 4-8 cells from at least 3 experiments were determined, and their frequencies () plotted (left panel). (A-E) stage + or.5; (A) animal cap, (B) prospective neuroectoderm of non-involuting marginal zone, (C) dorsal blastopore lip, (D) head mesoderm, (E) vegetal base endoderm. (F-H) stage 12; (F) dorsalanterior prospective endoderm, (G) posterior head mesoderm and anterior axial/paraxial mesoderm, (H) posterior axial/paraxial mesoderm. G H AM anterior AM posterior S12 S12

4 1934 S. Wacker and others HM A C 4 2 AC B * HM * Table 1. Frequency of head mesoderm-like cells in anterior to posterior regions of the dorsal axis Head mesoderm-like cells Stage Stage 12 HM 91.9 (4.4) 92.5 (5.7) aam 82.2 (6.) pam 55.6 (5.3) BPL 53.9 (5.3) 7. (3.4) NIMZ 1.8 (1.5) 1.7 (1.2) The percentage of head mesoderm-like cells in a given region was determined by counting about phalloidine-stained cells per experiment. Only spread cells were counted which could be classified as being either head mesoderm-like or BCR-like. Each entry represents the mean (±s.d.) from 3 experiments. HM, head mesoderm; BPL, blastopore lip region; NIMZ, noninvoluting marginal zone, i.e. BCR directly above the blastopore lip region; aam, pam, anterior and posterior axial/paraxial mesoderm, respectively. Combined aam, pam and BPL of stage 12 correspond to the BPL region of stage. D E F NIMZ AC VG Fig. 2. Polarity of non-attached cells (stage +). (A,B) Schematic diagrams of filopodia formed during a 15 minute interval in cells from (A) head mesoderm and (B) animal cap region. (C-F) For each region, 3- cells were recorded and angles between succeeding filopodia were measured. Frequencies of angles are plotted. (C) Head mesoderm (n=115), (D) non-involuting marginal zone (n=132), (E) animal cap (n=112), (F) vegetal cells (n=5). From the respective parameters of the nonpolar BCR cells, a confidence interval (α=.1) was calculated. Columns deviating significantly from an even distribution of angles by exceeding the upper confidence limit (24.9) are labeled with an asterisk. types of protrusions they form on FN. In head mesoderm cells, contact with FN induces within minutes the extension of lamellipodia, leading to bipolar cell spreading (Winklbauer and Selchow, 1992). In Fig. 3B, a typical spindle-shaped head mesoderm cell is shown. One protrusion is retracting, the opposite, active process is divided into a proximal particle zone and the distal hyaline lamellipodium. Rhodamine-phalloidin staining reveals a concentration of F- actin and radial actin bundles in the process (Fig. 3A), as is typical of lamellipodia of these cells (Selchow and Winklbauer, 1997). The stationary BCR cells respond differently to FN. They continue to extend filopodia when attached. Filopodia become stabilized by contact to the substrate, and the cell margin is drawn out along them (R. Winklbauer, unpublished observations). In this way, they spread to assume a polygonal shape without employing lamellipodia. Consequently, in their yolk-free cytoplasmic fringes, no hyaline zone is observed (Fig. 3D). Rhodamine-phalloidin staining confirms the absence of lamellipodia (Fig. 3C). As a third cell type, the large nonmigratory vegetal cells extend numerous protrusions which can be identified as small lamellipodia (Fig. 3E,F). Protrusive activity varies in the dorsal mesoderm from anterior to posterior, and does not change during involution In the early gastrula, the number of migratory cells in the mesoderm decreases from anterior to posterior. This is reflected in the morphology of cells (Table 1). In the head mesoderm, nearly all cells possess one or two large lamellipodia, whereas in the blastopore lip only half of the cells show this morphology. In the adjacent prospective neuroectoderm, which is part of the BCR, lamellipodia are almost absent. Thus, the head mesoderm-like morphology is correlated with migratory behavior. Moreover, the axial/paraxial mesoderm of the dorsal blastopore lip contains both head mesoderm-like migratory cells and BCR-like stationary cells. In the late gastrula, the number of cells with lamellipodia has remained unchanged in the head mesoderm and neuroectoderm. Of the mesoderm which has left the blastopore lip by involution, the anterior part consists mostly of head mesoderm-like cells, whereas in the more posterior part this cell type diminishes. The most posterior axial/paraxial mesoderm, still within the blastopore lip, contains almost no lamellipodia-bearing cells. Thus, like the early gastrula blastopore lip, the late gastrula axial/paraxial mesoderm is a

5 Cell motility in the Xenopus gastrula 1935 mixture of cell types, with the migratory type decreasing in frequency posteriorly. Overall, the fraction of migratory cells is about the same in the early and the late gastrula axial/paraxial mesoderm, suggesting that the motile behavior of cells does not change as they involute. In the gastrula, three types of motile cells can be distinguished We have shown previously that adhesion to FN differs between cells from different regions of the gastrula (Winklbauer, 1988, 199). Our present results, combined with these previous observations, make it possible to distinguish three cell types in the gastrula, which are defined by motility-related characters (Fig. 4). (1) BCR-like cells. All BCR cells exhibit non-polarized protrusive activity. Spreading occurs with filopodia, and adhesion requires high concentrations of FN. Cells are completely stationary. (2) Head mesoderm-like cells. Cells from the head mesoderm, anterior axial/paraxial mesoderm, and dorsalanterior endoderm are the migratory cells of the dorsal side. They show polarized protrusive activity, spread on low concentrations of FN, and extend lamellipodia. The posterior axial/paraxial mesoderm comprises BCR-like and head mesoderm-like cells. (3) Vegetal base cells. These mostly non-migratory prospective endoderm cells are non-polarized, but adhere at low FN concentrations and form lamellipodia. In all regions, but most consistently in the posterior mesoderm and the prospective neuroectoderm, cells may perform circus movements. This blebbing type of movement is apparently absent in the embryo, but could be an in vitro manifestation of a distinct mode of motility (Symes et al., 1994). Nevertheless, since these cells cannot be classified with respect to polarity or spreading behavior, they were omitted from our analysis. cells do not migrate. The velocity profiles show narrow peaks indistinguishable from that of control animal cap cells (Fig. 5A,B). During the third hour, most cells become migratory (Fig. 5C), and during the fourth hour, virtually all cells migrate (Fig. 5D). Thus, with a minute exposure to activin being sufficient for induction (Green et al., 199), it takes about 2 hours to translate the activin signal into migratory behavior. Apparently, migration is a late response to induction. Moreover, transient cycloheximide treatment delays the onset of activin-induced migration further, suggesting that it is not an immediate effect of induction (Wacker, 1997). Migration is also induced by bfgf When BCR is induced with bfgf, cells migrate (Fig. 5). However, there are differences in the response to bfgf, as compared to activin. First, to migrate, cells are best treated at the mid blastula stage. Induction at later stages is less effective (not shown). Second, bfgf has to be removed from the medium after 1-2 hours. BCR cells do not migrate but start blebbing when continuously exposed to bfgf as single cells (not shown). Third, bfgf-treated cells start migration at least Migration is a late response to induction by activin Activin-induced BCR cells become migratory. A revealing characteristic is the timing of the response. Spreading of cells cannot occur before the initial gastrula stage (Smith et al., 199a; Howard and Smith, 1993; Ramos et al., 1996). Therefore, to determine the time required to respond to the inductive signal by migration, activin was added to initial gastrula stage BCR cells, and cell behavior in the presence of activin was recorded (Fig. 5). During the first 2 hours, induced Fig. 3. Morphology of cells on FN. (A,C,E) TRITC-phalloidin staining of F-actin, (B,D,F) same cells under phase contrast optics. (A,B) Head mesoderm cell; edge of lamellipodium (arrowheads), particle zone (black arrow) and retracting tail (white arrow) are indicated. (C,D) Animal cap cell with filiform processes. (E,F) Vegetal cell with lamellipodia (arrowheads). All same magnification, bar = 25 µm.

6 1936 S. Wacker and others A 4 2 activin, -1 hr Fig. 4. Motile cell types in the dorsal half of the gastrula. Light shading, BCR-like cells (1); dark shading, migratory cells (2); intermediate, vegetal cells (3). From left to right: the position of different cell types in the gastrula; polarity of non-attached cells; FN concentrations required for adhesion (µg/ml); morphology on FN and cell trajectory for a 1 hour interval. 4 hours later than activin-induced cells. When treated at the mid blastula stage, and plated at an early to mid gastrula stage, cells do not migrate 2 hours after plating (Fig. 5E), although at late gastrula stages, cells translocate (Fig. 5F). Apparently, there is a constitutive gap of 5-6 hours between bfgf induction and the onset of migration. A similarly delayed response was observed for BCR cell spreading induced by BMP-4 (Howard and Smith, 1993). Lamellipodium formation is induced by activin and by bfgf Activin-induced transition to migratory behavior is paralleled by the acquisition of a head mesoderm type of morphology (Fig. 6A-C). After 1 hour in activin, cells have spread on FN, but despite being induced, they have retained their BCR cell morphology (Fig. 6A). Their appearance has not changed much after 2 hours (Fig. 6B), but after 3 hours, most of the now migratory cells possess lamellipodia and are of head mesoderm type (Fig. 6C). At this time, cycloheximide-treated BCR cells in activin still appear BCR cell-like (Fig. 6D), in agreement with the delayed onset of migration in these cells (Wacker, 1997). Continuous presence of activin is not necessary to maintain a head mesoderm-like morphology. BCR cells induced at blastula stages and then cultured without activin appear the same as cells kept permanently in activin (Fig. 6E). Treatment with bfgf also changes BCR cell morphology. When induced at the middle blastula stage and then cultured on FN to the late gastrula stage in the absence of bfgf, cells exhibit large lamellipodia (Fig. 6F). In summary, altered cell morphology is also a late and stable response to induction, and it exactly parallels the acquisition of migratory behavior. An activin-induced change in adhesive behavior is reproduced by expressing the immediate early genes Mix.1 and goosecoid On substrate coated with -2 µg/ml FN, head mesoderm cells adhere extensively, whereas BCR cells attach poorly (Winklbauer, 199; Fig. 7). Activin, but not bfgf treatment promotes the spreading of BCR cells on FN (Smith et al., B C D E F activin, 1-2 hr activin, 2-3 hr activin, 3-4 hr bfgf, ~ st bfgf, ~ st Fig. 5. Activin- and bfgf-induced migration. (A-D) Stage -+ animal cap cells were seeded on FN in MBS containing 2 units/ml of activin. Movement was continuously recorded for 5 hours in 3 experiments. Velocities of cells (; n=6-8) were determined during the 1st hour (A), 2nd hour (B), 3rd hour (C), and 4th hour (D). (E,F) Stage 8.5 animal caps were incubated in ng/ml of bfgf for 2 hours, cultured in MBS, and dissociated. Cells (n=114, 6 experiments) were seeded on FN at stage.5. Velocities were determined during the 2nd (E) and 3rd hour (F) after plating. 199a; Howard and Smith, 1993; Ramos et al., 1996), implying a change in adhesive behavior. This can be directly demonstrated (Fig. 7). Activin treatment raises the adhesion of BCR cells at low FN density to a level typical of head mesoderm cells, whereas bfgf does not. The transcription factors gsc and Mix.1 are both expressed in animal caps in response to activin, but not to bfgf. To

7 Cell motility in the Xenopus gastrula 1937 see whether they could be involved in mediating the effect of activin on adhesion, the respective mrnas were injected into the animal blastomeres at the 8-cell stage, and the BCR cells derived from them were tested for FN adhesion. gsc mrna does not affect cell attachment when injected at 8 or 16 pg per embryo (Fig. 7), although -6 of embryos formed secondary axes after control injections into the ventral marginal zone. Higher amounts of RNA were deleterious to BCR cells. Mix.1 mrna is without effect when injected at - pg, but a strong increase in adhesion is attained with 2-8 pg of RNA (Fig. 7). Apparently, Mix.1 mrna is able to mimick the effect of activin on BCR cell adhesion to FN. Although gsc has no effect when injected alone, it can cooperate with Mix.1 to promote adhesion. Coinjection of 4 pg of gsc mrna and 8 pg of Mix.1 mrna raises adhesion of BCR cells most efficiently (Fig. 7). These amounts of RNA are completely ineffective when injected separately. Since both Mix.1 and gsc are induced by activin in animal caps, and since their expression overlaps in the embryo (Lemaire et al., 1995; Artinger et al., 1997), this cooperative effect may be of biological significance. Expression of Mix.1 in response to activin requires FGF signaling (LaBonne and Whitman, 1994, 1997). If activininduced adhesiveness were indeed mediated by Mix.1, blocking the endogenous FGF signal should prevent activin stimulated attachment. This is observed. When a dominant inhibitory FGF receptor is expressed in BCR cells, activin no longer promotes adhesion at low FN density (Fig. 7), although spreading at higher FN density is not affected (Cornell and Kimelman, 1994; own unpublished results). Expression of a non-functional receptor does not interfere with activin function (Fig. 7). Our results suggest that activin and FGF may cooperate to change, through Mix.1 and gsc, cell interaction with FN. even inhibits Mix.1-promoted polarization (Fig. 8E). Apparently, polarity is controlled differently from FN adhesion. This conclusion is reinforced by the finding that polarized protrusive activity is also induced by bfgf, i.e. in the absence of Mix.1 (Fig. 8F). Thus, at least two pathways affect polarity. Mix.1-expressing BCR cells translocate without lamellipodia When expressed in BCR cells, Mix.1 mimicks two of the effects of activin, by changing adhesion behavior and cell polarity. However, lamellipodium formation, which is also induced by activin, is not promoted by Mix.1 injection. At 16 pg of mrna, cells spread on FN and may elongate, but instead of lamellipodia, filopodia form at the poles of the cells (Fig. 9A,B). At pg of RNA, cells appear like control BCR cells (not shown). Injection of gsc mrna at 16 pg (Fig. 9C) or 8 pg (not shown) has no effect on morphology. Coinjection of gsc and Mix.1 mrna (16 pg each) also yields a morphology indistinguishable from untreated BCR cells (Fig. Cell polarity is affected by multiple factors Activin treatment polarizes protrusive activity of BCR cells (Fig. 8A). Also, injection of an amount of Mix.1 RNA (16 pg) which is just sufficient to promote adhesion, and also migration (see below), has a strong polarizing effect (Fig. 8B), whereas the same amount of gsc RNA has none (Fig. 8C). However, contrary to the control of adhesion, Mix.1 and gsc do not cooperate to polarize BCR cells. When coinjected at low concentrations, as a mixture which synergistically promotes adhesion at low FN density (see above), polarity is not affected (Fig. 8D). Moreover, coinjection of gsc mrna Fig. 6. Morphology of induced cells. Stage animal cap cells were plated on FN in MBS at 2 units/ml activin and fixed after 1 hour (A), 2 hours (B), and 3 hours (C), in parallel to the experiments of Fig. 5, and stained with TRITC-phalloidin. (D) Animal cap cells treated with 2 µg/ml cycloheximide (9 minutes) before induction, after 3 hours in activin (compare to C). (E) Animal caps induced by blastocoelic injection of activin at stage 9, dissociated at stage. Cells were cultured on FN for 3 hours without activin. (F) bfgfincuced animal cap cell on FN, 6 hours after induction. Arrowheads, lamellipodia. Same magnification, bar = 25 µm.

8 1938 S. Wacker and others Fig. 7. FN adhesion. Untreated animal cap cells (1) and head mesoderm cells (2) served as controls. Animal cap cells were treated with activin (3) or bfgf (4), or were loaded with 8 (5) or with 16 pg (6) of gsc mrna per embryo, with - pg (7) or with 2-8 pg (8) of Mix.1 mrna, or with a mixture 4 pg gsc/8 pg Mix.1 RNA (9). Animal blastomeres were also injected with mrna encoding a dominant inhibitory FGF receptor (XFD) (), or a nonfunctional FGF receptor (d) (11). Derived BCRs were induced with activin. Each column represents 3 to 6 cells adhering AC HM activin bfgf gsc 8pg gsc 16pg mix.1 -pg mix.1 2-8pg experiments, except for columns (1) and (2). Since these controls were included in each series of experiments, the total numbers are 22 (AC) and 15 (HM), respectively. Bars indicate standard deviations. In each experiment, about 6 cells in 6 fields were counted. gsc/mix.1 4/8pg XFD+ activin d+ activin 9D), which is consistent with Mix.1-induced polarization being inhibited by gsc. Lower amounts of gsc and Mix.1 mrna (4-8 pg) coinjected in varying combinations (e.g. Fig. 9E,F) do also not affect BCR cell morphology. Altogether, we were not able to induce lamellipodium formation in BCR cells by Mix.1 or gsc. BCR cells injected with an amount of Mix.1 RNA sufficient to promote adhesion and polarization translocate on FN despite their lack of lamellipodia (Fig. A). Although velocities are lower than after activin induction, many cells move faster than 1 ute. 8 pg of Mix.1 RNA are not effective (preliminary result). gsc injected cells are also not migratory, although some are more motile than normal, forming a minor peak around 1 ute (Fig. B). Unlike adhesion to low density FN, migration is not stimulated by coinjection of sub-threshold amounts of gsc and Mix.1 RNA (Fig. C). On the contrary, like cell polarity, Mix.1-promoted migration is even inhibited by coinjection of gsc mrna (Fig. D). DISCUSSION Motility-related properties of Xenopus gastrula cells Among the different motile behaviors shown by gastrula cells, a particularly striking one is the migration of anterior mesoderm and endoderm cells on FN. To study the control of migratory behavior in more detail, we selected for analytical purposes three motility-related characters which can each be expressed in different states, which are easily assayed, and which are potentially related to the ability of cells to move. These are adhesion to FN (at low or high density of FN), symmetry of protrusive activity (polarized or non-polarized), and structure of protrusions formed on FN (lamellipodia or filopodia). In different cells, the alternative states of the motilityrelated parameters can be expressed in different combinations. Possible combinations may be depicted as the corners of a cube (Fig. 11). Three combinations are represented by embryonic cell types: migrating mesoderm and endoderm cells, BCR cells and vegetal cells. Treatment of BCR cells yielded three additional types not present in the embryo. Altogether, six of eight possible combinations have been observed, indicating that the respective features are controlled more or less independently. This was not obvious initially. For example, one might have expected that adhesion at low FN were related to FN-dependent lamellipodia formation. Both features describe an interaction with FN, and are correlated in the embryo. However, FGF stimulates lamellipodia formation without changing FN adhesion, whereas Mix.1 injection promotes adhesion at low FN density in the absence of lamellipodia. The finding that the motility-related features are independently controlled justifies treating them as elementary motility parameters. Of the different combinations of parameter states, some are migratory and some not. This provides an opportunity to examine which features are essential for translocation. For example, the difference uncovered in our FN adhesion assay is not decisive for migration. Both behaviors, cell attachement at low and at high FN density, are compatible with translocation, but also with stationary motility. Possession of lamellipodia is also neither sufficient nor necessary for migration. Vegetal cells form lamellipodia, but do not migrate, whereas Mix.1- injected cells translocate, but extend filopodia only. Polarized protrusive activity is the only feature correlated with translocation (Fig. 11). That the polarity of non-attached cells may be related to polarity on FN is seen during spreading of head mesoderm cells: one lamellipodium always extends at the site of former filopodia formation, a second one opposite. During subsequent translocation when lamellipodia divide or shift along the cell margin, this relationship is obscured (Winklbauer and Selchow, 1992). But still, a migrating cell typically possesses only one or two large lamellipodia. This is in contrast to the non-polarized vegetal cells, which extend many small lamellipodia in all directions. Perhaps, the polarized appearance of non-attached cells reveals a property which is also expressed on FN, i.e. the ability to concentrate protrusive activity. Of course, other parameters not assayed in our study, e.g. detachment of the trailing edge, could also be essential for migration, and be accordingly affected by the various treatments. Control of gastrula cell motility The earliest signs of differentiation in the mesoderm appear some time after the first wave of gene expression initiated at the mid blastula transition. These are mesoderm-specific

9 Cell motility in the Xenopus gastrula 1939 morphogenetic movements, among them migration (Smith and Howard, 1992). Activin and BMP-4 have previously been shown to confer migratory behavior on animal cap cells (Smith et al., 199a; Smith and Howard, 1992; Howard and Smith, A B C D E F * activin * mix.1 16pg gsc/mix.1 4pg/8pg * gsc 16pg gsc/mix.1 16pg/16pg * bfgf ; Ramos and DeSimone, 1996). We add bfgf to the list of mesoderm inducers that promote migration. One may ask how the signals involved in mesoderm induction control morphogenetic processes, in particular migration. One conclusion from our results is that the transitions in motile behavior associated with migration are mediated, late responses to induction. The most direct evidence is that activin-induced changes in FN adhesion and cell polarity can be mediated by the immmediate early gene Mix.1. Also, migration of BCR cells and the accompanying change in cell morphology start more than 2 hours after activin induction and even later after FGF treatment, and are delayed by transient cycloheximide treatment (Wacker, 1997). Ramos et al. (1996) found evidence for immediate effects of activin on BCR cell behavior, suggesting that a contribution of immediate, but persistent effects of activin or FGF receptor activation should not be excluded. We propose that two of the genes, that respond to activin, but not to FGF, Mix.1 and gsc, have a role in the control of cell motility. Both transcription factors possess a paired class homeodomain (Wilson et al., 1993), like Siamois, which is also expressed in the early embryo (Lemaire et al., 1995). On binding to DNA, these proteins can form homo- or heterodimers (Wilson et al., 1993; Mead et al., 1996). gsc is a transcriptional repressor whose function seems independent of dimer formation (Smith and Jaynes, 1996; Artinger et al., 1997). Siamois appears to work as an activator (Fan and Sokol, 1997). All three genes are implicated in the regionalization of the embryo (Cho et al., 1991; Niehrs et al., 1994; Lemaire et al., 1995; Carnac et al., 1996; Mead et al., 1996). Mix.1 expression is sufficient to promote BCR cell adhesion at low FN density, polarization of protrusive activity, and some degree of migration. The thresholds for adhesion and for migration will be somewhere between - 2 pg of RNA, i.e. similar to that for producing an embryonic phenotype. The threshold for polarization has yet to be determined. gsc modulates the functions of Mix.1. It acts synergistically to promote adhesion, lowering the amount of Mix.1 RNA required, but it antagonizes polarization and migration. The latter effects of gsc can be seen at 16 pg of RNA, which yields a mild embryonic phenotype when injected marginally, but in general, lower amounts seem to be sufficient. We do not know whether the interactions between gsc and Mix.1 are by heterodimer formation, or indirect. We never observed lamellipodia formation in Mix.1 or gsc injected BCR cells. Treating BCR cells with inducing factors leads to the development of mesodermal tissues, and migration is just one Fig. 8. Polarity of BCR cell protrusive activity after various treatments. cells from at least 2 experiments were recorded. (A) Stage + animal cap cells from embryos injected at stage 9 with activin (n=12); (B-E) Stage + animal cap cells derived from blastomeres injected at the 8-cell stage with: (B) 16 pg of Mix.1 mrna per embryo (n=97); (C) 16 pg gsc per embryo (n=3); (D) Mix.1 and gsc mrna (4 and 8 pg, respectively; n=178); (E) gsc and Mix.1 mrna (16 pg/embryo each; n=95). (F) Animal caps incubated at stage 8 for 2 hours in ng/ml of bfgf, cultured in MBS for 5 hours, dissociated and evaluated (n=94). Columns exceeding upper confidence limit are labeled with an asterisk.

10 194 S. Wacker and others Fig. 9. Morphology of Mix.1- and gscinjected BCR cells. (A,B) Animal blastomeres were injected with 16 pg of Mix.1 mrna/embryo. Derived animal cap cells were seeded on FN at stage + and fixed 1 hour later. Arrowheads indicate the spread region behind filopodia at advancing front; arrows, retraction fibers. (C) Same kind of experiment, injected with 16 pg of gsc mrna/embryo. (D-F) Blastomeres coinjected with (D) 8 pg of gsc and 16 pg of Mix.1 mrna/embryo, (E) 4 pg of gsc and 8 pg of Mix.1 RNA, (F) 8 pg of gsc and 4 pg of Mix.1 RNA. TRITC-phalloidin staining. All same magnification, bar = 25 µm. expression of mesoderm differentiation. Injection of an immediate early gene like Xbra also evokes mesoderm formation, apparently by triggering a positive feedback loop which involves efgf (Cunliffe and Smith, 1992; Isaacs et al., 1994). In contrast, loading BCR cells with Mix.1 (Ruiz i Altaba and Melton, 1989; Smith et al., 1991; Smith and Harland, 1991; Mead et al., 1996) or with gsc mrna (Niehrs et al., 1993) does not lead to mesoderm development or expression of mesodermal markers, i.e. injected cells are not identical to activin-treated cells in this case. Moreover, Mix.1 and gsc affect only certain aspects of motility when expressed in BCR cells. This suggests that the genes may be directly involved in the control of morphogenesis. For gsc, this has been previously suggested from the finding that its overexpression in the mesoderm leads to increased gastrulation movements (Niehrs et al., 1993). However, it remains to be shown that Mix.1 and gsc are actually necessary for the control of cell motility in the embryo. Motility domains in the gastrula At the onset of gastrulation, Mix.1 is expressed in the complete vegetal half of the embryo, i.e. in the prospective endoderm and in the lower part of the marginal zone consisting of prospective anterior mesoderm (Rosa, 1989; LaBonne and Whitman, 1997). The activin-like and the FGF signaling pathways are activated not only in prospective mesoderm, but also in the vegetal base (Hemmati-Brivanlou and Melton, 1994; Henry et al., 1996; LaBonne and Whitman, 1997). From this, and our present results, one might expect that all cells in the lower half of the embryo adhere at low FN density, extend lamellipodia, and are polarized, thus resembling activininduced BCR cells. With respect to adhesion and lamellipodium formation, the cells from the vegetal half differ from those of the animal hemisphere as expected. This introduces a basic subdivision of the embryo into an animal and a vegetal motility domain (Fig. 12). The vegetal motility domain is further subdivided. Polarized protrusive activity and the correlated ability to migrate are restricted to a small marginal region of this domain (Fig. 12). Our results suggest that paired class homeodomain transcription factors may be involved in cell polarity control, but the details remain to be clarified. For example, abundance of Mix.1 mrna in the vegetal base may be just too low for polarization, but sufficient for adhesion (vegetal base cells migrate sporadically, suggesting that they are close to being polarized). In the marginal zone, Siamois expression overlaps with that of Mix.1 (Lemaire et al., 1995). Since Siamois also polarizes BCR cells (M. Rieger and R. Winklbauer, unpublished), it could locally raise the combined activity of these factors above the threshold for polarization. The border between the animal and vegetal motility domains, which may correspond to the sharp late blastula expression boundary of Mix.1 (Rosa, 1989), runs straight across the mesoderm to separate it into an anterior migratory

11 Cell motility in the Xenopus gastrula 1941 A B mix.1 16pg gsc 16pg C D gsc/mix.1 4pg/8pg gsc/mix.1 16pg/16pg Fig.. Migration velocity () of Mix.1 and gsc loaded BCR cells. Animal blastomeres were injected as in Fig. 9 and derived cells seeded on FN at stage +. (A) Mix.1 (16 pg; n=75; 3 experiments), (B) gsc (16 pg; n=3; 6 experiments), (C) coinjection of gsc and Mix.1 (4 pg and 8 pg, respectively; n=46; 3 experiments), (D) gsc and Mix.1 (16 pg each; n=137; 7 experiments). moiety and a posterior non-migratory part (Fig. 12). The latter, i.e. the most posterior axial/paraxial mesoderm, expresses mesodermal markers like Xbra, participates in convergent Fig. 11. Summary of motile cell types. Normally occurring types (BCR, BCR-like cells; VG, vegetal cells; HM, head mesoderm-like cells) are shaded. New combinations are indicated by treatments generating them. All migratory cells are in the top plane. Fig. 12. Motility domains. (A) Late blastula. Mix.1 expression domain (dark shading; from Rosa, 1989) defines vegetal motility domain. Border separates anterior (am) from posterior mesoderm (pm). The latter belongs to the animal domain (light shading). Region of polarized cells (dashed area) extends into dorsal-anterior endoderm (DAE). (B) Early gastrula. Mesoderm involution deforms regions (see arrow) to yield pattern shown in Fig. 4. Mixing in lip region (hatched). extension, and differentiates into somites or notochord (Slack and Tannahill, 1992). However, the morphology of its cells is mostly BCR-like, although some cell mixing occurs during gastrulation (Vodicka and Gerhart, 1995). This mixing may explain why in the mesoderm, adhesion to FN changes in a graded manner from anterior to posterior, and not in a step (Winklbauer, 199). Likewise, the border between polarized and non-polarized cells in the vegetal motility domain lies apparently within the prospective endoderm, and does not separate mesoderm from endoderm, as judged from the migratory behavior of dorsalanterior cells (Fig. 12). Thus, in general, the boundaries separating regions of distinct types of cell motility in the gastrula do not coincide with the borders between prospective germ layers or tissues. In contrast to suggestions from previous work (Winklbauer, 199), we could not demonstrate changes in cell motility to be associated with mesoderm involution. In particular, posterior mesoderm cells do not acquire migratory potential as they internalize. Nevertheless, since much of the posterior mesoderm seems to consist of a mixture of migratory and stationary cells, whole explants from this region are able to translocate on FN in vitro (Winklbauer and Nagel, 1991). Altogether, it appears that a fixed spatial pattern of cell motility is laid out at the initial gastrula stage. During gastrulation, regions move relative to each other, and limited cell mixing occurs, but individual cells do not change appreciably their motile behavior. The majority of this work forms part of a dissertation by S. Wacker. FGF receptor plasmids, generated by Enrique Amaya and Marc Kirschner, were kindly provided by Stephan Schneider. We thank Christine Dreyer for providing activin, Martina Nagel for help with mrna synthesis, and Günther Plickert and his laboratory for hospitality and advice. This work was supported by the DFG.

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