fibronectin, mesoderm migration, and gastrulation in xenopus

DEVELOPMENTAL BIOLOGY 177, 413–426 (1996)ARTICLE NO. 0174

Fibronectin, Mesoderm Migration,and Gastrulation in Xenopus

Rudolf Winklbauer1 and Ray E. Keller2

Department of Zoology, University of California, Berkeley, Berkeley, California 94720

The role of fibronectin in mesoderm cell migration and and the importance of mesoderm migration for gastrulation inXenopus are examined. To allow for migration, a stable interface must exist between migrating mesoderm cells and thecells of the substrate layer, the blastocoel roof. We show that maintenance of this interface does not depend on fibronectin.We further demonstrate that fibronectin contributes to, but is not essential for, mesoderm cell adhesion to the blastocoelroof. However, interaction with fibronectin is necessary for cell spreading and the formation of lamelliform cytoplasmicprotrusions. Apparently, the specific role of fibronectin in mesoderm migration is to control cell protrusive activity.Consequently, when fibronectin function is blocked by GRGDSP peptide or antibodies, mesoderm cell migration is inhib-ited. Nevertheless, gastrulation proceeds nearly normally in inhibitor-treated embryos. It appears that in Xenopus, meso-derm migration is not essential for gastrulation. q 1996 Academic Press, Inc.

INTRODUCTION ments are most strongly expressed in the axial and paraxialmesoderm (Keller and Danilchik, 1988). In contrast, all re-

Amphibian gastrulation has been extensively studied in gions of the mesoderm have the capacity to migrate on thethe clawed toad, Xenopus laevis. In Xenopus, most of gas- BCR (Winklbauer and Nagel, 1991).trulation is ultimately driven by the activities of the meso- In the amphibian embryo, a network of fibronectin (FN)derm. In the blastula, the ring of prospective mesoderm fibrils covers the inner surface of the BCR (Boucaut andsurrounds the embryo below the equator. At the vegetal Darribere, 1983a,b; Nakatsuji and Johnson, 1983b; Lee etmargin of the mesoderm mantle, a blastopore invaginates, al., 1984; Nakatsuji et al., 1985; Collazo, 1994). In a classicalfirst dorsally and then also laterally and ventrally. The series of papers, Boucaut and co-workers showed that inhib-mesoderm above it begins to involute. It rolls over the blas- iting cell interaction with FN arrests gastrulation in thetopore lip, attaches to the blastocoel roof (BCR), and moves urodele Pleurodeles. Antibodies against FN or FN receptors,toward the animal pole. On the dorsal side, prospective head or peptides which mimic the RGD cell-binding site of FNmesoderm is the first to involute, followed by prospective and competitively inhibit the FN receptor all block gastrula-axial mesoderm. Movement of the mesodermal layer rela- tion (Boucaut et al., 1984a,b; Darribere et al., 1988, 1990;tive to the BCR is associated with migratory cell behavior Riou et al., 1990). Since mesoderm cells move on FN inon the side of the mesoderm, the BCR layer serving as the vitro (e.g., Nakatsuji, 1986; Darribere et al., 1988; Riou etsubstrate (for review see Keller, 1986; Keller and Winkl- al., 1990; Smith et al., 1990; Winklbauer, 1990), it was con-bauer, 1992). This process of mesoderm cell migration is cluded that FN is essential for mesoderm cell migration andaddressed in the current article. A different morphogenetic that mesoderm migration in turn is necessary for gastrula-movement is convergence and extension, where cell inter- tion in Pleurodeles.calation leads to a substrate-independent, tissue-autono- In Xenopus, the situation is more complicated. RGD pep-mous distortion of the mesodermal layer (Keller et al., 1985; tides have been reported not to arrest gastrulation in thisKeller and Danilchik, 1988). Convergent extension move- species (Winklbauer, 1989; Smith et al., 1990). On the other

hand, translocation of mesoderm cells on explanted BCRis severely impeded when interaction with FN is blocked1 To whom correspondence should be addressed at present ad-(Winklbauer, 1990). This suggests that migration is FN-de-dress: Universitat zu Koln, Zoologisches Institut, Weyertal 119,pendent, but not essential for gastrulation. The latter view50931 Cologne, Germany. Fax: 0221 470 5171. E-mail: rwinkl@is also supported by the observation that much of Xenopusnovell.biolan.uni-koeln.de.gastrulation occurs normally even when the whole BCR is2 Present address: Department of Biology, Gilmer Hall, Univer-

sity of Virginia, Charlottesville, VA 22903-2477. removed (Keller and Jansa, 1992). However, this tentative

413

0012-1606/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

AID DB 8250 / 6x0f$$$501 06-25-96 10:18:54 dbal AP: Dev Bio

414 Winklbauer and Keller

conclusion has been challenged by a report that antibodies the BCR was examined (Fig. 1). When seeded onto the innersurface of untreated BCR, virtually all cells bind to their into FN or to b1 integrin inhibit gastrulation in Xenopus

(Howard et al., 1992). In the current paper, we first examine vivo substrate (Fig. 1, 1). This substrate consists of FN fibrilsand of the surface of BCR cells exposed between them (Na-the role of FN in mesoderm cell migration. We show that

FN is indeed essential for migration, and we suggest a spe- katsuji and Johnson, 1983b; Nakatsuji et al., 1985). To testfor the relative contribution of the two components, thecific function for FN in this process. Second, on the basis

of the above result, we reexamine the role of mesoderm adhesiveness of each was assayed. In a first series of experi-ments, BCR extracellular matrix was transferred to a plasticmigration in Xenopus gastrulation. We demonstrate that

even when migration is inhibited, gastrulation proceeds surface by applying BCR explants to the bottom of a culturedish (Nakatsuji and Johnson, 1983a; Winklbauer and Nagel,nearly normally.1991). Substantial adhesion to such conditioned substrateis observed (Fig. 1, 2). It can be inhibited by Fab fragments

MATERIALS AND METHODS of FN antibody (Fig. 1, 3) or 1 mg/ml of GRGDSP peptide(Fig. 1, 5), but not by preimmune antibody (Fig. 1, 4) or

Xenopus laevis embryos were obtained as described (Winklbauer,control peptide (Fig. 1, 6). This strongly suggests that adhe-1990) and staged according to Nieuwkoop and Faber (1967). Also,sion to the BCR extracellular matrix is mediated by thetechniques of operation and the buffers used (Danilchik’s solution,RGD cell-binding site of FN.modified Barth’s solution (MBS), dissociation buffer) have been de-

Since interaction of cells with the BCR matrix can bescribed previously (Winklbauer, 1990).specifically blocked, the respective inhibitors can be usedto test whether FN-independent adhesion contributes toPeptide and Antibody Inhibition Experimentsmesoderm attachment to the BCR. With GRGDSP peptide,

HPLC-purified GRGDSP and GRGESP hexapeptides were from adhesion to the BCR is significantly reduced (Fig. 1, 7 and 8),Novabiochem (Laufelfingen, Switzerland). Stage 9 blastulae were compared both to untreated BCR (Fig. 1, 1) and to GRGESPplaced in Danilchik’s solution or MBS containing peptide at the control peptide (Fig. 1, 9). However, even at extremely highdesired concentration. The blastocoel was punctured and flushed peptide concentrations, there is substantial attachment ofwith peptide solution. Shortly before gastrulation, solution was

cells (Fig. 1, 8), suggesting FN-independent adhesion to thereplaced by 1/10-strength MBS, also containing peptide. Thus, em-BCR. This is confirmed by the finding that Fab fragment ofbryos developed in the continuous presence of the peptide and con-FN antibody has an effect similar to RGD peptide (Fig. 1,centration could be exactly controlled. The same type of experi-10 and 11).ment was performed using Fab fragments of a polyclonal rabbit

FN-independent adhesion could be due to cadherin-medi-antibody against Xenopus plasma FN (Winklbauer, in preparation).As a control, IgG from preimmune serum was used. ated interaction between mesoderm cells and BCR cells.

Therefore, FN antibody was complemented with a func-tional antibody against EP/C- and XB/U-cadherin. BothAdhesion Assaycadherins are expressed in the early embryo (Muller et al.,

Prospective head mesoderm cells from stage 10 1/2 gastrulae 1994). Preincubation in antibody mixture dissociates meso-were dissociated (Winklbauer, 1990) and seeded on explanted pieces derm explants into single cells (not shown), but does notof BCR of the same age. To prevent curling, explants were secured

inhibit adhesion to the BCR (Fig. 1, 11). Apparently, FN-under glass bridges supported by silicone grease. After 15 to 60 minindependent adhesion is not mediated by any of these cad-of incubation in Danilchik’s solution or MBS, explants were turnedherins. As a negative control for our assay, binding of meso-upside down in the incubation medium and shaken vigorously forderm cells to the nonadhesive apical surface of the BCR1–2 min. Cells remaining attached to the BCR and cells coming

off were counted. Adhesion to conditioned substrate was tested as was tested. As expected, cells do not attach to this substratedescribed (Winklbauer and Nagel, 1991). (Fig. 1, 12). In summary, we conclude that both an interac-

tion with the RGD cell-binding site of FN and an FN-inde-pendent interaction with the BCR contribute to the sub-Microscopystrate attachment of mesoderm cells.

For light microscopy, cells on the opaque BCR were visualizedby indirect illumination. For scanning electron microscopy, speci-mens were fixed in 2% glutaraldehyde in 0.125 M sodium cacodyl- FN Requirement for Protrusive Activityate (pH 7.4) overnight at 47C, washed in cacodylate buffer, dehy- of Mesoderm Cellsdrated in ethanol, critical point dried, mounted on stubs, and coated

FN was proposed to induce in mesoderm cells the forma-with gold-palladium in a sputter coater.tion of lamellipodia, which are the main translocatory or-ganelles of these cells (Winklbauer and Selchow, 1992). In

RESULTS agreement with this, single mesoderm cells which attachto the BCR in the absence of FN interaction remain spheri-

FN-Mediated and FN-Independent Adhesion cal and do not extend lamelliform protrusions (Winklbauerof Mesoderm Cells to the Blastocoel Roof et al., 1991). To demonstrate this effect in the intact em-

bryo, interaction with FN in situ was inhibited by continu-To identify the role of FN in substrate attachment ofmesoderm, adhesion of prospective head mesoderm cells to ous incubation in GRGDSP peptide or FN antibody from the

Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.


415Mesoderm Migration in Xenopus Gastrula

FIG. 1. Adhesion of prospective head mesoderm cells to the blastocoel roof (BCR) and to conditioned substrate (CS). The percentage ofcells binding to the inner surface of the BCR at stage 10 1/2 or to CS was determined in MBS or Danilchik’s solution alone (C) or in thepresence of Fab fragment of FN antibody (aFN), preimmune serum (ps), GRGDSP peptide (RGD), or GRGESP control peptide (RGE) atthe indicated concentrations (in mg/ml for antibodies and in mg/ml for peptides). Adhesion to the apical surface of the BCR (ap) was alsotested. Each column represents the average from 6–9 experiments involving 50–100 cells each; bars indicate standard deviations. Columnsare numbered; these numbers are referred to in the text.

late blastula stage onward. As shown below, the mesoderm mesodermal tissue, we examined the effect of RGD peptideor antibodies on the migration of mesoderm explants.becomes normally apposed to the BCR under these condi-

tions. When the BCR of inhibitor-treated and control em- On BCR, pieces of head mesoderm migrate directionallytoward the animal pole (Winklbauer and Nagel, 1991).bryos is removed after fixation at the middle gastrula stage,

the morphology of mesoderm cells that had been in contact Movement is inhibited by 4 mg/ml GRGDSP peptide (Fig.3a), but not by control peptide (Fig. 3b). Likewise, mesodermwith the substrate can be compared (Fig. 2). In the presence

of control peptide (Fig. 2a) or preimmune antibody (Fig. 2c), migration is blocked by FN antibody (Fig. 3c). Average speedis significantly lower than in the presence of preimmunemost cells possess lamellipodia and filipodia which prefer-

entially point in the direction of mesoderm movement and antibody (Fig. 3d). Altogether, pieces of mesoderm seem tobe moved by the migration of their constituent cells, andwhich are indicative of active cell migration (Winklbauer

and Nagel, 1991; Winklbauer and Selchow, 1992). In con- blocking FN interaction arrests translocation on the BCRof both single cells and mesoderm aggregates.trast, with GRGDSP peptide (Fig. 2b) or Fab fragment of FN

antibody (Fig. 2d), mesoderm cells are globular and extendprotrusions very rarely. Moreover, processes are always fili-

FN Is Not Involved in Stabilizing the Involutedform and point in any direction. Apparently, they are anMesoderm–Blastocoel Roof Interfaceexpression of the constitutive, substrate-independent for-

mation of filopodia which has been described for mesoderm During migration across the BCR, mesoderm cells mustcells (Winklbauer and Selchow, 1992). However, for the ex- not mix with cells of the substrate layer. Otherwise, direc-tension of lamellipodia across the substrate, interaction tional mesoderm movement would be impossible. FN couldwith FN is necessary. in principle, from its location between BCR and involuted

mesoderm, be involved in the formation or maintenance ofa stable interface between the two cell populations. In theCell–Fibronectin Interaction Is Essential forFN inhibition experiments described above, involutedMesoderm Translocation on the Blastocoel Roofmesoderm cells, though firmly attached, remained alwayson the surface of the BCR, which argues against such a roleThe decrease of protrusive activity upon inhibition of

cell–FN interaction suggests that FN may be required for for FN fibrils.To analyze this more explicitly, small pieces of involutedmesoderm translocation. Migration of single mesoderm

cells on explanted BCR is in fact severely impaired by head mesoderm and of preinvolution axial mesoderm wereplaced on isolated BCR (Fig. 4). Preinvolution mesoderm isGRGDSP peptide (Winklbauer, 1990). To confirm that in-

hibitor treatment also blocks movement of large pieces of part of the BCR. It becomes apposed to the BCR only later,




FIG. 2. In situ morphology of mesoderm cells on the BCR. Stage 11 embryos were fixed, and the BCR was removed to expose thesubstrate side of the moving mesoderm. Movement is to the top in all figures. Incubation in 4 mg/ml of GRGESP control peptide (a);same concentration of GRGDSP peptide (b); 25 mg/ml of IgG fraction from preimmune serum (c); and 25 mg/ml of Fab fragments of FNantibody (d). Arrows, examples of lamellipodia; arrowheads, filopodia. All same magnification; bar, 20 mm.


AID DB 8250 / 6x0f$$8250 06-25-96 10:18:54 dbal AP: Dev Bio


FIG. 3. Migration of mesoderm explants on the BCR in the presence of peptides or antibodies. A piece of head mesoderm was placed onexplanted BCR, secured under a glass bridge to prevent curling of the explant, and viewed under indirect illumination. The distance alongthe dorsal lip–animal pole axis between the starting position and the center of the mesoderm explant was determined from time-lapsevideo recordings and drawn as a function of time. A positive slope indicates movement away from the dorsal lip. Each line represents aseparate explant. Average velocities and standard deviations and the number of explants tested are indicated. (a) Explants in 4 mg/ml ofGRGDSP peptide, (b) in 4 mg/ml of GRGESP control peptide, (c) in 25 mg/ml of Fab fragments of FN antibody, (d) in 50 mg/ml of IgGfrom preimmune serum. Average velocity in (a) is not significantly different from zero (significance level a Å 0.05) and is significantlylower than velocity in (b) (a Å 0.001). Although average velocity in (c) is significantly higher than zero (a Å 0.001), it is neverthelesssignificantly lower than the average velocity in (d) (a Å 0.001).



At stage 13, the blastopore is usually closed in untreatedembryos, though a small yolk plug may protrude (Fig. 6a).Abnormalities typical of peptide-treated embryos werenever observed among controls (Table 1). At 2 mg/ml ofRGD-containing peptide, blastopore closure is slightly de-layed, but embryos are otherwise normal (Table 1). At 4mg/ml of GRGDSP peptide, the morphology of the gastrulais affected. Most embryos are pear-shaped and taper towarda noninternalized yolk plug (Fig. 6b; Table 1). This is alsothe predominant phenotype at 8–16 mg/ml of the peptide.A few more severely affected embryos are also found atthese very high concentrations (Table 1). They show evenless internalization of yolk and absence of the ventral blas-topore groove (Fig. 6c). However, total arrest of gastrulationFIG. 4. Interaction of pre- and postinvolution mesoderm with thewas never observed, and moreover, some normal embryosBCR. Experimental design. Stage 10 1/2 BCR was excised andare found at all concentrations (Table 1).placed in a dish with the apical side down (large twisted arrow).

At the apparent threshold concentration of the GRGDSPSmall pieces of involuted prospective head mesoderm (HM) andpreinvolution mesoderm (PM) were placed side by side on the BCR peptide (4 mg/ml), control peptide produces only few abnor-explant (solid arrows). The explant was secured under a glass bridge mal gastrulae. The incidence of defects increases stronglyand incubated in the absence (a) or presence (b) of 4 mg/ml of above 8 mg/ml of GRGESP peptide (Table 1). Most impor-GRGDSP peptide. Pieces of mesoderm were also placed on the tantly, however, these abnormalities differ from those ob-matrix-free outer surface of the inner BCR layer (dashed arrows), tained with GRGDSP peptide. Though gastrulation also ap-after removal of the apical layer (c).

pears incomplete, embryos do not taper toward the yolkplug, but instead toward the anterior end. A ring-like blasto-pore groove is always present (Fig. 6d).

Fab fragments of FN antibodies lead also to the develop-in the process of involution. The behavior of the small ex-ment of pear-shaped embryos with protruding yolk plugsplants was followed with the light microscope under indi-(Fig. 6e; Table 1), whereas control IgG has no effect in halfrect illumination (Fig. 5). As expected, involuted mesodermof the cases and causes incomplete gastrulation or exogas-remains on the surface of the BCR. In contrast, preinvolu-trulation in the other half (Fig. 6f; Table 1). Since it has beention mesoderm reintegrates into the BCR within less thanreported that whole FN antiserum inhibited gastrulation in30 min of contact, resuming its normal position within theXenopus (Howard et al., 1992), we also incubated embryosBCR (Fig. 5a). These findings are in agreement with previousin polyclonal anti-FN IgG. We obtained the same range ofresults (Keller R., unpublished data). Apparently, FN doeseffects as with Fab fragment (Table 1).not form a simple mechanical barrier to the exchange of

Altogether, these findings confirm our results from thecells between layers. Another possibility would be that FNpeptide inhibition experiments. Apparently, blocking FNacted specifically on involuted mesoderm cells to preventfunction always causes a distinct phenotype: (1) embryostheir reintegration. However, the same behavior as in Fig.are pear-shaped, tapering posteriorly; (2) a mass of vegetal5a was observed when the experiment was carried out incells is protruding from the blastopore; (3) a blastopore inva-the presence of GRGDSP peptide (Fig. 5b) or when meso-gination may be absent ventrally. Above a threshold level,derm pieces were applied to the matrix-free side of the innerthe severeness or frequency of this syndrome does not in-BCR layer (Fig. 5c). Thus, maintenance of the interface be-crease with inhibitor concentration. In contrast, the differ-tween involuted mesoderm and BCR does not depend onent type of abnormality caused by GRGESP peptide be-the FN matrix normally present between the two layers.comes more frequent at higher concentrations. Moreover,The principal role of FN seems to be the promotion of lamel-similar malformations are also obtained with control anti-lipodium formation in mesoderm cells, which is essentialbody. Presumably, these effects are nonspecific. Most strik-for mesoderm migration.ingly, however, the consequences of inhibiting FN functiondo not include an arrest of gastrulation.

Gastrulation in the Absence of Mesoderm To confirm this notion, the internal morphology of inhib-Migration itor-treated gastrulae was compared to that of controls.

Scanning electron microscopy of untreated embryos (Fig.To see how gastrulation proceeds in the absence of meso-derm cell migration, late blastulae were continuously incu- 7a) and embryos incubated in RGD-containing peptide (Fig.

7b) or control peptide (Fig. 7c) shows that involution atbated in GRGDSP peptide or GRGESP control peptide untilgastrulation was completed in untreated embryos. The the dorsal blastopore lip is not affected and that involuted

mesoderm advances on the BCR. At the end of gastrulation,range of phenotypes obtained is shown in Fig. 6 and quanti-fied in Table 1. The main result is that RGD-containing the most obvious abnormality in RGD-treated embryos is

the noninternalized yolk mass. Mesoderm has involutedpeptide may produce a distinct aberrant morphology, butdoes not arrest gastrulation. and seems to occupy its normal position, and a normal-sized




FIG. 5. Interaction of preinvolution and involuted mesoderm with the BCR. Pieces of prospective head mesoderm (arrowheads) andpreinvolution mesoderm (arrows) on the inner surface of the BCR in the absence (a) or presence (b) of 4 mg/ml of GRGDSP peptide or onthe outer, matrix-free surface of the inner BCR layer (c). (a–c) Top: a few minutes after explantation (left) and after reintegration of thepreinvolution mesoderm into the BCR (between 20 and 40 min) (right). Bottom: higher magnification view of top right figure in eachpanel. Top and bottom rows are all the same magnification, respectively; bars, 100 mm.

archenteron has formed (Fig. 7d). The same morphology is development of the tail could indicate disturbed mesodermmovement in the ventral regions of the gastrula. In summary,obtained with FN antibody (Fig. 8a). On the ventral side of

treated gastrulae, a blastopore groove is often not present inhibition of cell–FN interaction does not interfere with nor-mal translocation of most parts of the mesoderm. From the(Fig. 8a), in contrast to that in control embryos (Fig. 8b).

At the end of gastrulation, the archenteron is of equal length morphology of mesoderm cells in inhibitor-treated embryos(Fig. 2), we conclude that interaction with FN was effectivelyin control and inhibitor-treated embryos (Figs. 7 and 8), sug-

gesting normal movement and positioning of the dorsal meso- blocked and mesoderm migration was in fact abolished in ourexperiments. Any movement of the mesoderm which occursderm in the absence of cell–FN interaction. Whether the ven-

tral mesoderm is also correctly dispatched is less clear (Figs. under these conditions must therefore be caused by other fac-tors.7d and 8a). To display the presence or absence of mesoderm

regions, we raised GRGDSP-treated pear-shaped embryos tothe larval stage (Figs. 9b–9d) and compared them to GRGESP DISCUSSIONcontrols (Fig. 9a). A normal axis and anterior structures de-

The Role of Fibronectin in Mesoderm Cellvelop in treated embryos, but the amount of yolk in the gutMigrationis reduced, and the tail is truncated (Figs. 9b–9d). This sup-

ports our assumption that dorsal head and trunk mesoderm Interaction with FN is a necessary condition for meso-derm cell migration on the BCR of Xenopus. This has beenare normally positioned in FN-inhibited embryos. The weak




FIG. 6. Effect of peptides and FN antibody on gastrulation. (a) Dorsolateral view of stage 13 control embryos, with closed blastopore(arrow) or small remnant of yolk plug (arrowhead). (b) Pear-shaped embryos incubated in 4 mg/ml of GRGDSP peptide, with protrudingvegetal yolk mass (arrowheads); lateral view with dorsal side down (left) or up (right) at stage 13. (c) Embryo in 8 mg/ml of GRGDSPpeptide, with large external vegetal yolk mass (arrowhead), dorsolateral view, stage 13. (d) Embryos incubated in 16 mg/ml of GRGESPcontrol peptide, showing varying degrees of gastrulation abnormalities at stage 13, ranging from a slight delay of blastopore (arrow) closure(left), to a medium-sized (right), to a large (center) protruding yolk mass (arrowheads). (Arrows) Blastopore lip. Left and right embryo inapproximately dorsal view, and center embryo in lateral view, dorsal side to the left. (a–d) All same magnification; bar, 500 mm. (e) Pear-shaped stage 13 embryo incubated in 25 mg/ml of Fab fragments of FN antibody. Protruding yolk mass (arrowhead); blastopore lip (arrow).(f) Control embryo incubated in 50 mg/ml of IgG from preimmune serum, blastopore lip (arrow) nearly closed, small internalized yolkplug visible (arrowhead). (e and f) Same magnification; bar, 500 mm.




TABLE 1Effects of RGD Peptides and FN Antibody on Gastrulation

IncompleteNormal gastrulation Pear-shaped No. of cases

Control 27 0 0 27

GRGDSP peptide (mg/ml) (2) 14 2 0 16(4) 4 0 12 16(8) 3 0 9 12

(16) 3 0 13 16

GRGESP peptide (mg/ml) (4) 9 3 0 12(8) 10 4 0 14

(16) 2 15 0 17

aFN, Fab (mg/ml) (25) 5 0 18 23(50) 3 1 15 19

aFN, IgG (50) 2 0 10 12

ps, IgG (50) 10 5 1 16

Note. Embryos were incubated from stage 9 to stage 13 at the concentrations indicated in parentheses. aFN, anti-FN antibody, Fabfragments or whole IgG. ps, preimmune serum, IgG. See text and Fig. 7 for definition of categories.

shown previously for isolated head mesoderm cells (Winkl- tact with a FN substrate, this activity is modulated in tworespects. First, protrusive activity is relocalized, such thatbauer, 1990) and in the current article for whole mesoderm

explants. However, the essential function of FN is not to processes extend now exclusively along the substrate sur-face. Second, filopodia are largely replaced by lamelliformpromote cell adhesion to the BCR substrate, but to control

protrusion formation and hence the motility of mesoderm protrusions (Winklbauer et al., 1991; Winklbauer and Sel-chow, 1992; this article). In the absence of cell–FN interac-cells. Although FN improves adhesion to the BCR, strong

FN-independent binding can largely substitute for FN-medi- tion, mesoderm cells attach to the BCR, but do not formlamellae (Winklbauer et al., 1991; this article), showing thatated attachment.

FN-independent adhesion of mesoderm cells could be due substrate adhesion per se is not sufficient to modulate pro-trusive activity. Apparently, interaction with FN is re-to direct attachment to BCR cells. Nakatsuji (1976) ob-

served close apposition of mesoderm and BCR cell mem- quired. Interestingly, these effects are caused not only bydense in vitro FN substrates, but also by widely spaced FNbranes, suggestive of an adhesive interaction. At least two

maternal cadherins, EP/C- and XB/U-cadherin, are present fibrils on the BCR. Dependence of protrusion formation onFN may suffice to explain the observed inhibition of meso-on Xenopus early embryonic cells and could potentially me-

diate mesoderm cell adhesion to the BCR (Muller et al., derm migration in the absence of cell–FN interaction.1994). However, an antibody which inhibits both cadherins(Muller et al., 1994) does not reduce FN-independent adhe-

The Role of Mesoderm Cell Migration in Xenopussion to the BCR.GastrulationIf FN-independent adhesion were nonspecific, in the

sense that it could not be eventually traced back to a dis- A main finding of this work is that gastrulation proceedsessentially unimpaired in the absence of mesoderm cell mi-tinct receptor–ligand interaction, it could nevertheless be

functional. Thus, vermiform amphibian embryonic cells are gration. In a total of 98 embryos treated with effective con-centrations of GRGDSP peptide or FN antibody, no case ofable to translocate in vitro under conditions of apparently

weak and unspecific substrate attachment (Holtfreter, 1946; complete gastrulation arrest was observed. The mesodermoccupies approximately its normal position after gastrula-Kubota, 1981). Also, isolated head mesoderm cells show

residual migratory ability on the BCR in the absence of FN- tion, and larvae developing from treated embryos formheads and axes and have only minor deficiencies.mediated adhesion (Winklbauer, 1990). Most importantly,

however, FN-independent interaction with the BCR is suf- That RGD peptides do not block Xenopus gastrulationhas been noted previously (Winklbauer, 1989; Smith et al.,ficient to rescue gastrulation defects which are observed in

BCR-less Xenopus embryos (see below). 1990; Yost, 1992). In contradiction to this, antibodies to FNor b1 integrin have been reported to inhibit gastrulation inAlthough not essential for adhesion to the BCR, FN is

required for the proper formation of locomotory processes Xenopus (Howard et al., 1992). In the respective experi-ments, antisera containing polyvalent antibodies were used.on mesoderm cells. Nonattached head mesoderm cells ex-

tend filopodia spontaneously from their surface. Upon con- Our own findings with Fab fragments of antibodies to Xeno-




06-25-96 10:18:54 dbal AP: Dev Bio


pus FN, but also with the IgG fraction of the polyclonal irregular bending of the mesodermal axis (Keller and Jansa,1992). Again, FN-independent adhesion to the BCR is suffi-antiserum, are consistent with the outcome from the RGD

inhibition experiments. Moreover, we showed that locomo- cient to prevent bending. In amphibian species with a verydense FN matrix, these functions of the BCR may by neces-tory cytoplasmic protrusions are absent from mesoderm

cells of inhibitor-treated embryos. We conclude that mecha- sity depend on FN-mediated adhesion and would then besensitive to inhibition.nisms other than cell migration must be able to generate

nearly normal mesoderm movement in the Xenopus gas- Second, in contrast to BCR-less embryos, movement ofthe deep zone mesoderm is not strongly affected in FN-trula.

Two general types of mesoderm behavior can be dis- inhibited embryos. During development, spreading of thedeep zone appears largely independent of convergence andcerned, each characteristic of a subregion of the early gas-

trula mesoderm. At the onset of gastrulation, the prospec- extension. The anterior mesoderm starts moving towardthe animal pole before the posterior mesoderm involutes,tive mesoderm consists of an inner deep zone and the invo-

luting marginal zone peripheral to it. The deep zone converges, and extends behind it (Nieuwkoop andFlorschutz, 1950; Nakatsuji, 1975). In exogastrulae of Xeno-represents the future anterior mesoderm and includes, on

the dorsal side, the prospective head and heart mesoderm. pus, anterior mesoderm seems to move inside and to spreadon the BCR, although the posterior mesoderm does not in-It leads movement by spreading across the BCR surface.

The involuting marginal zone will form the more posterior volute, but converges and extends on the outside (Nieuw-koop and Koster, 1995). Spreading of the deep zone meso-mesoderm, including most of the axial and paraxial meso-

derm. In contrast to the deep zone mesoderm, this part derm, which is of course not observed in BCR-less embryos(Keller and Jansa, 1992), was assumed to be driven by activeconverges and extends dorsally (Keller, 1976; Gerhart and

Keller, 1986). cell migration (Gerhart and Keller, 1986). However, our re-sults suggest that deep zone spreading can also occur in theConvergent extension is a substrate-independent shape

change of mesodermal tissue which is driven by active cell absence of mesoderm migration.We showed that absence of locomotory protrusion fromrearrangement (Shih and Keller, 1992). It is not inhibited by

RGD peptides (Smith et al., 1990; Winklbower, R., unpub- mesoderm cells is compatible with approximately normalpositioning of the mesoderm during gastrulation. Moreover,lished observations). The relative importance and power of

this process are demonstrated when the BCR is removed inhibitor-treated embryos develop complete heads, im-plying correct movement and spreading of the head meso-before gastrulation, such that no substrate for mesoderm

spreading and migration remains. In these embryos, mesoderm. Also, RGD peptide does not inhibit heart formationfrom deep zone mesoderm at the normal site (Yost, 1992).derm involutes normally, the blastopore closes, and dorsal

convergence and extension generate an embryonic axis (Kel- Apparently, FN-independent interaction with the BCR issufficient for spreading movement of the deep zone meso-ler and Jansa, 1992). Thus, gastrulation is remarkably well

sustained in BCR-less embryos, and one might assume that derm. This important result is not easily explained fromour current knowledge of gastrulation mechanisms and cer-inhibition of FN function should be compatible with at least

the same degree of normal gastrulation movement. This tainly deserves further investigation.Although mesoderm movements are nearly normal, de-could in part explain the lack of gastrulation arrest after

inhibitor treatment. fined effects of inhibiting cell–FN interaction are observed.Most notably, the vegetal base of inhibitor-treated embryosHowever, mesoderm behavior is much more natural in

FN-inhibited embryos than in BCR-less ones. First, axial is not completely internalized. Since FN is present betweenvegetal cells (Danker et al., 1993), a direct effect of inhibi-mesoderm movement in roofless embryos shows deficienc-

ies which can be rescued by FN-independent interaction tors on the cell mass, e.g., a weakening of its cohesion, isconceivable. Alternatively, incomplete yolk internalizationwith the BCR. For example, without BCR, the involuted

mesodermal axis sinks into the endodermal cell mass (Kel- may be caused indirectly. Slowing down anteriorly directedmesoderm translocation relative to the rate of convergenceler and Jansa, 1992). This seems to be prevented in the em-

bryo by attachment of the mesoderm to the BCR. Since could possibly upset the normal pattern of forces in thegastrula, constricting the blastopore too fast with respectmesoderm is normally positioned in inhibitor-treated em-

bryos, FN-independent adhesion to the BCR is sufficient for to the inward movement of the yolk mass. Interestingly, alocal increase in cell adhesiveness in the dorsal marginalthis function. Another defect of BCR-less gastrulae is an

FIG. 7. Scanning electron micrographs of peptide-treated gastrulae. (a–c) Dorsal blastopore lip regions of sagittally fractured stage 11–11 1/2 gastrulae. (a) Control embryo; (b) embryo incubated in 4 mg/ml of GRGDSP peptide; and (c) embryo incubated in 4 mg/ml ofGRGESP control peptide. Arrowhead in archenteron is pointing anteriorly. Elongated bottle cells line the anterior end of archenteron.Above the archenteron, involuted mesoderm (m) is in contact with BCR. (d) Sagittally fractured stage 13 embryo incubated in 4 mg/mlof GRGDSP peptide. Large protruding vegetal yolk mass (y) to the right, dorsal side to the top. (Arrowhead) Dorsal blastopore lip. Involuteddorsal mesoderm (dm) with columnar cells. Ventral mesoderm (vm) forming an internal blastopore lip. bc, Remnant of blastocoel. (a–d)Bars, 100 mm.




FIG. 8. Scanning electron micrographs of antibody-treated gastrulae. (a) Sagittally fractured stage 13 embryo continuously incubated in25 mg/ml of anti-FN Fab fragments from stage 9 onward. Protruding vegetal yolk mass (y) to the left, dorsal side to the top. (Arrowhead)Dorsal blastopore lip. dm, involuted dorsal mesoderm; a, archenteron cavity; vm, ventral mesoderm. (b) Sagittally fractured control embryoincubated in 25 mg/ml of IgG from preimmune serum. y, yolk plug; dm, dorsal mesoderm; vm, ventral mesoderm; a, archenteron cavity.Same orientation as in (a). Bars, 200 mm.




indirect consequences. In amphibians, a possible proximaleffect is inhibition of cell migration. Whether this leadssecondarily to gastrulation arrest depends on additional fac-tors.

ACKNOWLEDGMENTS

This work was supported in part by an EMBO long-term fellow-ship to R. Winklbauer and by NIH Grant HD 18979 to R. Keller.Part of the work was performed by R. Winklbauer at the Max-Planck-Institut fur Entwicklungsbiologie, Tubingen. Still anotherpart, carried out by R. Winklbauer at the Zoologisches Institut,Universitat zu Koln, was supported by the Deutsche Forschungs-gemeinschaft. Antibody to Xenopus cadherins was a gift from Drs.FIG. 9. Larvae developing from peptide-treated gastrulae. Em-Arno Muller and Brigitte Angres. We thank Paul Tibbetts and Jur-bryos that gastrulated in the continuous presence of 4 mg/ml ofgen Sauter for help with the SEM and Andrea Belkacemi for techni-GRGESP control peptide (a) or 4 mg/ml of GRGDSP peptide (b–d)cal assistance.were raised to the larval stage. Deficiencies after GRGDSP peptide

treatment show a graded series (b–d).

REFERENCES

zone, generated by the overexpression of EP/C-cadherin (Lee Boucaut, J. C., and Darribere, T. (1983a). Presence of fibronectinand Gumbiner, 1995), yields the same phenotype that we during early embryogenesis in the amphibian Pleurodeles waltlii.have obtained by FN inhibition. Cell Differ. 12, 77–83.

Boucaut, J. C., and Darribere, T. (1983b). Fibronectin in early am-phibian embryos: Migrating mesodermal cells contact fibronec-tin established prior to gastrulation. Cell Tissue Res. 234, 135–Fibronectin, Mesoderm Migration, and145.Gastrulation in Other Amphibian Embryos

Boucaut, J. C., Darribere, T., Boulekbache, H., and Thiery, J. P.(1984a). Prevention of gastrulation but not neurulation by anti-The role of FN-dependent processes in gastrulation hasbodies to fibronectin in amphibian embryos. Nature 307, 364–been most extensively studied in the urodele Pleurodeles.367.Here, inhibition of FN function leads to an arrest of gastrula-

Boucaut, J. C., Darribere, T., Poole, T. J., Aoyama, H., Yamada,tion movements (Boucaut et al., 1984a,b; Darribere et al.,K. M., and Thiery, J. P. (1984b). Biologically active synthetic

1988, 1990; Riou et al., 1990). A possible interpretation is peptides as probes of embryonic development: A competitive pep-that interaction with FN is necessary for mesoderm migra- tide inhibitor of fibronectin function inhibits gastrulation in am-tion and that, in contrast to Xenopus, mesoderm migration phibian embryos and neural crest cell migration in avian em-is indispensable for gastrulation in this species. However, bryos. J. Cell Biol. 99, 1822–1830.

Boucaut, J. C., Darribere, T., Shi, D.-L., Boulekbache, H., Yamada,it has not been excluded that other effects contribute toK. M., and Thiery, J. P. (1985). Evidence for the role of fibronectinthe observed gastrulation arrest. For example, the FN fibrilin amphibian gastrulation. J. Embryol. Exp. Morphol. 89(Suppl.),network is more dense in these embryos (Boucaut et al.,211–227.1985), and therefore mesoderm adhesion to the BCR could

Collazo, A. (1994). Molecular heterochrony in the pattern of fibro-depend wholly on FN. Without this adhesion, internalizednectin expression during gastrulation in amphibians. Evolutionmesoderm may not be able to assume its normal position,48, 2037–2045.

as is also observed in Xenopus BCR-less embryos. Also, Danker, K., Hacke, H., Ramos, J., DeSimone, D., and Wedlich, D.ingression of mesoderm at the blastopore lip, which is im- (1993). V/-fibronectin expression and localization prior to gastru-portant in urodele gastrulation, could be affected by FN lation in Xenopus laevis embryos. Mech. Dev. 44, 155–165.inhibition. In inhibitor-treated Pleurodeles embryos, meso- Darribere, T., Yamada, K. M., Johnson, K. E., and Boucaut, J.-C.

(1988). The 140-kDa fibronectin receptor complex is required forderm remains on the surface of the gastrula and is thereforemesodermal cell adhesion during gastrulation in the amphibiannot available for translocation across the BCR by whateverPleurodeles waltlii. Dev. Biol. 126, 182–194.mechanism.

Darribere, T., Guida, K., Larjava, H., Johnson, K. E., Yamada,In the anuran Rana pipiens, blocking FN function withK. M., Thiery, J.-P., and Boucaut, J.-C. (1990). In vivo analyses ofantibodies or RGD peptides leads to an intermediate pheno-integrin b1 subunit function in fibronectin matrix assembly. J.type. Although mesoderm does not reach the animal pole, itCell Biol. 110, 1813–1823.

shows substantial translocation (Johnson et al., 1993; Saint- Gerhart, J., and Keller, R. (1986). Region-specific cell activities inJeannet and Dawid, 1994). In summary, it appears that FN’s amphibian gastrulation. Annu. Rev. Cell Biol. 2, 201–229.role in gastrulation is not directly revealed by inhibition Holtfreter, J. (1946). Structure, motility and locomotion in isolatedexperiments. In analyzing this role, it is essential to distin- embryonic amphibian cells. J. Morphol. 79, 27–62.

Howard, J. E., Hirst, E. M. A., and Smith, J. C. (1992). Are b1guish between proximal effects of blocking FN function and




integrins involved in Xenopus gastrulation? Mech. Dev. 38, 109– oriented locomotion by amphibian gastrula mesodermal cells. J.Cell Sci. 59, 43–60.120.

Nakatsuji, N., and Johnson, K. E. (1983b). Comparative study ofJohnson, K. E., Darribere, T., and Boucaut, J.-C. (1993). Mesodermalextracellular fibrils on the ectodermal layer in gastrulae of fivecell adhesion to fibronectin-rich fibrillar extracellular matrix isamphibian species. J. Cell Sci. 59, 61–70.required for normal Rana pipiens gastrulation. J. Exp. Zool. 265,

Nakatsuji, N., Smolira, M. A., and Wylie, C. C. (1985). Fibronectin40–53.visualized by scanning electron microscopy immunocytochemis-Keller, R. E. (1976). Vital dye mapping of the gastrula and neurulatry on the substratum for cell migration in Xenopus laevis gastru-of Xenopus laevis II. Prospective areas and morphogenetic move-lae. Dev. Biol. 107, 264–268.ments of the deep layer. Dev. Biol. 51, 118–137.

Nieuwkoop, P. D., and Florschutz, P. A. (1950). Quelques caracteresKeller, R. E. (1986). The cellular basis of amphibian gastrulation.speciaux de la gastrulation et de la neurulation de l’oeuf de Xeno-In ‘‘Developmental Biology: A Comprehensive Synthesis Vol. 2:pus laevis, Daud. et de quelques autres Anoures. Premiere partie.The Cellular Basis of Morphogenesis’’ (L. Browder, Ed.), pp. 241–Etude descriptive. Arch. Biol. 61, 113–150.327. Plenum, New York.

Nieuwkoop, P. D., and Faber, J. (1967). ‘‘Normal Table of XenopusKeller, R. E., Danilchik, M., Gimlich, R., and Shih, J. (1985). Con-laevis (Daudin).’’ North-Holland, Amsterdam.vergent extension by cell intercalation during gastrulation of

Nieuwkoop, P. D., and Koster, K. (1995). Vertical versus planarXenopus laevis. In ‘‘Molecular Determinants of Animal Form,’’induction in amphibian early development. Dev. Growth Differ.UCLA Symposia on Molecular and Cellular Biology, New Series37, 653–668.31 (G. M. Edelman, Ed.), pp. 111–141. A. R. Liss, New York.

Riou, J.-F., Shi, D.-L., Chiquet, M., and Boucaut, J.-C. (1990). Exoge-Keller, R. E., and Danilchik, M. (1988). Regional expression, patternnous tenascin inhibits mesodermal cell migration during am-and timing of convergence and extension during gastrulation ofphibian gastrulation. Dev. Biol. 137, 305–317.

Xenopus laevis. Development 103, 193–210.Saint-Jeannet, J.-P., and Dawid, I. B. (1994). Vertical versus planar

Keller, R., and Winklbauer, R. (1992). Cellular Basis of Amphibian neural induction in Rana pipiens embryos. Proc. Natl. Acad. Sci.Gastrulation. Curr. Topics Dev. Biol. 27, 39–89. USA 91, 3049–3053.

Keller, R., and Jansa, S. (1992). Xenopus gastrulation without a Shih, J., and Keller, R. E. (1992). Cell motility driving mediolateralblastocoel roof. Dev. Dyn. 195, 162–176. intercalation in explants of Xenopus laevis. Development 116,

Kubota, H. Y. (1981). Creeping locomotion of the endodermal cells 901–914.dissociated from gastrulae of the Japanese newt, Cynops pyrrho- Smith, J. C., Symes, K., Hynes, R. O., and DeSimone, D. (1990).gaster. Exp. Cell Res. 133, 137–148. Mesoderm induction and the control of gastrulation in Xenopus

Lee, G., Hynes, R., and Kirschner, M. (1984). Temporal and spatial laevis: The roles of fibronectin and integrins. Development 108,regulation of fibronectin in early Xenopus development. Cell 36, 229–238.729–740. Winklbauer, R. (1988). Differential interaction of Xenopus embry-

Lee, C.-H., and Gumbiner, B. M. (1995). Disruption of gastrulation onic cells with fibronectin in vitro. Dev. Biol. 130, 175–183.movements in Xenopus by a dominant-negative mutant for C- Winklbauer, R. (1989). A reinvestigation of the role of fibronectincadherin. Dev. Biol. 171, 363–373. in amphibian gastrulation. J. Cell Biol. 107, 815a.

Muller, H.-A. J., Kuhl, M., Finnemann, S., Schneider, S., van der Winklbauer, R. (1990). Mesodermal cell migration during XenopusPoel, S. Z., Hausen, P., and Wedlich, D. (1994). Xenopus cadher- gastrulation. Dev. Biol. 142, 155–168.

Winklbauer, R., and Nagel, M. (1991). Directional mesoderm cellins: The maternal pool comprises distinguishable members ofmigration in the Xenopus gastrula. Dev. Biol. 148, 573–589.the family. Mech. Dev. 47, 213–223.

Winklbauer, R., Selchow, A., Nagel, M., Stoltz, C., and Angres, B.Nakatsuji, N. (1975). Studies on the gastrulation of amphibian em-(1991). Mesodermal cell migration in the Xenopus gastrula. Inbryos: Cell movement during gastrulation in Xenopus laevis em-‘‘Gastrulation: Movements, Patterns and Molecules’’ (R. Keller,bryos. Wilhelm Roux Arch. 178, 1–14.W. Clark, and F. Griffin, Eds.), pp. 147–168. Plenum, New York.Nakatsuji, N. (1976). Studies on the gastrulation of amphibian em-

Winklbauer, R., and Selchow, A. (1992). Motile behavior and protru-bryos: Ultrastructure of the migrating cells of anurans. Wilhelmsive activity of migratory mesoderm cells from the Xenopus gas-Roux Arch. 180, 229–240.trula. Dev. Biol. 150, 335–351.Nakatsuji, N. (1986). Presumptive mesoderm cells from Xenopus

Yost, H. J. (1992). Regulation of vertebrate left–right asymmetrieslaevis gastrulae attach to and migrate on substrata coated withby extracellular matrix. Nature 357, 158–161.fibronectin or laminin. J. Cell Sci. 86, 109–118.

Nakatsuji, N., and Johnson, K. E. (1983a). Conditioning of a culture Received for publication December 28, 1995Accepted April 22, 1996substratum by the ectodermal layer promotes attachment and



fibronectin, mesoderm migration, and gastrulation in xenopus

Documents