Initiation of mesodermal cell migration and spreading ... · different cleavage stages and dorsal marginal zone (DMZ) explants of early gastrula stage were cultured either on fibronectin

Development 105, 351-363 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

351

Initiation of mesodermal cell migration and spreading relative to gastrulation

in the urodele amphibian Pleurodeles waltl

DE-LI SHI1, THIERRY DARRIBERE1, KURT E. JOHNSON2 and JEAN-CLAUDE BOUCAUT1

1 Laboratoire de Biologie Experimentale, UA-CNRS 1135, Universite Pierre et Marie Curie, Batiment C-30, 9 Quai Saint-Bernard, ',France'Department of Anatomy, The George Washington University, Medical Center, Washington, DC 20037, USA

Summary

We have investigated the autonomous migration ofmarginal cells and their interactions with extracellularmatrix (ECM) located on the inner surface of theblastocoel roof in the urodele amphibian, Pleurodeleswaltl, using a novel in vitro migration assay. Animalhemispheres containing equatorial cells removed atdifferent cleavage stages and dorsal marginal zone(DMZ) explants of early gastrula stage were culturedeither on fibronectin (FN)-coated or ECM-conditionedsubstrata. In explanted animal hemispheres, dorsalmarginal cells showed autonomous migration on FN-coated substratum at the same time as the onset ofgastrulation in control embryos. They acquired thiscapacity at least at the 32-cell stage, whereas lateral andventral marginal cells acquired it after the 64-cell stage.DMZ outgrowths of early gastrula stage exhibited auton-omous spreading on both substrata. In addition, weshowed that they spread preferentially toward the ani-

mal pole when deposited on substratum conditioned bythe dorsal roof of the blastocoel. By culturing dissociatedmarginal cells on ECM-conditioned substratum, we alsofound that increased spreading capacity of marginalcells was related to the initiation of their migration. Acomparative study of the migration of marginal cells inultraviolet (u.v.)-irradiated and normal embryos wasalso made. The results indicate that dorsal marginal cellmigration was absent or dramatically reduced by u.v.-irradiation. These results suggest that the differentialacquisition in the spreading capacity both in timing andin intensity around the marginal zone was correlatedwith the sequential involution of mesodermal cells in thecourse of gastrulation.

Key words: cell migration, marginal zone, gastrulation,substratum, urodele, Pleurodeles waltl.

Introduction

The basic body plan of the amphibian embryo arisesfrom sequentially programmed cellular activities. Thedorsoventral axis is established after fertilization as aresult of cortical/cytoplasmic rotation (Vincent & Ger-hart, 1987). The cytoplasmic localizations responsiblefor the formation of axial structures are initially local-ized in the subequatorial region of uncleaved egg(Gurdon et al. 19856). Alternatively, during cleavagestages, mesoderm induction which involves cell interac-tions between the presumptive endoderm and theadjacent animal hemisphere leads to the formation ofan equatorial zone of mesoderm (Nieuwkoop, 1969a;Sudarwati & Nieuwkoop, 1971; Nieuwkoop, 1977; Gur-don et al. 1985a). Mesoderm induction is known to haveregional specificity because there are different signals tospecify the dorsal and ventral mesodermal structures(Nieuwkoop, 19696; Smith & Slack, 1983; Dale et al.1985; Dale & Slack, 19876).

The most immediate consequence of mesoderm in-

duction is the extensive morphogenetic cell movementsthat occur during gastrulation. These involve variousregion-specific cellular activities (reviewed by Gerhart& Keller, 1986). The dorsal mesoderm plays an import-ant role in this process in both anuran and urodeleamphibians. In Xenopus laevis, cells of the DMZundergo convergent extension by the mechanism ofactive cell intercalation which alone can account for itsinvolution and blastopore closure (Keller et al. 1985).Unlike Xenopus, the DMZ of the Pleurodeles waltl (Shiet al. 1987), and also other urodele amphibians such asCynops pyrrhogaster (Nakatsuji, 1975) and Ambystomamexicanum (Lundmark, 1986), consists of two or threelayers of presumptive mesodermal cells between thelate blastula and the early gastrula stages. The DMZbecomes a single layer of cells soon after involution hasbegun. We have previously found that migration of theinvoluted cells on the inner surface of the blastocoelroof ensures complete gastrulation, whereas cell inter-calation plays only a limited role in this process.

Recently, several lines of evidence have demon-

352 D.-L. Shi and others

strated the presence of ECM fibrils covering the innersurface of the blastocoel roof both in anuran andurodele amphibians (Nakatsuji & Johnson, 19836).These fibrils have been shown to contain both FN(Boucaut & Darribere, 1983; Lee etal. 1984; Darribereet al. 1985; Nakatsuji et al. 1985a) and laminin (LM)(Nakatsuji et al. 19856; Darribere et al. 1986). ECM-fibrils serve as a contact-guidance system for mesoder-mal cell migration toward the animal pole (Nakatsuji etal. 1982; Nakatsuji & Johnson, 1983a, 1984a,6; Nakat-suji, 1984). The role of FN in this contact-guidancesystem has been well established (Boucaut et al.1984a,b, 1985). Also, it has been shown that Xenopusgastrula mesodermal cells adhere to and migrate onLM-coated substratum (Nakatsuji, 1986). More re-cently, integrin (INT), a 140xl03Mr glycoprotein com-plex involved in cell adhesion to FN and LM, has beenreported to be present and play a functional role duringgastrulation in Pleurodeles embryos (Darribere et al.1988; reviewed by Johnson et al. 1988). Thus studies ofmesodermal cell interactions with ECM componentsare important to our understanding of the control ofgastrulation movements. The search for intrinsic factorsgoverning mesodermal cell migration is equally import-ant because it can provide information about thetemporal-spatial regulation of cellular movements aswell as the relationship between the time of onset andthe level of morphogenetic cell movements and theirdirected migration toward the animal pole during gas-trulation.

Here we report on the initiation and temporal-spatial regulation of mesodermal cell migration inPleurodeles gastrulae. The study was undertaken pri-marily to examine the idea that intrinsic factors governthe initiation of mesodermal cell migration. For thatpurpose, we devised an in vitro migration assay byculturing animal hemispheres containing the equatorialcells from different cleavage stages and DMZ explantsfrom early gastrulae on FN-coated substratum. We alsoinvestigated the migratory autonomy of mesodermalcells and their interactions with ECM components. Ourresults indicate that marginal zone cells show auton-omous migration and spreading on ECM-conditionedsubstratum.

Materials and methods

EmbryosEmbryos of the urodele amphibian Pleurodeles waltl Michahwere obtained and staged as described previously (Shi et al.1987).

U. v. irradiationEggs were collected from spawning females. Jelly wasmanually removed with forceps under a dissecting micro-scope. The time period between fertilization and the firstcleavage division is about 6h at 21-23 °C. Irradiation wascarried out at 2h after fertilization. Dejellied eggs weretransferred into a quartz chamber placed 7-5 cm above a254 nm u.v. lamp (15 W; Vilbert Lourmat, France) andirradiated for 1 min. The energy was measured using a 254 nm

u.v. dosimeter (Vilbert Lourmat, France) asl-8-2-0xlO4ergsmm~2. The development of axial structureswas determined according to the descriptions of Scharf &Gerhart (1980).

Preparation of the migration substrataFor FN-coated substratum, plastic culture dishes (35 mm;Nunc, Denmark) were incubated overnight at 4°C withhuman plasma FN (Sigma Chemical Co.) at SO^gml"1 insterile distilled water. ECM-conditioned substratum was pre-pared according to Nakatsuji & Johnson (1983a, 1984«,£>).Blastocoel roofs were removed from early gastrula embryosusing a tungsten needle and a platinum loop. They were thenexplanted onto culture dishes containing Steinberg's solution(SS) with their basal surface against the plastic. After 2 h, theexplants were flushed away with a Pasteur pipette. For bothkinds of substrata, residual protein-binding sites on theculture dishes were blocked by incubation with 5% bovineserum albumin (BSA) (Fraction V; Sigma Chemical Co.) inSS for 1 h. The culture dishes were rinsed with SS before use.

Migration assaysDissection was carried out in 1 % agar-coated culture dishescontaining SS. A rectangular explant of DMZ and adjacentectoderm was removed from the early gastrula stage and thendeposited onto FN-coated or ECM-conditioned substrata.Animal hemispheres containing equatorial cells from differ-ent cleavage stages were obtained by cutting circumferentiallybelow the equatorial line. Next, they were deposited onto theculture dishes with their basal surface against the substratum.Both kinds of explants were cultured in SS supplemented withpenicillin (100i.u. ml"1), streptomycin (lfjgml"') and genta-mycin (25^gml~1) for appropriate periods.

Cell cultureDissociated cell spreading was performed on ECM-con-ditioned substratum. Explants of DMZ and ventral marginalzone (VMZ) were dissociated in Ca2+-/Mg2+-free Barth'smedium (88mM-NaCl, lmM-KCl, 2-4mM-NaHCO3, 2MM-Na2HPO4, 0-lmM-KH2PO4, 0-5niM-EDTA, pH7-8). Com-pletely dissociated cells were rinsed in SS for 10 min and thenseeded onto the ECM-conditioned areas (300-400 cells foreach area). Spread cells were flattened, formed lamellipodialattachments and assumed a bipolar, triangular or polygonalshape. The percentage of spread cells was determined at 30,60, 90 and 120min in culture.

Inhibition of mesodermal cell migrationThe interactions of spreading mesodermal cells with ECMcomponents were investigated using different antibodies andsynthetic peptides. Antibodies to Ambystoma mexicanumplasma FN have been previously described (Boucaut &Darribere, 1983). Antibodies to avian INT that cross-reactwith Pleurodeles (Darribere et al. 1988), the FN cell-bindingpeptide Gly-Arg-Gly-Asp-Ser (GRGDS), FN collagen-bind-ing peptide Cys-Gln-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr(CQNSETRTFY) and LM cell-binding peptide Tyr-Iso-Gly-Ser-Arg (YIGSR) were kind gifts from Dr Kenneth M.Yamada (NIH, Bethesda, MD) and Dr Jean-Paul Thiery(ENS, Paris). Affinity-purified antibodies to highly purifiedL M from mouse Engelbreth-Holm-Swarm sarcoma have beenobtained and characterized previously (Riou et al. 1987).Monovalent Fab' fragments were prepared according toBrackenbury et al. (1977). Their final concentrations in theculture medium were: antibodies to FN, O-Smgml"1; anti-bodies to INT, lmgml"1; antibodies to LM, lmgrnP1;GRGDS, 0-2mgml - l . YIGSR, lmgml" and

Mesodermal cell migration in amphibian gastrulation 353

CQNSETRTFY, lrngml" . A control experiment was per-formed using antibodies to FN absorbed with the antigen in aratio of 1:2.

ImmunofluorescenceECM-conditioned substratum was monitored for the presenceof FN and LM. After removing the blastocoel roofs, con-ditioned substrata were fixed in 3-7 % formaldehyde in SS for30min. They were then rinsed in SS and incubated sequen-tially with antibodies to FN (S^gmP1) or LM (20iigml~l),and fluorescein isothiocyanate (FITC)-conjugated sheep IgGanti-rabbit IgG (5^gml~'; Biosys, France). After rinsing inSS, conditioned substrata were covered by a coverslip in thepresence of Mowiol (Hoeshst, RFG). They were examinedwith a Leitz Dialux-20 epifiuorescent microscope and photo-graphed using Kodak Tri-X film.

Scanning electron microscopyControl embryos at the midgastrula stage and u.v.-irradiatedembryos that displayed a circular blastopore at the vegetalpole were selected. Vitelline membranes were removed usingsharpened forceps and, at the same time, a small area ofanimal pole cells was removed to make a hole for penetrationof fixatives. Embryos were then processed as describedpreviously (Shi et al. 1987).

Results

Temporal-spatial acquisition of cell migration in themarginal zoneThe developmental capacity for autonomous migrationof mesodermal cells was investigated by culture ofanimal hemispheres containing equatorial cells from the32-cell stage onward on the FN-coated substratum. Atthe 32-cell stage, the 8 vegetal cells were removed andthe remaining 24 animal and equatorial cells werecultured. From the 64-cell stage onward until the 512-cell stage, dissection of embryos was performed belowthe equatorial region in order to discard only thepresumptive endodermal cells (see Fig. 1 andFig. 2A-D). As judged by shaking the culture dishes,1-2 h was necessary for such explants to attach firmly tothe FN substratum.

When the respective control embryos reached theearly gastrula stage (8a), i.e. about 30h after fertiliz-ation at 21-23°C, where involution is initiated in thedorsal region, cell migration begins at the margin of theexplants. Cells migrated away from the explants largelyas a cohesive cell sheet but with some isolated pioneer

cells (Fig. 2E-H). At each stage, the results show thatthe average culture time required for the initiation ofcell migration corresponds approximately to thatneeded for control embryos to start gastrulation(Table 1) except that a 3-4h delay was observed. Thisdelay may be due to the healing process and the initialretardation of cell adhesion to the substratum. Thereare significant differences in cell migration in themargin of explants at different developmental stages(Fig. 2I-L). In explants derived from the 32-cell stage,cell migration was restricted to a limited zone (Fig. 21).The area covered by the migrating mesodermal cellsheet was enlarged in explants derived from the 64-cellstage (Fig. 2J). A contracted region developed betweenmigrating mesodermal cells and the adjacent ectodermwas observed in explants cultured from early embryos(32- and 64-cell stages). In explants derived from the128-cell stage onward, mesodermal cell migration be-gins first in a small region but later extends all aroundthe explant margin (Fig. 2K,L).

In order to see if the region where cell migration firstoccurs corresponds to the future dorsal blastoporal lipof the embryos, vital dye staining was performed. InPleurodeles waltl, a discordance between the position of

32-cell

64-cell

,FN-cdatedsubstratum

Early gastrula

Fig. 1. Schematic representation of the dissection ofembryos for cell migration assay. At the 32-cell stage, the24-animal and equatorial blastomeres were isolated. Fromthe 64-cell to the late blastula stages, explants included theentire blastocoel roof. Explants of DMZ and the adjacentectoderm were excised at the early gastrula stage when thedorsal blastopore was just visible (stage 8a).

Table 1. Time of the initiation of DMZ cell migration in explanted animal hemispheres from different cleavagestages on FN-coated substratum

StagesNo. of explants

analysed% of explants showing

cell migration*

86-3

90-7100100100

* Animal hemispheres of each stage with the same pattern of cell migration as presented in Fig. 2I-L were scored at late gastrula stage.tThe average time was determined as that when 50% of the explants or control embryos showed cell migration or gastrulation.

32-cell64-cell

128-cell256-cell512-cell

5143363829

Average timetto migration

19 h17 h16 hl l h6h

Average timetto gastrulation

15 h14 h12 h8h3h


Fig. 2. Culture of animal hemispheres containing ectoderm and equatorial cells on FN-coated substratum. (A-D) Animalhemispheres isolated from the 32- to the 256-cell stages, lOmin after explantation onto FN-coated substratum. (E-H) Whencontrol embryos for explants of each stage reached the early gastrula stage, progeny cells of the dorsal equatorialblastomeres become less cohesive and start to migrate away from the explants. (I-L) Control embryos for explants of eachstage were at the end of gastrulation. Mesodermal cell migration is restricted to the dorsal region in explants removed at the32- and the 64-cell stages (I,J). At the 128- and the 256-cell stages (K,L), mesodermal cell migration can be observed allaround the margin of explants. Scale bar, lmm.


grey crescent and the future dorsal region of theembryos could be observed frequently. For this reason.80 eggs from the same batch were vital-dye marked inthe grey crescent region. When they reached the 32-cellstage, at which time the dye markers were localized inthe tier-3 equatorial blastomeres, 30 were used for thein vitro migration assay as described above, the remain-ing 50 were allowed to develop until the early gastrulastage in order to score the percentage of embryos withdorsal region stained. In 62% of the controls, stainingwas localized in the dorsal blastoporal lip. In othercontrol embryos, vital dye staining was observed onboth sides of the forming blastopore. In 60% of theexplants excised at 32-cell stage and cultured on FN-coated substratum, the initiation of cell migrationoccurred in the stained region. As a result, we canconclude that DMZ cells begin to migrate first in bothcultured explants and in intact embryos.

In vitro migration of early gastrula DMZ cellsExplants of DMZ and the adjacent ectoderm weredissected just when the blastoporal depression wasvisible (stage 8a), they were then cultured on the FN-coated substratum (see Fig. 1). Both DMZ and theadjacent ectoderm adhered to the substratum within30min (Fig. 3A) as assessed by gently shaking theculture dishes. One hour later, migration of cells wasoccurring. Mesodermal cells that were initially near thedorsal blastoporal lip became less cohesive and some ofthem detached from the explants to migrate as isolatedcells (Fig. 3B). In contrast, explants deposited onto

BSA-coated or uncoated plastic culture dishes showedneither adhesion nor migration. The actively migratingpioneer mesodermal cells are bipolar and extend lamel-lipodia and filopodia at opposing ends of the cell body.These cells migrate both forward and backward. Cellsbehind these pioneer cells also undergo spreadingmigration. In the explant, a contracted region wasobserved between migrating mesodermal cells and theadjacent ectodermal cells (Fig. 3B,C) just as observedin explants cultured from early embryos. The appear-ance of this contracted region is concurrent with theinitiation of DMZ spreading as can be discerned inFig. 3B. By the end of gastrulation in control embryos,the spreading of mesodermal cells reached its maximallevel as a fan-shaped cohesive cell sheet (Fig. 3C). Weestimated the extent of outgrowth of mesodermal cellsfrom the dorsal lip of DMZ explants to the outer edgeof outgrowth. Isolated cells were not taken in account.During the 24 h period from the time of initiation ofcultures until the end of gastrulation in control em-bryos, we found that there was a migration of1-4mm ±0-1S .D . (n = 28), giving a migration rate ofljummin"1. After the gastrula stage, the rate of DMZspreading slowed down. There was no obvious increaseof this distance when the explants were cultured untilcontrol embryos reached the late neurula stage. More-over, these cells begin to differentiate according to theirprospective fate such as notochord and somitic cells(results not shown).

DMZ migration was also assayed on ECM sub-stratum conditioned by the blastocoel roof of early

Fig. 3. In vitro mesodermal cell migration from DMZ on FN-coated substratum. Explant (10 mm in length, 0-8 mm inwidth) of DMZ together with adjacent ectoderm was cultured for 24 h. (A) 30min after explantation, the explant has healedand adhered to the substratum. (B) 2h after explantation, cells initially near the dorsal lip of the blastopore detach from theexplant and migrate. A contracted region (arrows) appears concurrently with the initiation of DMZ spreading. (C) 24 h ofculture, DMZ has fully expanded forming a fan-shaped cohesive cell sheet with isolated cells which migrate randomly. Theregion between migrating DMZ and the adjacent ectoderm becomes more contracted (arrows). Scale bar, 0-5 mm.


gastrulae. A cohesive cell sheet developed after 24 h ofculture. However, unlike on FN-coated substratumwhere isolated cells migrated randomly (see Fig. 3C),we rarely observed isolated migrating cells on ECM-conditioned substratum (see Fig. 6). In fact, on thelater substratum, a few cells of the dorsal blastoporal lipdetached from the explants and migrated as isolatedcells during the first 3-4 h of culture. Later, all migrat-ing cells fused together as a cohesive cell sheet. Thisphenomenon is observed during normal developmentwhere isolated pioneer mesodermal cells appeared onlyat the early gastrula stage (results not shown). Theextent of DMZ outgrowth was the same on bothsubstrata.

Interactions of migrating mesodermal cells with ECMcomponentsIn order to ascertain that the in vitro spreading andmigration of the DMZ cell sheet depends on specificinteractions between mesodermal cells and ECM com-ponents, antibodies and synthetic peptides were addedto the culture medium. We cultured DMZ explants onFN-coated and also on ECM-conditioned substratahoping to mimic the conditions found in vivo. On thelatter substratum, immunofluorescent studies showedthat, at the front of migration, mesodermal cells extendlamellipodia and filopodia interacting with the ECMcomponents present in the deposited fibrils(Fig. 4A,B). Each reagent was tested for 2h on dupli-cate cultures including ten DMZ explants.

When monovalent Fab' fragments of anti-FN wereadded to culture of fully spread DMZ cell sheet,isolated migrating cells no longer adhered to the sub-stratum. The cohesive DMZ cell sheet was disruptedinto isolated cultures of cells (Fig. 4C). These cellsbecame rounded but still attached to the substratum(Fig. 4E). In contrast, the coherence of the ectodermalsheet was not affected (Fig. 4C). Antibodies to INT andalso the FN cell-binding peptide GRGDS gave the sameresults (not shown). On the other hand, neither themonovalent antibodies to LM nor the LM cell-bindingpeptide YIGSR had such effects (Fig. 4D). As can beseen in Fig. 4F, the cohesive cell sheet remains intactand the filopodia of cells at the leading edge of theoutgrowth are still adherent to the substratum. Controlexperiments using anti-FN absorbed with the antigenand the FN collagen-binding peptide CQNSETRTFY,indicated that these reagents had no inhibitive effectson mesodermal cell migration. All DMZ explants gavethe same results for a given reagent tested.

Furthermore, if antibodies to FN, INT and thepeptide GRGDS were added to the medium at thebeginning of DMZ culture, cell spreading was pre-vented. Removal of the antibodies or peptide from theculture before gastrulation had ended in control em-bryos, restores mesodermal cell migration and allowsthe development of DMZ cell sheet. Antibodies to LMand the peptide YIGSR did not prevent DMZ fromspreading on ECM-conditioned substratum. It appearsthat the autonomous spreading and migration of DMZcells involves specific interactions with FN.

Involvement of ECM in the orientation of DMZoutgrowthsIn experiments involving cultures of dissociated meso-dermal cells on ECM-conditioned substratum, it wasshown that these cells migrated preferentially towardthe animal pole (Nakatsuji & Johnson, 1983a, 1984a,b).We made use of the in vitro migration assay to see ifECM provides a similar guidance for DMZ outgrowth.ECM substratum was conditioned by rectangularexplants excised in the area extending between thedorsal blastoporal lip and the animal pole of the earlygastrula (stage 8a). After conditioning, a small explantcorresponding to DMZ and the adjacent ectoderm wasexcised from another early gastrula and then depositedon the ECM-conditioned substratum. The DMZexplant orientation was perpendicular to that of theECM-depositing explant (Fig. 5). After a cultureperiod of 24 h, the orientations of DMZ outgrowthswere scored.

Results based on 78 explants are summarized inTable 2. 68 % of explants had DMZ outgrowths towardthe animal pole (AP+); in such cases, a cohesive DMZcell sheet was deflecting 90° with respect to its animalpole to blastopore axis (Fig. 6). In this direction, DMZcells migrated only until they reached the edges of theconditioned area. By contrast, only two explants (3%)had DMZ outgrowths oriented toward the blastopore(BP+). Spreading in both directions on the substratum(Neutral) was observed in 19 % (15 out of 78) of theexplants, with outgrowth toward the animal pole notsignificantly more marked. Finally, total absence ofoutgrowths was observed in 10 % (8 out of 78) of theexplants (no migration). Control experiments werecarried out by coating the same area as in experimentswith ECM-conditioned substratum with plasma FNinstead. Under these conditions, DMZ outgrowthsalways spread regularly at the periphery of the explantswithout preferential orientation (results not shown).

Increase of the spreading capacity on ECM bymarginal cells at the onset of their involutionThe in vitro migration assays have indicated thatspreading capacity was acquired differentially aroundthe marginal zone. Dorsal cells have acquired thiscapacity as early as 32-cell stage. In explants, they beginto migrate only at the beginning of gastrulation incontrol embryos. These observations suggest that theinitiation of migration involves some regulative processthat is intrinsic to mesodermal cells. Examination of thespreading capacity of dissociated DMZ and VMZ cellson ECM-conditioned substratum provides a way to testthis hypothesis.

DMZ cells were dissociated at early gastrula stage(stage 8a), and VMZ cells dissociated at early gastrula,

Table 2. Orientation of DMZ outgrowths onconditioned substrata

Animalpole Neutral

Nomigration Blastopore Total

53 (68 %) 15 (19%) 8 (10%) 2(3%) 78(100%)


Fig. 4. Mesodermal cell interactions with ECM-conditioned substratum. (A) Mesodermal cells at the front of migrationextend lamellipodia and filopodia which interact with ECM fibrils (arrows) revealed by rabbit anti-FN antibodies (5figmr')followed by FITC sheep anti-rabbit IgG (5/igml"1). (B) Mesodermal cells interaction with ECM fibrils (arrows) revealed byrabbit anti-LM antibodies (20^gml ) followed by FITC sheep anti-rabbit IgG (SjUgmF1). (C) Effect of monovalent Fab'fragments of anti-FN on DMZ cell migration. Explant of DMZ and adjacent ectoderm of the early gastrula was cultured onECM-conditioned substratum, monovalent Fab' fragments of anti-FN were added after 24 h of culture as a finalconcentration of 0-5mgml~\ DMZ cell sheet is completely disintegrated into clusters of cells which become rounded. Theadjacent ectoderm cell sheet is not affected. (D) Monovalent Fab' fragments of anti-LM at the final concentration of1 mgml"1 has no effect on mesodermal cell migration. (E) Phase contrast corresponding to C showing the rounding up ofdisrupted DMZ cells. (F) Phase contrast of the leading edge of DMZ outgrowth in D. Cells remain cohesive and adherent tothe substratum, m, Mesodermal cells. Scale bars: (A.B), 50fim; (C,D), 0-5mm; (E,F), 100j<m.

early midgastrula (stage 9) and late midgastrula (stage10) stages, respectively. Once spread on ECM-con-ditioned substratum, cells flattened and extendedlamellipodia, assuming a bipolar, triangular or polyg-onal shape (see Fig. 3C). On unconditioned plastic

culture dishes (control), cells had a rounded shape anddid not extend lamellipodia (not shown). Significantdifferences in cell spreading were noticed betweenDMZ and VMZ cells dissociated at early gastrula stage(Fig. 7). A high percentage of spread cells (58 % ± 8 %)


AP

BP

Fig. 6. Orientation of DMZ outgrowth toward the animalpole (AP+) on ECM-conditioned substratum. Explantcontaining DMZ and adjacent ectoderm was initiallydeposited in the centre of the substratum (see Fig. 5). Itsoriginal animal pole to blastopore axis (thick arrow) wasperpendicular to that of the substratum which is marked byfine arrows in the corners of the micrograph. After 24h ofculture, DMZ outgrowth has fully spread toward the animalpole, a cohesive cell sheet developed on this side. The frontof migration reached the edge of animal pole and stoppedthere because ECM-conditioned substratum was no longeravailable. Scale bar, 0-5 mm.

was recorded for DMZ whereas only 10 % ± 2 % VMZcells spread. As gastrulation proceeds, the percentageof spread VMZ cells increased progressively, it attained15 % ± 2 % at the early midgastrula stage and reached26% ± 3 % at the late midgastrula stage, but neverreached the same level as the early gastrula DMZ cells,

Fig. 5. Experimental design to test theinvolvement of ECM in the orientation of DMZoutgrowths. To make the conditionedsubstratum, rectangular explants (1-2 mm inlength, 0-8 mm in width) between the blastopore(BP) and the animal pole (AP) were dissectedfrom the early gastrula stage. They were thendeposited onto a culture dish with the blastocoelroof against the plastic (upper part). The edgesof the explant were marked with forceps. After2h in culture, the explant was flushed away. Asmaller explant (0-6 mm in length, 0-4 mm inwidth) of DMZ and adjacent ectoderm wasdissected from another gastrula (lower part) anddeposited in the centre of the ECM substratum(represented by crosses) with its animal pole toblastopore axis (arrow) perpendicular to that ofthe substratum. It was then cultured for 24h.

DMZ 8a

60Time of culture

90

VMZ 10

VMZ 9VMZ 8a

120 (min)

Fig. 7. Spreading of dissociated DMZ and VMZ cells onECM-conditioned substratum. DMZs were dissociated atthe early gastrula stage (stage 8a); VMZs dissociated fromthe early gastrula stage, the early midgastrula stage (stage9) and late midgastrula stage (stage 10). They were thenseeded on ECM substratum conditioned by early gastrulablastocoel roof, 300-400 cells for each conditioned area.Attached cells with a bipolar, triangular or polygonal shapewere counted at 30, 60, 90 and 120min in culture. Thevalues represent the mean of triplicate determinations± S.D.

even at the stage where involution would normally takeplace (stage 10). These results suggest that DMZpossesses a stronger inherent spreading capacity than itsventral counterpart in the course of gastrulation. It isalso true that there are fewer mesodermal cells in theVMZ than in the DMZ. These results were confirmedby the observation that VMZ explants cultured oneither FN-coated or ECM-conditioned substrataexhibited the same behaviour as DMZ explants exceptthat spreading of mesodermal cell sheet was less exten-sive (results not shown).


Fig. 8. Cellular organization and mesodermal cell migration in u.v.-irradiated embryos. (A,B) Scanning electron microscopyof cell rearrangement at the midgastrula stage. (A) Normal embryo fractured midsagittally, involuted cells attach to theinner surface of the blastocoel roof and migrate toward the animal pole. (B) Embryo irradiated at 1-8-2-OxlO4 ergsmm"2 at2h after fertilization and fractured when control embryos were at the midgastrula stage. A circular blastopore was formed atthe vegetal pole, endodermal cells were pushed upward by the invaginated bottle cells. No cells attached to the inner surfaceof the blastocoel roof. (C,D) Pattern of mesodermal cell migration in animal hemispheres of irradiated embryo of the 256-cell stage on FN-coated substratum. (C) Mesodermal cell migration can be observed to occur initially at multiple sites. (D)When control embryos were at the late gastrula stage, the irradiated explant has a discontinuous mesodermal cell sheet withmultiple regions devoid of migrating cells (arrowheads), bl, Blastocoel; bp, blastopore; m, mesodermal cells. Scale bars:(A,B), 250/mi; (C,D) lmm.

Reduced dorsal cell migration in u.v.-irradiatedembryosU.v. irradiation was performed to investigate furtherthe dorsal-ventral polarity of mesodermal cell mi-gration in relation to gastrulation. In each experiment,half of the irradiated embryos were used for migrationassays and the other half was allowed to develop inorder to control the alteration of dorsal polarityinduced by u.v. irradiation. We observed that, at thegastrula stage, 80% of irradiated embryos bear acircular blastopore in the vegetal pole. Later, theydeveloped as aneural or acephalic embryos.

For comparison with unirradiated control embryos,the cellular organization of irradiated embryos thatdisplayed a circular blastopore was first observed byscanning electron microscopy (Fig. 8A,B). Fracturedsections show clearly a regular distribution of bottlecells all around the vegetal pole (Fig. 8B). They wereradially oriented with their contracted apices borderingthe blastopore. No cells adhere to the inner surface ofthe blastocoel roof. Endodermal cells were pushedupward by the invaginated bottle cells. The samecellular organization was observed in all sagittal sec-tions made through the animal-vegetal axis of u.v.-


c

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Fig. 9. Histogram of the effect of u.v. irradiation onmesodermal cell migration. Embryos were irradiated at 2hafter fertilization at l-8-2-0xl04ergsmm~2. Animalhemispheres were excised from the 32-cell (n = 41), the64-cell (n = 46), the 128-cell (n = 22) and the 256-cell(n = 24) stages and were cultured on FN-coated substratum.The pattern of mesodermal cell migration was scored whencontrol embryos were at late gastrula stage using thefollowing criteria: (1) explants without any migrating cellsat all; (2) abnormal cell migration, explants with either fewmigrating cells or an interrupted mesodermal cell sheet atmultiple regions and (3) explants with normal cell migrationwith respect to control explants.

irradiated embryos forming a circular blastopore. Theentire marginal zone of these irradiated embryos wassimilar to the ventral region of unirradiated controlembryos (Fig. 8A). This homology between VMZ ofnormal embryo and the entire marginal zone of u.v.-irradiated embryos was also reflected by the in vitromigratory behaviour of equatorial cells. When animalhemispheres of u.v.-irradiated 256-cell-stage embryoswere cultured on FN-coated substratum, in most cases,mesodermal cell migration was observed simul-taneously in multiple regions (Fig. 8C). Later, whencontrol embryos were at the late gastrula stage, adiscontinuous mesodermal cell sheet had developed inu.v.-irradiated explants. In contrast to control cultureswhere the spreading is regular around the margin ofexplants, the margin of irradiated explants had multipleregions devoid of migrating cells (Fig. 8D).

Therefore, u.v. irradiation has transformed thespreading migration of dorsal cells into ventral type.This effect can be observed more clearly if we repeatthe same experiment as shown in Fig. 2 while usingu.v.-irradiated animal hemispheres. Spreading of meso-dermal cells in explanted animal hemispheres excisedfrom u.v.-irradiated and unirradiated embryos wascompared. A dramatic reduction and/or disappearanceof the capacity of mesodermal cells to migrate wereobserved in irradiated explants (Fig. 9). We dis-tinguished three kinds of explants for each stage: (1) nomigrating cells at all; (2) reduced cell migration whereirradiated explants presented either few migrating cells

or a discontinuous mesodermal cell sheet at the marginof explant and (3) normal cell migration. A significantpercentage of the irradiated explants derived from the32- (77%) and the 64-cell (52%) stages were totallydevoid of migrating cells (Fig. 9). This result indicatesthat u.v. irradiation considerably reduces dorsal meso-dermal cell migration. There were progressively fewerexplants devoid of migrating cells when they wereremoved from the 128-(36%) and the 256-cell (26%)stages (Fig. 9), however, most explants derived fromthese two stages (45 % and 52 %, respectively)exhibited abnormal marginal cell migration as de-scribed above and as illustrated in Fig. 8C,D.

Discussion

In a previous study, we showed that mesodermal cellmigration plays a major role in gastrulation of urodeleamphibian embryos (Shi et al. 1987). The DMZ ofPleurodeles waltl gastrula is initially composed of two orthree layers of mesodermal cells and becomes one-layered soon after involution has begun (see alsoFig. 8A). In the course of gastrulation, involuted cellsmigrate toward the animal pole and form the archen-teron roof as one sheet of cohesive cells. In the presentstudy, we have observed that, in vitro, the spreadingand migration of mesodermal cells as a cohesive cellsheet mimics the conditions found in vivo, and thusprovides a useful model to study mesodermal cellbehaviour in amphibian gastrulation. This paper dealswith four new aspects of this process by using an in vitromigration assay. First, the autonomous spreading andmigration of early gastrula marginal zones was estab-lished using animal hemispheres taken from the 32-cellstage onward. Second, the involvements of FN in cellmigration and of ECM in the orientation of earlygastrula DMZ outgrowths were investigated. Third, thespreading capacity of dissociated cells on ECM fromDMZ and VMZ was analysed at gastrula stages.Finally, the migration of mesodermal cells in normaland u.v.-irradiated embryos was compared.

We have found that the capacity for mesodermal cellsto undergo autonomous migration on FN-coated sub-stratum was acquired as early as the 32-cell stage forDMZ, and between the 64- to the 128-cell stages forlateral and ventral marginal zones. A similar differencein the time at which DMZ and VMZ become specifiedto form their appropriate mesodermal cell types wasalso noticed in Xenopus laevis (Nakamura, 1978).When animal hemispheres containing equatorial cellswere cultured, mesodermal cells derived from thedorsal equatorial blastomeres began to migrate at thetime of initiation of gastrulation in control embryos ifone allows for a delay in healing and attachment ofexplants. These results are consistent with thosereported by Gimlich (1986), who showed that, inXenopus, dorsal equatorial cells acquired their develop-mental autonomy at the 32-cell stage. When these cellswere transplanted to u.v.-irradiated recipients, theypromoted normal gastrulation and later formed axial


structures. Consequently, on the basis of in vitromigration assays in Pleurodeles and transplantationexperiments in Xenopus, it seems that DMZ cells of the32-cell embryo have already acquired the capacity tomigrate autonomously at gastrulation. Nevertheless,the ability of both DMZ and VMZ cells to migrate mayalso be related to an increased number of specifiedmesodermal cells. In this respect, the possibility thatvegetal inducing cells are included in the progeny of thedorsal equatorial blastomeres and may cause mesoderminduction cannot be completely excluded. It can beargued that there is little or no mesoderm on the ventralside of the explanted animal hemisphere from 32-cellstage. At this stage, mesoderm induction has probablynot yet occurred (Jones & Woodland, 1987). Examin-ation of the fate map of the 32-cell stage of Xenopusembryo shows that, on the dorsal side, the subequator-ial cells give rise to a significant amount of endoderm,which may be able to cause mesoderm induction on thisside. However, much less endoderm is derived fromventral subequatorial cells (Dale & Slack, 1987a).Therefore, there were probably few mesodermal cellspresent in the ventral side at gastrulation. Furtherexperiments should be done to test this importantpoint.

In other respects, the initiation of mesodermal cellmigration is probably due to a programmed develop-mental 'clock' because dorsal equatorial cells of differ-ent developmental stages begin to migrate at the sametime both in vitro and in control embryos. Symes &Smith (1987) reported that, in Xenopus, animal poleexplants exposed to mesoderm-inducing activity atdifferent developmental stages exhibit gastrulation-likemovements (convergent extension) at about the sametime. They also showed that the gastrulation clock is notdependent upon the number of cell divisions, numberof DNA replication cycles or the ratio of nuclear tocytoplasmic volume. In this study, we found that thespreading capacity of VMZ cells on ECM-conditionedsubstratum increases as gastrulation proceeds. Thus acorrelation exists between the rate of spreading and theamplitude of mesodermal cell migration. Work on otheramphibian species has shown a change in cell contactduring gastrulation (Johnson, 1970) and an increasingability of gastrula cells to adhere to FN (Johnson, 1985;Johnson & Silver, 1986; Komazaki, 1988). Therefore,the migratory capacity displayed by marginal cells at theonset of their involution is attributable primarily to anincrease in the adherence and spreading on ECM-substratum.

While this paper was in preparation, Keller & Danil-chik (1988) reported that, in Xenopus, the degree ofextension of marginal zone is greater on the dorsal sideof the gastrula. They also showed that convergentextension of the involuting marginal zone (IMZ) is anautonomous process and begins at a stage when theIMZ has involuted. These results agree well with theobservations made in this study. This consistency inresults suggests that expression of regional cellularactivities during gastrulation in Xenopus and Pleuro-deles shares common characteristics. However, mar-

ginal cells of these two species may use differentmechanisms to accomplish gastrulation. An importantobservation made in this study is that DMZ cells ofPleurodeles need an adequate substratum to exerciseautonomous migration. It may explain the previousobservation that sandwich explants of DMZ showedlittle convergent extension until the late gastrula stage(Shi etal. 1987).

We showed also that the marginal mesodermal cellspossess the capacity to undergo autonomous attach-ment, spreading and migration on both FN-coated andECM-conditioned substrata. If antibodies to FN, INTor the cell-binding peptide GRGDS were added to themedium, spreading of DMZ cells was inhibited. Theseresults support an important role of FN and INT inmesodermal cell migration, which has been establishedboth in vivo (Boucaut etal. 1984a,b, 1985; Darribere etal. 1988) and in vitro using other experimental systems(Johnson, 1985; Johnson & Silver, 1986; Nakatsuji,1986). Although antibodies to LM or the cell-bindingpeptide YIGSR failed to inhibit migration and spread-ing of DMZ cells on ECM-conditioned substratum,differences exist between outgrowth from DMZexplants on FN-coated and ECM-conditioned sub-strata. On the latter substratum, we rarely observed anyisolated migrating cells as we did on the former sub-stratum. These observations may suggest that theassemblage of ECM components such as LM and FNinto ECM fibrils suppresses the random migration ofmesodermal cells, and thus serves as a contact-guidancesystem. However, we cannot exclude the possibilitythat other extracellular molecules not identified pre-sently can also fulfil this function.

The preferential spreading of DMZ outgrowthstoward the animal pole observed in this study suggeststhat their strong autonomous spreading capacity associ-ated with the orientation modulated by ECM are majormechanisms for gastrulation movements in Pleurodeles.These observations have two important implications.First, they may imply that differences exist in the ECMalong the blastopore-animal pole axis. The substratumis more favourable for migrating mesodermal cellsapproaching the animal pole. Second, concerning theDMZ explants per se, they are capable of responding tothe guidance of ECM in spite of the discrepancybetween the direction of migration and their originalaxis. They also reinforce our previous experimentsshowing that grafted rotated DMZ explants were ableto migrate according to the animal-vegetal axis of hostembryos (Shi et al. 1987). Both in vivo and in vitrostudies have shown that ECM fibrils are predominantlyoriented along the blastopore-animal pole axis andprovide a contact-guidance system for migrating meso-dermal cells (Nakatsuji et al. 1982; Nakatsuji & John-son, 1983a, 1984a,b; Nakatsuji, 1984). Moreover, dur-ing the gastrulation process, epibolic movement of theprospective ectoderm could be involved in the orien-tation of such ECM fibrils (Nakatsuji, 1984). However,this process does not rule out the possibility thatregional differences in ECM components might existand play a role in directing the oriented movement of


migrating mesodermal cells. Further microsurgical ex-periments and biochemical analysis are needed tounderstand this important mechanism of cell guidanceduring amphibian gastrulation.

The u.v. irradiation of fertilized amphibian eggsinterferes with the determination of axial structures(Grant & Wacaster, 1972; Malacinski et al. 1975, 1977;Scharf & Gerhart, 1980, 1983; Youn & Malacinski,1981). It also inhibits grey crescent formation. Thisearly effect may be involved in the subsequent develop-mental abnormalities observed in u.v.-irradiated em-bryos (Manes & Elinson, 1980). In Pleurodeles, wehave found that u.v. irradiation was associated with acomplete inhibition or a pronounced decrease in DMZcell migration in animal hemispheres excised at eitherthe 32- or the 64-cell stage. In later stages, there is anobvious synchrony in the migratory capacity of meso-dermal cells everywhere in the margins of explants.Thus, changes in DMZ cell migration may explain theabsence of dorsal blastoporal lip formation and thedevelopment of the circular blastopore observed inu.v.-irradiated embryos (see Fig. 8B). The results fromscanning electron microscopic analysis and in vitromigration assays indicate that the entire marginal zoneof u.v.-irradiated embryos behaves like the VMZ ofnormal embryos. Moreover, it has been shown that allmeridians of an irradiated embryo produce mesodermfounder cells in a fashion that is typical of the slowest-developing posterior/ventral sector of the normal em-bryo (Cooke & Smith, 1987). An alternative example isthe dorsoanterior-enhanced embryos produced by lith-ium treatment. In this case, the entire mesodermalmantle is composed of chordomesoderm and representsthe long axis of the embryo (Kao & Elinson, 1988). Incontrast to u.v.-irradiated embryos which form rudi-mentary gut (grade 5), the invagination in lithium-treated embryos leads to the elongation of a symmetri-cal archenteron. Therefore, our present results supportthe proposition that the predominance of cell migrationin DMZ over lateral and ventral zones accounts for thegraded times of onset of gastrulation found in normalembryos.

In conclusion, we have provided evidence that mi-gration of mesodermal cells appears to be an auton-omous process, their interactions with FN and ECM area crucial factor causing dorsal cells to migrate towardthe animal pole. The initiation of mesodermal cellmigration is correlated with an increase in the adher-ence and subsequent spreading on the ECM. Thus, itwill be of interest to investigate the developmentalexpression of molecules involved in these processes inorder to understand the molecular control of mesoder-mal cell migration in amphibian embryos.

We are grateful to Dr J. P. Thiery and Dr K. Yamada forgenerous gifts of antibodies and synthetic peptides. We wouldlike to thank Dr H. Boulekbache and F. Meury for SEMobservations, J. Derosiers for illustrations and P. Greve forhis help. This work was supported by grants from the CNRSand FRM.

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(Accepted 29 September 1988)

Documents

Initiation of mesodermal cell migration and spreading ... · different cleavage stages and dorsal marginal zone (DMZ) explants of early gastrula stage were cultured either on fibronectin