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, Germany), not overlap with that of BCR cells (Fig. 1D). Occasionally, cultured in MBS as required, and dissociated. head mesoderm preparations are contaminated by large cells mRNA synthesis and injection from the adjacent endoderm. These are also migratory (not Full length goosecoid mRNA was synthesized from EcoRI linearized shown). In contrast, cells from the vegetal base endoderm are pSPgsc (Niehrs et al., 1994) by transcription with SP6 RNA mostly stationary (Fig. 1E). polymerase, Mix.1 mRNA from Sfi1 linearized pBSRN3mix.1 In the late gastrula, the most anterior part of the cell mass (Lemaire et al., unpublished) by transcription with T3 RNA advancing on the BCR consists of large endoderm cells which polymerase. Dominant negative (XFD) and nonfunctional (d50) FGF are separated from the BCR by a thin layer of small cells receptor clones constructed by Enrique Amaya and Marc Kirschner extending up to the leading edge (e.g. Hausen and Riebesell, (Amaya et al., 1991) were used. Linearized plasmids were transcribed 1991), presumably prospective head mesoderm. Although not with SP6 RNA polymerase. At the 8-cell stage, 8 nl of RNA solution in contact with the BCR substrate in the embryo, the dorsal- was injected in each of the 4 animal blastomeres. anterior prospective endoderm cells migrate well on FN in vitro TRITC-phalloidin staining (Fig. 1F). Also, head mesoderm cells migrate vigorously (not Cells on FN were fixed in 4% formaldehyde in MBS for 5 minutes, shown), as before (see Fig. 1D). permeabilized by the addition of 0.1% Triton X-100 for another 10 By the late gastrula stage, most of the axial/paraxial minutes, blocked with 0.1% BSA, and stained with 1 µg/ml of mesoderm has left the blastopore lip and become apposed to TRITC-phalloidin for 20 minutes. the BCR. Anteriorly, these cells are mainly migratory (Fig. Cell motility in the Xenopus gastrula 1933

1G), but posteriorly, cells are AC predominantly stationary (Fig. 1H). 50 S10.25 Super-imposing the velocity 40 distributions of anterior and 30 % posterior axial/paraxial mesoderm 20 10 would yield a distribution similar to 0 that of the early gastrula blastopore 0 0.5 1 1.5 2 2.5 3 lip. This suggests that involution of A µm/min the axial/paraxial mesoderm is not 50 NIMZ accompanied by a transition from S10.25 40 stationary to migratory behavior. 30 In summary, we conclude that the % 20 cells of the anterior mesoderm, 10 including the head mesoderm and 0 the anterior axial/paraxial 0 0.5 1 1.5 2 2.5 3 µm/min mesoderm, and the adjacent dorsal- B 50 BPL anterior endoderm cells are S10.25 migratory on FN. In contrast, most of 40 30

the posterior mesoderm cells, and all % cells of the BCR, are stationary. 20 10 Also, most vegetal cells do not 0 translocate on FN. 0 0.5 1 1.5 2 2.5 3 C µm/min Protrusive activity differs 50 HM between migratory mesoderm S10.5 40 cells, BCR cells, and vegetal 30 cells % 20 On non-adhesive substrate, all 10 gastrula cells extend filopodia, but 0 0 1 2 3 the pattern differs between cell 0.5 1.5 2.5 D µm/min types. In head mesoderm cells, most 50 VG filopodia form within a narrow zone, S10.25 giving cells a polarized appearance 40 30

(Fig. 2A; Winklbauer and Selchow, % 1992). Consequently, angles 20 10 between successively appearing 0 filopodia are typically small (Fig. 0 0.5 1 1.5 2 2.5 3 2C). In contrast, cells from the BCR E µm/min are non-polar. Filopodia extend in all 50 DAE S12 directions (Fig. 2B), leading to an 40 even distribution of angles (Fig. 30 2D,E). The same non-polarized % 20 distribution is observed for vegetal 10 cells (Fig. 2F). 0 0 1 2 3 Gastrula cells differ also in the 0.5 1.5 2.5 F µm/min

AM anterior Fig. 1. FN-dependent migration of 50 S12 gastrula cells. For defined regions 40 30

(boxed region in right panel), average % µ 20 velocities ( m/min) of 40-80 cells from 10 at least 3 experiments were determined, 0 and their frequencies (%) plotted (left 0 0.5 1 1.5 2 2.5 3 panel). (A-E) stage 10+ or 10.5; G µm/min (A) animal cap, (B) prospective neuroectoderm of non-involuting 50 AM posterior S12 marginal zone, (C) dorsal blastopore lip, 40 (D) head mesoderm, (E) vegetal base 30 % endoderm. (F-H) stage 12; (F) dorsal- 20 anterior prospective endoderm, (G) 10 0 posterior head mesoderm and anterior 0 0.5 1 1.5 2 2.5 3 axial/paraxial mesoderm, (H) posterior H µm/min axial/paraxial mesoderm. 1934 S. Wacker and others

HM AC Table 1. Frequency of head mesoderm-like cells in anterior to posterior regions of the dorsal axis % Head mesoderm-like cells Stage 10 Stage 12 HM 91.9 (4.4) 92.5 (5.7) aAM − 82.2 (6.0) pAM − 55.6 (5.3) BPL 53.9 (5.3) 7.0 (3.4) A B NIMZ 1.8 (1.5) 1.7 (1.2)

50 * HM The percentage of head mesoderm-like cells in a given region was 40 determined by counting about 300 phalloidine-stained cells per experiment. Only spread cells were counted which could be classified as being either head 30 * mesoderm-like or BCR-like. Each entry represents the mean (±s.d.) from 3 % 20 experiments. HM, head mesoderm; BPL, blastopore lip region; NIMZ, non- involuting marginal zone, i.e. BCR directly above the blastopore lip region; 10 aAM, pAM, anterior and posterior axial/paraxial mesoderm, respectively. 0 Combined aAM, pAM and BPL of stage 12 correspond to the BPL region of C 30° 60° 90° 120° 150° 180° stage 10.

50 NIMZ is retracting, the opposite, active process is divided into a 40 proximal particle zone and the distal hyaline lamellipodium. 30 Rhodamine-phalloidin staining reveals a concentration of F- % 20 actin and radial actin bundles in the process (Fig. 3A), as is 10 typical of lamellipodia of these cells (Selchow and 0 Winklbauer, 1997). D 30° 60° 90° 120° 150° 180° The stationary BCR cells respond differently to FN. They continue to extend filopodia when attached. Filopodia become 50 AC stabilized by contact to the substrate, and the cell margin is 40 drawn out along them (R. Winklbauer, unpublished 30 observations). In this way, they spread to assume a polygonal % 20 shape without employing lamellipodia. Consequently, in their 10 yolk-free cytoplasmic fringes, no hyaline zone is observed 0 (Fig. 3D). Rhodamine-phalloidin staining confirms the absence ° ° ° ° ° ° of lamellipodia (Fig. 3C). As a third cell type, the large non- E 30 60 90 120 150 180 migratory vegetal cells extend numerous protrusions which can be identified as small lamellipodia (Fig. 3E,F). 50 VG 40 Protrusive activity varies in the dorsal mesoderm 30 from anterior to posterior, and does not change % 20 during involution 10 In the early gastrula, the number of migratory cells in the 0 mesoderm decreases from anterior to posterior. This is F 30° 60° 90° 120° 150° 180° reflected in the morphology of cells (Table 1). In the head mesoderm, nearly all cells possess one or two large Fig. 2. Polarity of non-attached cells (stage 10+). (A,B) Schematic lamellipodia, whereas in the blastopore lip only half of the cells diagrams of filopodia formed during a 15 minute interval in cells show this morphology. In the adjacent prospective from (A) head mesoderm and (B) animal cap region. (C-F) For each neuroectoderm, which is part of the BCR, lamellipodia are region, 3-10 cells were recorded and angles between succeeding almost absent. Thus, the head mesoderm-like morphology is filopodia were measured. Frequencies of angles are plotted. (C) Head correlated with migratory behavior. Moreover, the mesoderm (n=115), (D) non-involuting marginal zone (n=132), (E) axial/paraxial mesoderm of the dorsal blastopore lip contains animal cap (n=112), (F) vegetal cells (n=105). From the respective parameters of the nonpolar BCR cells, a confidence interval (α=0.01) both head mesoderm-like migratory cells and BCR-like was calculated. Columns deviating significantly from an even stationary cells. distribution of angles by exceeding the upper confidence limit In the late gastrula, the number of cells with lamellipodia (24.9%) are labeled with an asterisk. 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 types of protrusions they form on FN. In head mesoderm mesoderm-like cells, whereas in the more posterior part this cells, contact with FN induces within minutes the extension cell type diminishes. The most posterior axial/paraxial of lamellipodia, leading to bipolar cell spreading mesoderm, still within the blastopore lip, contains almost no (Winklbauer and Selchow, 1992). In Fig. 3B, a typical lamellipodia-bearing cells. Thus, like the early gastrula spindle-shaped head mesoderm cell is shown. One protrusion blastopore lip, the late gastrula axial/paraxial mesoderm is a Cell motility in the Xenopus gastrula 1935 mixture of cell types, with the migratory type decreasing in cells do not migrate. The velocity profiles show narrow peaks frequency posteriorly. Overall, the fraction of migratory cells indistinguishable from that of control animal cap cells (Fig. is about the same in the early and the late gastrula 5A,B). During the third hour, most cells become migratory axial/paraxial mesoderm, suggesting that the motile behavior (Fig. 5C), and during the fourth hour, virtually all cells of cells does not change as they involute. migrate (Fig. 5D). Thus, with a 30 minute exposure to activin being sufficient for induction (Green et al., 1990), it takes In the gastrula, three types of motile cells can be about 2 hours to translate the activin signal into migratory distinguished behavior. Apparently, migration is a late response to We have shown previously that adhesion to FN differs between induction. Moreover, transient cycloheximide treatment cells from different regions of the gastrula (Winklbauer, 1988, delays the onset of activin-induced migration further, 1990). Our present results, combined with these previous suggesting that it is not an immediate effect of induction observations, make it possible to distinguish three cell types in (Wacker, 1997). the gastrula, which are defined by motility-related characters (Fig. 4). Migration is also induced by bFGF (1) BCR-like cells. All BCR cells exhibit non-polarized When BCR is induced with bFGF, cells migrate (Fig. 5). protrusive activity. Spreading occurs with filopodia, and However, there are differences in the response to bFGF, as adhesion requires high concentrations of FN. Cells are compared to activin. First, to migrate, cells are best treated at completely stationary. the mid blastula stage. Induction at later stages is less effective (2) Head mesoderm-like cells. Cells from the head (not shown). Second, bFGF has to be removed from the mesoderm, anterior axial/paraxial mesoderm, and dorsal- medium after 1-2 hours. BCR cells do not migrate but start anterior endoderm are the migratory cells of the dorsal side. blebbing when continuously exposed to bFGF as single cells They show polarized protrusive activity, spread on low (not shown). Third, bFGF-treated cells start migration at least 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. 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., 1990a; 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 Fig. 3. Morphology of cells on FN. (A,C,E) TRITC-phalloidin staining of F-actin, (B,D,F) gastrula stage BCR cells, and cell same cells under phase contrast optics. (A,B) Head mesoderm cell; edge of lamellipodium behavior in the presence of activin (arrowheads), particle zone (black arrow) and retracting tail (white arrow) are indicated. was recorded (Fig. 5). (C,D) Animal cap cell with filiform processes. (E,F) Vegetal cell with lamellipodia During the first 2 hours, induced (arrowheads). All same magnification, bar = 25 µm. 1936 S. Wacker and others

50 activin, 0-1 hr 40 30 % 20 10 0 0 0.5 1 1.5 2 2.5 3 A µm/min

50 activin, 1-2 hr 40 30 % 20 10 0 0 0.5 1 1.5 2 2.5 3 Fig. 4. Motile cell types in the dorsal half of the gastrula. Light B µm/min shading, BCR-like cells (1); dark shading, migratory cells (2); activin, 2-3 hr intermediate, vegetal cells (3). From left to right: the position of 50 40 different cell types in the gastrula; polarity of non-attached cells; FN 30 concentrations required for adhesion (µg/ml); morphology on FN and % 20 cell trajectory for a 1 hour interval. 10 0 0 0.5 1 1.5 2 2.5 3 C µm/min 4 hours later than activin-induced cells. When treated at the activin, 3-4 hr mid blastula stage, and plated at an early to mid gastrula stage, 50 40 cells do not migrate 2 hours after plating (Fig. 5E), although 30 at late gastrula stages, cells translocate (Fig. 5F). Apparently, % 20 there is a constitutive gap of 5-6 hours between bFGF 10 0 induction and the onset of migration. A similarly delayed 0 0.5 1 1.5 2 2.5 3 response was observed for BCR cell spreading induced by D µm/min BMP-4 (Howard and Smith, 1993). 50 bFGF, ~ st. 11.5 Lamellipodium formation is induced by activin and 40 by bFGF 30 % 20 Activin-induced transition to migratory behavior is paralleled 10 by the acquisition of a head mesoderm type of morphology 0 (Fig. 6A-C). After 1 hour in activin, cells have spread on FN, 0 0.5 1 1.5 2 2.5 3 µm/min but despite being induced, they have retained their BCR cell E morphology (Fig. 6A). Their appearance has not changed 50 bFGF, ~ st. 12 much after 2 hours (Fig. 6B), but after 3 hours, most of the 40 30 now migratory cells possess lamellipodia and are of head % 20 mesoderm type (Fig. 6C). At this time, cycloheximide-treated 10 BCR cells in activin still appear BCR cell-like (Fig. 6D), in 0 agreement with the delayed onset of migration in these cells 0 0.5 1 1.5 2 2.5 3 F µm/min (Wacker, 1997). Continuous presence of activin is not necessary to maintain a head mesoderm-like morphology. BCR Fig. 5. Activin- and bFGF-induced migration. (A-D) Stage 10-10+ cells induced at blastula stages and then cultured without animal cap cells were seeded on FN in MBS containing 20 units/ml activin appear the same as cells kept permanently in activin of activin. Movement was continuously recorded for 5 hours in 3 (Fig. 6E). Treatment with bFGF also changes BCR cell experiments. Velocities of cells (µm/min; n=60-80) were determined morphology. When induced at the middle blastula stage and during the 1st hour (A), 2nd hour (B), 3rd hour (C), and 4th hour then cultured on FN to the late gastrula stage in the absence of (D). (E,F) Stage 8.5 animal caps were incubated in 50 ng/ml of bFGF bFGF, cells exhibit large lamellipodia (Fig. 6F). In summary, for 2 hours, cultured in MBS, and dissociated. Cells (n=114, 6 altered cell morphology is also a late and stable response to experiments) were seeded on FN at stage 10.5. Velocities were induction, and it exactly parallels the acquisition of migratory determined during the 2nd (E) and 3rd hour (F) after plating. behavior.

An activin-induced change in adhesive behavior is 1990a; Howard and Smith, 1993; Ramos et al., 1996), implying reproduced by expressing the immediate early a change in adhesive behavior. This can be directly genes Mix.1 and goosecoid demonstrated (Fig. 7). Activin treatment raises the adhesion of On substrate coated with 10-20 µg/ml FN, head mesoderm BCR cells at low FN density to a level typical of head cells adhere extensively, whereas BCR cells attach poorly mesoderm cells, whereas bFGF does not. (Winklbauer, 1990; Fig. 7). Activin, but not bFGF treatment The transcription factors gsc and Mix.1 are both expressed promotes the spreading of BCR cells on FN (Smith et al., in animal caps in response to activin, but not to bFGF. To Cell motility in the Xenopus gastrula 1937 see whether they could be involved in mediating the effect even inhibits Mix.1-promoted polarization (Fig. 8E). of activin on adhesion, the respective mRNAs were injected Apparently, polarity is controlled differently from FN into the animal blastomeres at the 8-cell stage, and the BCR adhesion. This conclusion is reinforced by the finding that cells derived from them were tested for FN adhesion. gsc polarized protrusive activity is also induced by bFGF, i.e. in mRNA does not affect cell attachment when injected at 80 the absence of Mix.1 (Fig. 8F). Thus, at least two pathways or 160 pg per embryo (Fig. 7), although 30-60% of embryos affect polarity. formed secondary axes after control injections into the ventral marginal zone. Higher amounts of RNA were Mix.1-expressing BCR cells translocate without deleterious to BCR cells. Mix.1 mRNA is without effect lamellipodia when injected at 50-100 pg, but a strong increase in adhesion When expressed in BCR cells, Mix.1 mimicks two of the is attained with 200-800 pg of RNA (Fig. 7). Apparently, effects of activin, by changing adhesion behavior and cell Mix.1 mRNA is able to mimick the effect of activin on BCR polarity. However, lamellipodium formation, which is also cell adhesion to FN. induced by activin, is not promoted by Mix.1 injection. At 160 Although gsc has no effect when injected alone, it can pg of mRNA, cells spread on FN and may elongate, but cooperate with Mix.1 to promote adhesion. Coinjection of 40 instead of lamellipodia, filopodia form at the poles of the cells pg of gsc mRNA and 80 pg of Mix.1 mRNA raises adhesion (Fig. 9A,B). At 50 pg of RNA, cells appear like control BCR of BCR cells most efficiently (Fig. 7). These amounts of RNA cells (not shown). Injection of gsc mRNA at 160 pg (Fig. 9C) are completely ineffective when injected separately. Since or 80 pg (not shown) has no effect on morphology. Coinjection both Mix.1 and gsc are induced by activin in animal caps, and of gsc and Mix.1 mRNA (160 pg each) also yields a since their expression overlaps in the embryo (Lemaire et al., morphology indistinguishable from untreated BCR cells (Fig. 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 activin- induced 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. 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 (160 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 Fig. 6. Morphology of induced cells. Stage 10 animal cap cells were plated on FN in MBS at 20 units/ml activin and fixed after 1 hour (A), 2 hours (B), and 3 hours (C), in parallel to cooperate to polarize BCR cells. When the experiments of Fig. 5, and stained with TRITC-phalloidin. (D) Animal cap cells treated coinjected at low concentrations, as a with 20 µg/ml cycloheximide (90 minutes) before induction, after 3 hours in activin mixture which synergistically promotes (compare to C). (E) Animal caps induced by blastocoelic injection of activin at stage 9, adhesion at low FN density (see above), dissociated at stage 10. Cells were cultured on FN for 3 hours without activin. (F) bFGF- polarity is not affected (Fig. 8D). incuced animal cap cell on FN, 6 hours after induction. Arrowheads, lamellipodia. Same Moreover, coinjection of gsc mRNA magnification, bar = 25 µm. 1938 S. Wacker and others

Fig. 7. FN adhesion. Untreated animal 100 cap cells (1) and head mesoderm cells 90 (2) served as controls. Animal cap cells 80 were treated with activin (3) or bFGF 70 (4), or were loaded with 80 (5) or with 60 160 pg (6) of gsc mRNA per embryo, with 50-100 pg (7) or with 200-800 pg 50 (8) of Mix.1 mRNA, or with a mixture 40

40 pg gsc/80 pg Mix.1 RNA (9). Animal % cells adhering 30 blastomeres were also injected with 20 mRNA encoding a dominant inhibitory 10 FGF receptor (XFD) (10), or a non- 1 2 3 4 5 6 7 8 9 10 11 0 functional FGF receptor (d50) (11). AC HM activin bFGF gsc gsc mix.1 mix.1 gsc/mix.1 XFD+ d50+ Derived BCRs were induced with 80pg 160pg 50-100pg 200-800pg 40/80pg activin activin activin. Each column represents 3 to 6 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 600 cells in 6 fields were counted.

9D), which is consistent with Mix.1-induced polarization observed, indicating that the respective features are controlled being inhibited by gsc. Lower amounts of gsc and Mix.1 more or less independently. This was not obvious initially. For mRNA (40-80 pg) coinjected in varying combinations (e.g. example, one might have expected that adhesion at low FN Fig. 9E,F) do also not affect BCR cell morphology. were related to FN-dependent lamellipodia formation. Both Altogether, we were not able to induce lamellipodium features describe an interaction with FN, and are correlated in formation in BCR cells by Mix.1 or gsc. the embryo. However, FGF stimulates lamellipodia formation BCR cells injected with an amount of Mix.1 RNA sufficient without changing FN adhesion, whereas Mix.1 injection to promote adhesion and polarization translocate on FN promotes adhesion at low FN density in the absence of despite their lack of lamellipodia (Fig. 10A). Although lamellipodia. The finding that the motility-related features are velocities are lower than after activin induction, many cells independently controlled justifies treating them as elementary move faster than 1 µm/minute. 80 pg of Mix.1 RNA are not motility parameters. effective (preliminary result). gsc injected cells are also not Of the different combinations of parameter states, some are migratory, although some are more motile than normal, migratory and some not. This provides an opportunity to forming a minor peak around 1 µm/minute (Fig. 10B). Unlike examine which features are essential for translocation. For adhesion to low density FN, migration is not stimulated by example, the difference uncovered in our FN adhesion assay is coinjection of sub-threshold amounts of gsc and Mix.1 RNA not decisive for migration. Both behaviors, cell attachement at (Fig. 10C). On the contrary, like cell polarity, Mix.1-promoted low and at high FN density, are compatible with translocation, migration is even inhibited by coinjection of gsc mRNA (Fig. but also with stationary motility. Possession of lamellipodia is 10D). 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. DISCUSSION Polarized protrusive activity is the only feature correlated with translocation (Fig. 11). That the polarity of non-attached Motility-related properties of Xenopus gastrula cells cells may be related to polarity on FN is seen during spreading Among the different motile behaviors shown by gastrula cells, of head mesoderm cells: one lamellipodium always extends at a particularly striking one is the migration of anterior the site of former filopodia formation, a second one opposite. mesoderm and endoderm cells on FN. To study the control of During subsequent translocation when lamellipodia divide or migratory behavior in more detail, we selected for analytical shift along the cell margin, this relationship is obscured purposes three motility-related characters which can each be (Winklbauer and Selchow, 1992). But still, a migrating cell expressed in different states, which are easily assayed, and typically possesses only one or two large lamellipodia. This is which are potentially related to the ability of cells to move. in contrast to the non-polarized vegetal cells, which extend These are adhesion to FN (at low or high density of FN), many small lamellipodia in all directions. Perhaps, the symmetry of protrusive activity (polarized or non-polarized), polarized appearance of non-attached cells reveals a property and structure of protrusions formed on FN (lamellipodia or which is also expressed on FN, i.e. the ability to concentrate filopodia). protrusive activity. Of course, other parameters not assayed in In different cells, the alternative states of the motility- our study, e.g. detachment of the trailing edge, could also be related parameters can be expressed in different combinations. essential for migration, and be accordingly affected by the Possible combinations may be depicted as the corners of a various treatments. cube (Fig. 11). Three combinations are represented by embryonic cell types: migrating mesoderm and endoderm Control of gastrula cell motility cells, BCR cells and vegetal cells. Treatment of BCR cells The earliest signs of differentiation in the mesoderm appear yielded three additional types not present in the embryo. some time after the first wave of gene expression initiated at Altogether, six of eight possible combinations have been the mid blastula transition. These are mesoderm-specific Cell motility in the Xenopus gastrula 1939 morphogenetic movements, among them migration (Smith and 1993; Ramos and DeSimone, 1996). We add bFGF to the list Howard, 1992). Activin and BMP-4 have previously been of mesoderm inducers that promote migration. shown to confer migratory behavior on animal cap cells (Smith One may ask how the signals involved in mesoderm et al., 1990a; Smith and Howard, 1992; Howard and Smith, 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 50 activin * is that activin-induced changes in FN adhesion and cell polarity 40 can be mediated by the immmediate early gene Mix.1. Also, 30 migration of BCR cells and the accompanying change in cell % 20 morphology start more than 2 hours after activin induction and even later after FGF treatment, and are delayed by transient 10 cycloheximide treatment (Wacker, 1997). Ramos et al. (1996) 0 found evidence for immediate effects of activin on BCR cell A 30° 60° 90° 120° 150° 180° behavior, suggesting that a contribution of immediate, but persistent effects of activin or FGF receptor activation should 50 mix.1 160pg not be excluded. 40 * We propose that two of the genes, that respond to activin, 30 but not to FGF, Mix.1 and gsc, have a role in the control of % 20 cell motility. Both transcription factors possess a paired class homeodomain (Wilson et al., 1993), like Siamois, which is 10 also expressed in the early embryo (Lemaire et al., 1995). On 0 binding to DNA, these proteins can form homo- or B 30° 60° 90° 120° 150° 180° heterodimers (Wilson et al., 1993; Mead et al., 1996). gsc is a transcriptional repressor whose function seems independent 50 gsc 160pg of dimer formation (Smith and Jaynes, 1996; Artinger et al., 40 1997). Siamois appears to work as an activator (Fan and 30 Sokol, 1997). All three genes are implicated in the % 20 regionalization of the embryo (Cho et al., 1991; Niehrs et al., 1994; Lemaire et al., 1995; Carnac et al., 1996; Mead et al., 10 1996). 0 Mix.1 expression is sufficient to promote BCR cell C 30° 60° 90° 120° 150° 180° adhesion at low FN density, polarization of protrusive activity, and some degree of migration. The thresholds for 50 gsc/mix.1 40pg/80pg adhesion and for migration will be somewhere between 100- 40 200 pg of RNA, i.e. similar to that for producing an 30 embryonic phenotype. The threshold for polarization has yet

% to be determined. gsc modulates the functions of Mix.1. It acts 20 synergistically to promote adhesion, lowering the amount of 10 Mix.1 RNA required, but it antagonizes polarization and 0 migration. The latter effects of gsc can be seen at 160 pg of D 30° 60° 90° 120° 150° 180° RNA, which yields a mild embryonic phenotype when injected marginally, but in general, lower amounts seem to be 50 gsc/mix.1 160pg/160pg sufficient. We do not know whether the interactions between 40 gsc and Mix.1 are by heterodimer formation, or indirect. We never observed lamellipodia formation in Mix.1 or gsc 30 *

% injected BCR cells. 20 Treating BCR cells with inducing factors leads to the 10 development of mesodermal tissues, and migration is just one 0 E 30° 60° 90° 120° 150° 180° Fig. 8. Polarity of BCR cell protrusive activity after various 50 bFGF treatments. 10 cells from at least 2 experiments were recorded. (A) Stage 10+ animal cap cells from embryos injected at stage 9 with 40 * activin (n=120); (B-E) Stage 10+ animal cap cells derived from 30 blastomeres injected at the 8-cell stage with: (B) 160 pg of Mix.1 % 20 mRNA per embryo (n=97); (C) 160 pg gsc per embryo (n=103); (D) Mix.1 and gsc mRNA (40 and 80 pg, respectively; n=178); 10 (E) gsc and Mix.1 mRNA (160 pg/embryo each; n=95). (F) Animal 0 caps incubated at stage 8 for 2 hours in 50 ng/ml of bFGF, cultured F 30° 60° 90° 120° 150° 180° in MBS for 5 hours, dissociated and evaluated (n=94). Columns exceeding upper confidence limit are labeled with an asterisk. 1940 S. Wacker and others

Fig. 9. Morphology of Mix.1- and gsc- injected BCR cells. (A,B) Animal blastomeres were injected with 160 pg of Mix.1 mRNA/embryo. Derived animal cap cells were seeded on FN at stage 10+ 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 160 pg of gsc mRNA/embryo. (D-F) Blastomeres coinjected with (D) 80 pg of gsc and 160 pg of Mix.1 mRNA/embryo, (E) 40 pg of gsc and 80 pg of Mix.1 RNA, (F) 80 pg of gsc and 40 pg of Mix.1 RNA. TRITC-phalloidin staining. All same magnification, bar = 25 µm. expression of mesoderm differentiation. Injection of an 1994; Henry et al., 1996; LaBonne and Whitman, 1997). From immediate early gene like Xbra also evokes mesoderm this, and our present results, one might expect that all cells in formation, apparently by triggering a positive feedback loop the lower half of the embryo adhere at low FN density, extend which involves eFGF (Cunliffe and Smith, 1992; Isaacs et al., lamellipodia, and are polarized, thus resembling activin- 1994). In contrast, loading BCR cells with Mix.1 (Ruiz i Altaba induced BCR cells. With respect to adhesion and and Melton, 1989; Smith et al., 1991; Smith and Harland, lamellipodium formation, the cells from the vegetal half differ 1991; Mead et al., 1996) or with gsc mRNA (Niehrs et al., from those of the animal hemisphere as expected. This 1993) does not lead to mesoderm development or expression introduces a basic subdivision of the embryo into an animal of mesodermal markers, i.e. injected cells are not identical to and a vegetal motility domain (Fig. 12). activin-treated cells in this case. Moreover, Mix.1 and gsc affect The vegetal motility domain is further subdivided. Polarized only certain aspects of motility when expressed in BCR cells. protrusive activity and the correlated ability to migrate are This suggests that the genes may be directly involved in the restricted to a small marginal region of this domain (Fig. 12). control of morphogenesis. For gsc, this has been previously Our results suggest that paired class homeodomain suggested from the finding that its overexpression in the transcription factors may be involved in cell polarity control, mesoderm leads to increased gastrulation movements (Niehrs but the details remain to be clarified. For example, abundance et al., 1993). However, it remains to be shown that Mix.1 and of Mix.1 mRNA in the vegetal base may be just too low for gsc are actually necessary for the control of cell motility in the polarization, but sufficient for adhesion (vegetal base cells embryo. migrate sporadically, suggesting that they are close to being polarized). In the marginal zone, Siamois expression overlaps Motility domains in the gastrula with that of Mix.1 (Lemaire et al., 1995). Since Siamois also At the onset of gastrulation, Mix.1 is expressed in the complete polarizes BCR cells (M. Rieger and R. Winklbauer, vegetal half of the embryo, i.e. in the prospective endoderm unpublished), it could locally raise the combined activity of and in the lower part of the marginal zone consisting of these factors above the threshold for polarization. prospective anterior mesoderm (Rosa, 1989; LaBonne and The border between the animal and vegetal motility Whitman, 1997). The activin-like and the FGF signaling domains, which may correspond to the sharp late blastula pathways are activated not only in prospective mesoderm, but expression boundary of Mix.1 (Rosa, 1989), runs straight also in the vegetal base (Hemmati-Brivanlou and Melton, across the mesoderm to separate it into an anterior migratory Cell motility in the Xenopus gastrula 1941

50 mix.1 160pg 40 30 % 20 10 0 0 0.5 1 1.5 2 2.5 3 A µm/min

50 gsc 160pg 40 30 % 20 10 0 0 0.5 1 1.5 2 2.5 3 B µm/min

50 gsc/mix.1 40pg/80pg 40 30 Fig. 12. Motility domains. (A) Late blastula. Mix.1 expression % 20 domain (dark shading; from Rosa, 1989) defines vegetal motility 10 domain. Border separates anterior (aM) from posterior mesoderm 0 (pM). The latter belongs to the animal domain (light shading). 0 0.5 1 1.5 2 2.5 3 Region of polarized cells (dashed area) extends into dorsal-anterior C µ m/min endoderm (DAE). (B) Early gastrula. Mesoderm involution deforms regions (see arrow) to yield pattern shown in Fig. 4. Mixing in lip 50 gsc/mix.1 160pg/160pg 40 region (hatched). 30 % 20 10 0 extension, and differentiates into somites or notochord (Slack 0 0.5 1 1.5 2 2.5 3 D and Tannahill, 1992). However, the morphology of its cells is µm/min mostly BCR-like, although some cell mixing occurs during gastrulation (Vodicka and Gerhart, 1995). This mixing may µ Fig. 10. Migration velocity ( m/min) of Mix.1 and gsc loaded BCR explain why in the mesoderm, adhesion to FN changes in a cells. Animal blastomeres were injected as in Fig. 9 and derived cells graded manner from anterior to posterior, and not in a step seeded on FN at stage 10+. (A) Mix.1 (160 pg; n=75; 3 experiments), (B) gsc (160 pg; n=103; 6 experiments), (C) coinjection of gsc and (Winklbauer, 1990). Mix.1 (40 pg and 80 pg, respectively; n=46; 3 experiments), (D) gsc Likewise, the border between polarized and non-polarized and Mix.1 (160 pg each; n=137; 7 experiments). 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 dorsal- moiety and a posterior non-migratory part (Fig. 12). The latter, anterior cells (Fig. 12). Thus, in general, the boundaries i.e. the most posterior axial/paraxial mesoderm, expresses separating regions of distinct types of cell motility in the mesodermal markers like Xbra, participates in convergent gastrula do not coincide with the borders between prospective germ layers or tissues. In contrast to suggestions from previous work (Winklbauer, 1990), 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 Fig. 11. Summary of motile cell types. Normally occurring types Kirschner, were kindly provided by Stephan Schneider. We thank (BCR, BCR-like cells; VG, vegetal cells; HM, head mesoderm-like Christine Dreyer for providing activin, Martina Nagel for help with cells) are shaded. New combinations are indicated by treatments mRNA synthesis, and Günther Plickert and his laboratory for generating them. All migratory cells are in the top plane. hospitality and advice. This work was supported by the DFG. 1942 S. Wacker and others

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