Animal and vegetal pole cells of early Xenopusembryos ...dev.biologists.org/content/develop/124/21/4275.full.pdf · Establishment of a correct vertebrate body plan relies on the

4275Development 124, 4275-4286 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV2180

Animal and vegetal pole cells of early Xenopus embryos respond differently

to maternal dorsal determinants: implications for the patterning of the

organiser

Sébastien Darras 1, Yusuke Marikawa 2, Richard P. Elinson 2 and Patrick Lemaire 1,*1Institut de Biologie du Développement de Marseille, Laboratoire de Génétique et Physiologie du Développement, UMR 6545CNRS-Université de la Méditerranée, Campus de Luminy, Case 907, F-13288 Marseille cedex 9, France2Department of Zoology, University of Toronto, 25 Harbord Street, Toronto M5S 3G5, Ontario, Canada*Author for correspondence (e-mail: [email protected])

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The maternal dorsal determinants required for the specifi-cation of the dorsal territories of Xenopusearly gastrulaeare located at the vegetal pole of unfertilised eggs and aremoved towards the prospective dorsal region of the fer-tilised egg during cortical rotation. While the molecularidentity of the determinants is unknown, there are dorsalfactors in the vegetal cortical cytoplasm (VCC). Here, weshow that the VCC factors, when injected into animal cellsactivate the zygotic genes Siamoisand Xnr3, suggesting thatthey act along the Wnt/β-catenin pathway.

In addition, Siamoisand Xnr3are activated at the vegetalpole of UV-irradiated embryos, indicating that these twogenes are targets of the VCC factors in all embryonic cells.However, the consequences of their activation in cells thatoccupy different positions along the animal-vegetal axisdiffer. Dorsal vegetal cells of normal embryos or VCC-treated injected animal cells are able to dorsalise ventral

mesoderm in conjugate experiments but UV-treated vegetalcaps do not have this property. This difference is unlikelyto reflect different levels of activation of FGF or activin-likesignal transduction pathways but may reflect the activationof different targets of Siamois. Chordin, a marker of thehead and axial mesoderm, is activated by the VCC/Siamoispathway in animal cells but not in vegetal cells whereascerberus, a marker of the anterior mesendoderm whichlacks dorsalising activity, can only be activated by theVCC/Siamoispathway in vegetal cells. We propose that theregionalisation of the organiser during gastrulationproceeds from the differential interpretation along theanimal-vegetal axis of the activation of the VCC/β-catenin/Siamoispathway.

Key words: Siamois, Xnr3, chordin, noggin, cerberus, Xenopus,regionalisation, Organiser, maternal dorsal determinants, Wnt, cor

SUMMARY

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INTRODUCTION

Establishment of a correct vertebrate body plan relies on formation of a functional Spemann’s organiser by the eagastrula stage (reviewed by Lemaire and Kodjabachian, 19Therefore, understanding the molecular mechanisms undeing the formation of this structure has been a major goamodern embryology. At the early gastrula stage, Spemanorganiser is located in the dorsal marginal zone of amphibembryos, in a territory fated to form dorsoanterior mesodeRecent evidence has indicated that two types of informatare involved in the formation of the organiser: mesodeinduction and a dorsal pathway triggered by cortical rotatio

Mesoderm induction is mediated by polypeptide growfactors which probably belong to the TGF-β or FGF families.These factors are required for the induction of the corramount of mesoderm (Hemmati-Brivanlou and Melton, 199Amaya et al., 1991) and cooperate with an independent dopathway to give rise to dorsal mesoderm (Kimelman et 1992).

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The dorsal pathway is likely to make use of components the Wnt signalling pathway (reviewed by Kuhl and Wedlich1997). Ventral overexpression of several Wnt family membeor of downstream components of their transduction pathwsuch as Xdsh and β-catenin, leads to the formation of anectopic organiser. Conversely, inactivation of β-catenin or ofthe maternal transcription factor Xtcf-3, homologous to thDrosophila Wg transducer pangolin (Brunner et al., 1997leads to the ventralisation of Xenopusembryos. Consistentwith these findings, β-catenin is translocated to the nucleus ia broad dorsal domain during the blastula stages (Schneideal, 1996; Larabell et al., 1997). Several zygotic targets of thpathway have been identified (Carnac et al., 1996; YanSnyder et al., 1996; Brannon and Kimelman, 1996; Ryan et 1996; Fagotto et al., 1997). Among these, Siamoisencodes ahomeodomain transcription factor with strong dorsalisinactivity (Lemaire et al., 1995), Xnr-3codes for a TGF-βfamilymember (Smith et al., 1995) and chordinand noggincode forinhibitors of Bmp4 protein (Smith and Harland, 1992; Sasai al., 1994; Piccolo et al., 1996; Zimmermann et al., 1996).

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Siamoisand Xnr3are activated very early and might be diretargets of the Wnt pathway. In addition, Fan and Sokol (19showed that a fusion protein between the N-terminal repredomain of Drosophilaengrailed and the DNA-binding domainof Siamoisacted as a dominant repressor mutant of Siamoisandsuppressed axis formation, thus suggesting that Siamoisactivityis required for the formation of the organiser.

In contrast, activation of chordinmay be indirect as this geneis activated later during development (Sasai et al., 1994; Ret al., 1996) and is a target of Siamois(Carnac et al., 1996; Fanand Sokol, 1997).

These results have greatly improved our understandingthe early events during axis formation in Xenopusbut have alsoleft several issues unresolved. The first one concerns identity of the endogenous maternal determinants that initthe cascade of events leading to the stabilisation and trancation of β-catenin. Overexpression of a dominant negatform of Xdsh, which acts upstream of β-catenin in the Wntpathway, does not prevent the formation of the organi(Sokol, 1996), suggesting that the endogenous maternal dminants that regulate β-catenin may not belong to the Wnfamily. These determinants are located at the vegetal pbefore cortical rotation and subsequently relocated to the fudorsal side of the embryo as cortical rotation proceeds (Yet al., 1990; Fujisue et al., 1993; Holowacz and Elinson, 19Kikkawa et al., 1996; Sakai, 1996). A more precise analysisthe subcellular localisation of the determinants has reveathat they are associated with the cortex (Kageura, 1997). thermore, transplantation of vegetal cortical cytoplasm (VCfrom fertilised eggs into ventral blastomeres is sufficient induce formation of a secondary set of axial structu(Holowacz and Elinson, 1993; Fujisue et al., 1993). In theexperiments, the VCC, like Wnts or noggin, has no mesodinducing activity but can synergise with FGF to give rise more dorsoanterior tissue (Holowacz and Elinson, 199These results suggest that the dorsal factors present in the are the maternal dorsal determinants. However, a direct between the VCC factors and the Wnt/β-catenin pathway hasnot been reported.

A second unresolved issue concerns the regionalisatiothe dorsal territories. Following cortical rotation, materndorsal determinants are translocated to a large domain onprospective dorsal side of the embryo (reviewed by Elinson Holowacz, 1995), a process that can be witnessed by translocation of β-catenin to the nucleus at the mid-blastustage. By the mid-gastrula stage, the dorsal territories hbeen regionalised in at least three functional domains: anteendomesoderm (Bouwmeester et al., 1996), head organand trunk/tail organiser (reviewed by Lemaire and Kojabachian, 1996). How the early broad dorsal domain is regalised is not known.

In this study, we have addressed these two issues. Weshow that the VCC factors activate the Wnt/β-catenin pathwayas demonstrated by the activation of Siamoisand Xnr3. Wethen present data indicating that the activation of the Wnβ-catenin pathway is interpreted differently along the animvegetal axis. In response to the activation of this pathwvegetal cells acquire an anterior endomesodermal fate, maby the expression of cerberus(Bouwmeester et al., 1996)while more animal cells adopt a head/trunk organiser famarked by the expression of chordin.

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MATERIALS AND METHODS

Embryo manipulations and injectionsEmbryos were in vitro fertilized, dejellied, UV treated and culturein 0.1× MBS as previously described (Lemaire et al., 1995). For clineage studies, 4.6 nl of fluorescein lysinated dextran or rhodamlysinated dextran (FLDx, 10 mg/ml in water; RLDx, 5 mg/ml in wateMolecular Probes, Inc., Eugene, OR) were injected into both bltomeres at the two-cell stage or into the two ventral blastomeres atfour-cell stage. Cytoplasmic transplantations were carried out esstially as described in Holowacz and Elinson (1993). Briefly, 30-40 of animal or vegetal cortical cytoplasm was taken from UV-treatfertilized eggs and injected into two animal blastomeres of eight-cembryos, which were used as donors of the animal caps. To ovepress noggin, chordinor Siamoisin tissue explants, 4.6 nl of syntheticmRNA diluted in water were injected into two animal or vegetal blatomeres at the two to four-cell stage. Synthetic mRNAs for Siamois,noggin and chordin, were prepared as described by Carnac et (1996). Synthetic mRNAs for v-rasand tARwere prepared accordingto the methods of Whitman and Melton (1992) and HemmaBrivanlou and Melton (1992).

Construction of pBSRN3 enR-SiapBSRN3 enR-Sia was constructed as follows: the region encodingN-terminal engrailed repressor domain (aa 1-298) was PCR-amplifiwith the oligonucleotides en-F (5′- CGG AAT TCA ACT TTG GCCATG GCC CTG GAG GAT CGC TGC -3′; the underlined sequencecontains a cloning EcoRI site placed in front of the initiator methio-nine of engrailed) and en-R (5′- GGA TCC CAG AGC AGA TTTCTC -3′). The region encoding amino acids 135-208 from Siamocorresponding to the homeodomain and a few flanking amino acwas PCR-amplified with the two oligonucleotides SiaEV-F (5′- CCAGAG AAA TCT GCT CTG GGA TCC TCT CCA GCC ACC AGTA -3′) and SiaEV-R (5′- ATA AGA ATG CGG CCG CTA CTG GGGAGA GTG GAA AGT GG -3′, the underlined sequence introduces cloning NotI site and an in frame stop codon). The two amplifiefragments were mixed and reamplified with the two oligos en-F aSiaEV-R. Because of the partially complementary sequences of eand SiaEV-F, a fragment of 1141 bp was amplified, correspondinga fusion protein between the N-terminal repressor domain of engraand the homeodomain of Siamois. This fragment was cloned into EcoRI/NotI sites of pBluescriptRN3 (Lemaire et al., 1995) to generapBSRN3 enR-Sia. To prepare mRNA for enR-Sia, pBSRN3 enR-Swas linearised with Sfi1 and synthetic capped mRNA was synthesifrom the T3 promoter.

Tissue explants combinationsAll explants were cut in 1× MBS with no. 5 Dumont forceps. Marginalzone explants were cut at stage 10-10.25. Vegetal explants wereout at stage 8.5 and comprised cells located within a 30° arc of vegetal pole. Animal caps were excised at stage 8.5. Recombinabetween ventral marginal zone explants and animal or vegetal cwere carried out according to Carnac et al. (1996). Conjugates wcultured in 1× MBS with 0.2% BSA until control sibling embryosreached the appropriate stage.

Immunohistochemistry and in situ hybridisationDetection of muscle with the monoclonal antibody 12/101 wperformed as described by Carnac et al. (1996) for Figs 5 and12/101 was detected (see Fig. 4) using lissamine rhodamine-cogated donkey anti-mouse IgG (1:100 dilution, BIO/CAN Sci. IncMississauga, Ontario). In situ hybridisation on 10 µm embryo sectionswere performed as previously described (Lemaire et al., 1995) excthat the posthybridisation RNase treatment was omitted. The Xnr3andchordin probes were synthesized using T7 polymerase from EcoRI-

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Fig. 1.Animal caps injected with vegetal cortical cytoplasm expressSiamoisand Xnr3but not Xbra. Animal caps from embryos injectedwith vegetal cortical cytoplasm (VCC) or animal cortical cytoplasm(ACC) were analysed by RT-PCR at stage 10.5 for the expression ofSiamois, Xnr3and Xbra. Endogenous FGFR1transcripts were usedhere as an internal standard. Analysis of the same samples by RNaseprotection gave similar results, as did independent northern analysisof Xnr3. + and – refers in this and the following figures to thepresence or absence of reverse transcriptase, as a control for DNAcontamination. WE, whole embryo; An, uninjected animal caps.

linearised pKSfug (Ecochard et al., 1995) and pSKChd (Sasai et1994) plasmids respectively.

RT-PCR analysisFor the experiments shown in Figs 1 and 6B, RNA was extractedthe proteinase K/LiCl method according to Sambrook et al. (198In all other experiments, embryos or tissue pieces were homogenin NETS buffer (0.3 M NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 1%SDS). Following two rounds of phenol/chloroform extractions, tonucleic acids were ethanol precipited and resuspended in wOligo(dT)-primed first strand cDNA was prepared from the equivlent of 4-12 caps or 1 embryo using Superscript II reverse transctase (Gibco-BRL). A negative control excluding enzyme wincluded for each sample. 1/3 to 1/20 of the cDNA obtained was ufor each PCR reaction which was carried out using 0.5 unit of TDNA polymerase (Promega), 10 pmol of each primer, 100µMdNTPs, 3 mM MgCl2, 0.5 µCi of [α-32P] dATP in a 25 µl volume of1× PCR buffer. During amplification the temperature profile of eacycle was: denaturation at 94°C for 30 seconds, annealing at 6for 30 seconds and extension at 72°C for 30 seconds. For all gstudied except cerberus, 29 amplification cycles were performed. Focerberus, 25 cycles were performed. In these conditions, the sigobtained was a linear function of the imput cDNA (data not showAs an internal loading control, PCR primers for the ubiquitousexpressed FGF-R1 gene were included in all PCR reactions (Lemand Gurdon, 1994). 1/8 to 1/3 of the PCR reaction was loaded o6% sequencing gel which was subsequently dried and autoragraphed.

To avoid confusion between amplification of genomic DNA ancDNA, we chose, for each gene, PCR primers that flanked an intThe sequences of the Xbra and FGF-R1 primers were described previously (Lemaire and Gurdon, 1994). The others primers werefollows: Siamoisforward 5′- AAA CCA CTG ATT CAG GCA GAGG -3′, reverse 5′- GTA GGG CTG TGT ATT TGA AGG G -3′; Xnr-3 forward 5′- GTG AAT CCA CTT GTG CAG TT-3′, reverse 5′-ACAGAG CCA ATC TCA TGT GC -3′; chordin forward 5′- CTG TACCAA CCC AAT CCG TGC C -3′, reverse 5′- CTT GGT GCA ACATCT GTC CCG C -3′; cerberusforward 5′- GCT TGC AAA ACCTTG CCC TT -3′, reverse 5′- CTG ATG GAA CAG AGA TCT TG-3′.

RESULTS

Vegetal cortical cytoplasm induces Siamois andXnr3 expression in animal cap cellsThe dorsal factor in vegetal cortical cytoplasm (VCC) behavas a competence modifier like Xwnt-8 or noggin rather thana dorsal mesoderm inducer (Holowacz and Elinson, 199suggesting that the dorsal factor in VCC may function activate a Wnt signalling cascade. If this is the case, injecof the VCC dorsal factor in animal caps should induce the tWnt targets genes Siamoisand Xnr3 but not the early trunkmesodermal marker Xbra (Smith et al., 1991).

To test this idea, 8-cell embryos were injected with VCCthe animal blastomeres and cultured until stage 10. Animcaps were excised and analysed for Xnr3, Siamois and Xbraexpression at stage 10.5 by semi-quantitative RT-PCR (Materials and Methods). As maternal mRNA for SiamoisandXnr3are present at very low levels (Lemaire et al., 1995; Smet al., 1995) the presence of Siamoisor Xnr3 transcripts ininjected animal caps reflects the activation of the genes rathan a possible carry-over of maternal mRNAs in the injeccortical cytoplasm. As shown in Fig. 1, Xnr3 and Siamois

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These experiments demonstrate that injection of VCC inanimal cap cells is sufficient to activate two targets of thWnt/β-catenin pathway, Siamoisand Xnr3in the absence ofdetectable trunk mesoderm.

Xnr3 and Siamois are activated in cortical cells atthe vegetal pole of UV irradiated embryosWe next wanted to ask whether the inheritance of the VCfactors by vegetal cells would lead to the activation of the sadorsal genetic programme at the early gastrula stage. To this, we UV-irradiated fertilised eggs, thus preventing corticrotation and causing the dorsal determinants to remain at vegetal pole of these embryos.

We first examined if Siamoisand Xnr3were expressed inwhole irradiated embryos at the early gastrula stage (Sta10.5) (Figure 2A). Like Siamois, which was previouslyreported to be expressed in UV-treated embryos (Brannon Kimelman, 1996; Cui et al., 1996), Xnr3 was also activated inthese embryos when assayed by semi-quantitative RT-P(Fig. 2). Activation of these two genes in UV-irradiateembryos did not reflect a general activation of all organis

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Fig. 2.Expression of Xnr3and Siamoisin UV-irradiated gastrulae.(A) Expression in whole embryos. Fertilised eggs were irradiated the expression of Xnr3, Siamoisand the FGFR1genes was assayedby RT-PCR at stage 10.25. In the experiment shown, the averagedorsoanterior index (DAI) of the embryos was 0.27 at the tadpolestage. C, control embryo; U.V., irradiated embryo. (B) Distributionthe transcripts in control (C) or UV-irradiated (U.V.) early gastrulae(DAI=0.26). Vegetal pole explants were dissected at stage 10, anRNA from both these explants and the rest of the embryos wasprepared at stage 10.25. Analysis of transcript abundance was caout by RT-PCR. Siamoisand Xnr3transcripts are enriched at thevegetal pole of UV-irradiated embryos. (C) In situ hybridisation wian Xnr3antisense RNA probe on sectioned control or UV-irradiateearly gastrulae. Due to a better penetration of the probe on sectioembryos, it can be observed that the distribution of Xnr3 transcriptsin normal early gastrulae is not restricted to the epithelial outer celayer, but extends into the deep marginal and vegetal cells. In UVtreated embryos, Xnr3 transcripts are detected in the perinuclearspace of cortical cells. A similar result was obtained with a Siamoisprobe (not shown). Note that in normal embryos, vegetal expressof Xnr3extends nearly down to the vegetal pole.

genes as chordin was not activated in the UV-irradiatedembryos. To determine the localisation of the transcripts fXnr3 and Siamois, vegetal poles of normal or UV-irradiatedembryos were explanted at stage 10 and analysed by RT-Pat stage 10.25 (Fig. 2B). Xnr3 transcripts, like those of Siamois(Brannon and Kimelman, 1996; Cui et al., 1996), were mainpresent at the vegetal pole of UV-irradiated embryos (Fig. 2BFurthermore, analysis by in situ hybridisation of the precisedistribution Siamoisand Xnr3 transcripts confirmed that theywere radially distributed at the vegetal pole and showed thXnr3, like Siamois, is most strongly activated in the cells othe surface layer of the embryo (Fig. 2C and not shown). these cells have probably inherited the vegetal corticcytoplasm, this distribution strengthens the idea thexpression of Xnr3and Siamoisis a consequence of the inheritance of factors present in the VCC. Since vegetal pole ceare larger than dorsal marginal zone cells, fewer cells inhethe dorsal determinants in UV-irradiated embryos than normal embryos, thus providing an explanation for threduced expression of Siamoisand Xnr3in irradiated embryos(Fig. 2A). Taken together, these results indicate that Xnr3 andSiamoisare targets of the VCC in cells of both animal anvegetal origin.

The activation of organiser genes by VCC factors in animand vegetal pole cells prompted us to test the dorsalising prerties conferred by dorsal determinants to animal and vegepole cells.

VCC-injected animal caps have the ability todorsalise ventral marginal zone explants but UV-irradiated vegetal caps do notCarnac et al. (1996) showed that animal cap cells injected weither SiamoisRNA or Xwnt-8 RNA secrete a dorsalisingsignal. As the vegetal cortical factor behaves similarly to Xwnt-8 RNA, we expected that the cortical dorsal factor would alsinduce animal cap cells to secrete a dorsalising signal.

To test this possibility, we injected VCC into animal blastomeres of eight-cell embryos and conjugated stage 8animal caps from these embryos to pieces of ventral margizone (VMZ) from early gastrulae which had been labellewith the fluorescent lineage tracer, fluorescein lysinatedextran (FLDx). The conjugates were cultured until thequivalent of stage 32 and analysed for the presencemuscle by immunostaining with the 12/101 antibod(Kintner and Brockes, 1984). Animal caps from embryoinjected with ACC or uninjected embryos were used acontrols. A schematic representation of this protocol presented in Fig. 3A.

As shown in Table 1 and Fig. 3B, 58.5% of the conjugatwith VCC-injected animal caps contained muscle in the VMexplant. However, muscle was rarely seen in the conjugawith ACC-injected or noninjected animal caps, and notochordal tissue was not morphologically detectable in eithcase. These results indicate that VCC-injected animal cap celike animal cap cells in which the Wnt/β-catenin pathway hasbeen activated, secrete a dorsalising signal which respeciVMZ into muscle cells.

Like VCC-injected animal caps, vegetal poles from UV-irradiated embryos and dorsal vegetal cells from normal embryexpress Siamoisand Xnr3 in response to the inheritance omaternal dorsal determinants. To test their ability to dorsal

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al cortical cytoplasm secrete a dorsalising signal. (A) Diagrammaticral marginal zone explants (composed mostly of ventral mesodermal cells)) previously injected with the lineage tracer FLDx. The explants wereimal caps (stage 8.5) derived from embryos previously injected withCC, VCC). The conjugates were cultured until their mesodermal

stage 32 and they were then immunostained with the muscle antibodyrough conjugates composed of a ventral marginal zone explant labelledDx) combined with an animal cap derived either from uninjected embryos

getal cortical cytoplasm (VCC), or from embryos injected with animal bright-field images; middle panels, position of FLDx-labelled cells; rightfic staining is seen only in the progeny of the FLDx-labelled marginal zoneC-injected animal cap.

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ventral marginal zone explants, we conjugated stage 10 Vexplants with (i) stage 8.5-9 vegetal poles from UV or contrembryos and (ii) stage 8.5-9 dorsal vegetal explants (schematic representation in Fig. 4A). As a positive control, dorsalising properties of stage 10 dorsal marginal zone (DMexplants was also analysed.

Unlike VCC-injected animal caps, we found that vegetpoles from UV-irradiated or control embryos were unable dorsalise the VMZ explants (Fig. 4B and Table 2). contrast, stage 8.5-9 dorsal vegetal explants secreted a faable to cause muscle differentiation in 52% of the VMexplants (Fig. 4B and Table 2). In the latter case, dormesoderm differentiation was limited to the cells derivefrom the VMZ explants, demonstrating that the dorsvegetal explants were not contaminated with mesodeThus, we find that, like DMZ cells, dorsal vegetal celsecrete a signal able to dorsalise ventral mesoderm wheUV-vegetal pole cells do not.

Cui and colleagues (1996) also assessed the inducing perties of UV-irradiated vegetal cells by analysing their abilito induce dorsal mesoderm in conjugated animal caps. Untheir experimental conditions, UV-vegetal cells could indumuscle and, in a minority of cases notochord, in the ectodeThis contrasts with our results. This discrepancy could resfrom differences in the assay system. In our assay, we meathe secretion of a dorsal-ising signal able todorsalise mesodermafter its induction, aproperty which themesoderm induceractivin lacks (Smith etal., 1993). Cui and col-leagues measure theability of vegetal cells tosecrete factors that willinduce dorsal mesodermin ectoderm. In theirassay, induction ofmuscle and notochordby the vegetal capsmight arise from thesecretion by the vegetalcaps of mesoderminducers rather than ofdorsalising molecules.Consistent with thisproposition, we find thatour UV-irradiatedvegetal pole explants,which lack dorsalisingactivity, can inducenotochord and muscle ina similar proportion ofectodermal explants tothat reported by Cui andcolleagues (not shown).

From these experi-ments, we conclude thatthe interpretation of theinheritance of maternal

Fig. 3. Animal caps injected with vegetrepresentation of the experiment. Ventwere taken from embryos (stage 10.25immediately combined with blastula ananimal or vegetal cortical cytoplasm (Acomponent reached the equivalent of 12/101. (B) Photographs of sections thwith fluoresceine lysinated dextran (FL(none), from embryos injected with vecortical cytoplasm (ACC). Left panels,panels, 12/101 staining. Muscle-specicells which were conjugated to the VC

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dorsal determinants depends on the cellular context: vegepole cells do not secrete dorsalisers while cells located highup along the animal-vegetal axis do.

Vegetal pole cells can secrete dorsalisers but fail todo so in response to SiamoisSeveral explanations could account for the failure of veget

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Table 1. Respecification of ventral mesoderm (VMZ) into muscle cells by a dorsalising signal from VCC-injected animalcaps

Number of Number of conjugates with muscle cells in

% of conjugates withInjected samples conjugates examined VMZ* AC† VMZ+AC‡ muscle cells in VMZ§

None 10 1 0 0 10.0Vegetal cortical cytoplasm 41 24 1 2 58.5Animal cortical cytoplasm 29 3 1 0 10.3

Stage 8.5 animal caps, uninjected or injected with vegetal or animal cortical cytoplasm (VCC or ACC), were conjugated to stage 10.25 ventral marginal zone(VMZ) explants. The conjugates were cultured until stage 32 before sectioning. Secretion of a dorsalising signal was detected by the presence of 12/101-positivemuscle cells in the VMZ explant.

*Muscle cells were derived only from the ventral marginal zone explants (VMZ).†Muscle cells were derived only from the animal cap (AC).‡ Muscle cells were derived from VMZ and AC.§The percentage of the conjugates which had muscle cells from VMZ.

Table 2. Dorsalising activity of dorsal marginal, dorsalvegetal and vegetal pole cells

Type of explant Number of conjugated to Number of conjugates with % of conjugatesVMZ tissue conjugates muscle cells with musclepieces analysed in VMZ cells in VMZ

none 47 2 4.2DMZ cells 19 16* 84.2Dorsal vegetal Cells 25 13* 52Veg. pole 22 3 13.6Veg. pole from UV emb. 34 0 0

Stage 8.5-9 animal cap, dorsal vegetal or vegetal pole explants from controlor UV-irradiated embryos were conjugated to stage 10 ventral marginal zone(VMZ) explants. The conjugates were cultured until stage 30-35 beforesectioning. Secretion of a dorsalising signal was detected by the presence of12/101-positive muscle cells in the VMZ explant. No muscle was everdetected in the other part of the conjugates. Notochord was present in DMZexplants (see Fig. 2B) but was never detected by morphological criteria in theVMZ part of the conjugates. *Large patches of muscle were present.

pole cells to secrete dorsalising factors in response maternal dorsal determinants. One of them is that tsecretory machinery of vegetal cells may not supposecretion of dorsalising polypeptides. To test this, we ovexpressed chordin or noggin, two genes coding for dorsalising polypeptides (Carnac et al., 1996) in vegetal pole ceand analysed the dorsalising properties of the resultexplants. Vegetal poles of embryos injected vegetally wchordin (900 pg) or noggin (100 pg) mRNA were explanteat stage 8.5-9, conjugated with stage 10 VMZ explants acultured until tailbud stages (Fig. 5A). Here, we chose to uvegetal pole cells from normal embryos, which also lack dosalising activity (Table 2) and withstand the mRNA injectiobetter than vegetal pole cells from UV-irradiated embryos. a positive control, animal caps from embryos injected in thanimal pole with the same amount of mRNA were conjugatwith stage 10 VMZ explants. Table 3 and Fig. 5B show thinjection of both chordin and nogginmRNA resulted in thesecretion of a dorsalising factor by both animal and vegecaps. Moreover, the proportion of conjugates containimuscle cells in the VMZ part was similar in animal cap/VMconjugates and in vegetal cap/VMZ conjugates. Thindicates that the ability of animal and vegetal cells to secra dorsalising polypeptide was not significantly different ancould not account for the observed difference in dorsalisiproperties.

As the level of expression of Siamoisis decreased in UV-irradiated embryos compared to controls, the lack of dorsaing activity of UV-vegetal pole cells could also be due to ainsufficient level of expression of this gene. To test this hypoesis, we conjugated stage 10 VMZ explants with stage 8.Siamois-injected vegetal poles from UV-irradiated embryoAs a positive control, stage 8.5-9 Siamois-injected animal capswere also conjugated with stage 10 VMZ explants (Fig. 5). previously reported (Carnac et al., 1996), injection of 100 of SiamoismRNA into animal pole cells conferred dorsalisinactivity to injected cells (Fig. 5B; Table 3). However, vegetpole cells from UV-irradiated embryos injected with 100 pg SiamoismRNA lacked dorsalising activity in our conjugateassay (Fig. 5B; Table 3). To rule out that the inability of UVirradiated vegetal pole cells to secrete a dorsaliser in respoto Siamoiswas due to the damaging effects of the ultraviolirradiation, we also conjugated Siamois-injected vegetal polesfrom control embryos to VMZs. In this case, injection of up

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250 pg of SiamoismRNA did not lead to the secretion of a dorsalising signal (Table 3). Hence, the lack of dorsalising activof vegetal pole cells is unlikely to result from the insufficienlevel of activation of Siamois. Rather it appears that the interpretation of the cue provided by Siamois depends on tcellular context.

Cornell et al. (1995) have suggested that the level of acvation of the FGF pathway may be lower in vegetal pole cethan in other embryonic cells, while work by Henry et a(1996) suggests that vegetal cells differ from other embryoncells due to their exposure to higher concentrations of activin-like mesoderm inducing factor. We therefore askewhether artificial activation of the FGF pathway or impairment of an activin-like pathway would confer dorsalisinactivity to Siamois-injected vegetal pole cells. In these expeiments, the FGF/MAP kinase pathway was activated by oveexpressing v-ras, the constitutively active oncogenic form othe protooncogene c-ras (Cornell et al., 1995), while inter-ference with an activin-like pathway was obtained by oveexpressing a truncated form of the type II activin receptotAR (Hemmati-Brivanlou and Melton, 1992). 100 pg oSiamoismRNA was injected into the vegetal pole of early 4cell embryos either singly or with mRNA encoding either v-ras (100 pg) or tAR (1 ng or 4.2 ng). The amounts of v-ras


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oe

and tARmRNA injected were similar to those used by Corneet al. (1995) and Henry et al. (1996) respectively. Vegepoles from injected embryos were dissected at stage 8.immediately conjugated to stage 10 VMZ and cultured unthe tailbud stages. Analysis of muscle formation in tinjected vegetal pole/VMZ conjugates demonstated tinjection of either v-rasor tARmRNA failed to enable vegetapole cells to secrete dorsalisers in response to Siamois(Table3). Therefore, differences in the levels of activation of tactivin-like or FGF pathways in vegetal pole and animal ccells are unlikely to explain the differential response of thecells to the VCC/β-catenin/Siamoispathway.

Activation of different Siamois targets in animal andvegetal cellsThe strong contrast in dorsalising activity conferred Siamoisto vegetal caps and to animal caps suggests that gene activates different targets in these two cell typChordin, coding for a Bmp4 antagonist (Piccolo et al., 199is one of the targets of Siamois: it is activated in animal capoverexpressing Siamois(Carnac et al., 1996) and is repressein embryos in which Siamoisfunction has been disrupted bthe dorsal overexpression of SE, a fusion protein betweenN-terminal repressor domain of Drosophila engrailed andthe DNA binding domain of Siamois (Fan and Sokol, 199Another organiser gene, cerberus, is expressed in a broadedorsal domain (Boumeester et al., 1996). To determine if tgene is also a target of Siamois, we overexpressed Siamoisin the vegetal ventral blastomeres of 4-cell embryoexplanted the ventral vegetal domain of stage 10 embrand cultured the explants until stage 11. Analysis of tabundance of cerberustranscripts by RT-PCR indicated thathis gene could be activated by Siamois(Fig. 6A). To testwhether the presence of Siamois was also required forcerberusexpression, we used enR-Sia, encoding an engrailerepressor domain/Siamois homeodomain fusion protsimilar to SE (Fan and Sokol, 1997), and which also antonises Siamois function (not shown). Dorsal injection oenR-SiamRNA in 4-cell embryos led to the repression o

Table 3. Dorsalising activity of animal and vegetal cells

Type of explant conjugatedto VMZ tissue pieces mRNA injected

None −Veg. pole from UV emb. Sia 100 pgVeg. pole from control embryos Sia 100 pg

Sia 250 pgAn Caps Sia 100 pgVeg. pole from control embryos chd 900 pgAn caps chd 900 pgVeg. pole from control embryos nog 100 pgAn. caps nog 100 pgVeg. pole from control embryos Sia 100 pg + v-ras 100 pgVeg. pole from control embryos Sia 100 pg + tAR 1 ng

Sia 100 pg + tAR 4,2 ng

Stage 8.5-9 animal cap or vegetal pole explants derived from control or v-rasor tARwere conjugated to stage 10 ventral marginal zone (VMZ) expduring normal development as assayed by the expression of Xbra, while idifferentiation (determined morphologically; not shown). The conjugates w12/101 antibody. When present, the muscle cells were always in the VMZVMZ part of the conjugates. Sia, Siamois; Chd, chordin; nog, noggin; tAR

lltal5-9,til

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7).rhis

s,yoshet

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cerberusat stage 11 (Fig. 6A), confirming that this gene a target of Siamois.

We then compared the expression of chordin and cerberusin VCC-injected animal caps and UV vegetal caps. Like Xnr3and Siamois, chordin was activated in VCC-injected animalcaps. However, the level of accumulation of chordin tran-scripts was lower in VCC-injected animal caps than in contrembryos (Fig. 6B). This result may reflect the fact thchordin, unlike Xnr3 or Siamois,may require the presence ofmesoderm inducers to be fully activated. In contrast chordin, cerberuswas not activated in animal caps injectewith either VCC or SiamoismRNA (Fig. 6B and data notshown).

The behaviour of chordin and cerberusalso differed inUV-irradiated embryos. Analysis of their expression pattein UV-irradiated or control embryos revealed that whilchordin was expressed at less than 2% of control levels irradiated embryos (Fig. 2A), the total amount of cerberusmRNA present in an embryo was barely affected by the Utreatment (Fig. 6C). RT-PCR analysis of the distribution cerberustranscripts in UV-treated embryos showed that thmajor site of expression was at the vegetal pole (Fig. 6CFurthermore, this vegetal expression was dependent Siamois as overexpression of enR-Siaat the vegetal pole ofUV-treated embryos led to the complete loss of cerberusexpression (Fig. 6C).

From these data, we conclude that whereas early genesSiamoisand Xnr3are activated by the VCC/β-catenin pathwayin both animal and vegetal cells, these two cell types differtheir ability to express cerberusand chordin, two targets ofSiamois. Chordin, which is activated in animal but not invegetal cells, is a potent dorsalising molecule. In contracerberus can only be activated in vegetal cells anBouwmeester et al. (1996) have shown that cerberus is nodorsaliser, as cerberusmRNA-injected VMZs do not formdorsal mesoderm (lack of dorsalising activity was alsconfirmed in our mRNA-injected animal caps/VMZ conjugatassay; data not shown). The differential activation of cerberusor chordin in response to Siamoiscould therefore account for

injected with Siamois, chordin, noggin, v-rasor tAR mRNANumber of % of explants

Number of conjugates with with muscle cellsconjugates analysed muscle cells in VMZ in the VMZ

56 4 7.132 4 12.512 2 16.6

8 0 030 27* 90

15 9* 6016 10* 62.5

11 8* 72.719 15* 78.9

18 0 011 0 0

22 0 0

UV-irradiated embryos injected as indicated with mRNA for Siamois, chordin noggin,lants. The levels of tARmRNA used were sufficient to block mesoderm inductionnjection of 100 pg of v-rasmRNA into animal caps led to ventral mesodermere cultured until stage 30-35 before sectioning and staining with the muscle-specific part of the conjugate. Notochord was never detected by morphological criteria in the, truncated activin type II receptor. *Large patches of muscle were present.

4282

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RLDx

V D

DMZ D. Veg Veg Pole

V D

VMZVMZVMZ

UVControl

Cultureuntil tadpole stages and stainwith 12/101

St. 8.5St. 8.5St. 10.25

St. 102-cell St.

g activity of dorsal marginal zone cells, dorsal vegetal cells or vegetalV-irradiated embryos. (A) Experimental strategy. Explants of ventralMZ) were taken at stage 10 from embryos injected with the lineage lysinated dextran (RLDx). They were combined with either vegetal

ated embryos (UV Veg Pole), dorsal vegetal cells from stage 8.5-9) or stage 10 dorsal marginal zone (DMZ). The conjugates werege 30-35 and immunostained with the muscle antibody 12/101. of sections through conjugates of ventral marginal zone explantsegetal pole (top), dorsal vegetal cells (middle) or dorsal marginal zone). While DMZ and dorsal vegetal cells have a dorsalising activity, UVl to dorsalise VMZ explants. Notochordal differentiation, monitored byhighly vacuolated cells, is found in the DMZ explants but never observedetal explants. Left panels, 12/101 staining; right panels, position oflls.

A

the different dorsalising properties of VCC-injected animcaps and UV-vegetal caps.

DISCUSSION

In this study, we show that the VCC dorsal factor is able activate the Wnt-responsive genes Siamoisand Xnr3in animalcaps as well as at the vegetal pole of embryos in which cortirotation has been blocked. This suggests that all cells tinherit maternal determinants express Siamois. However, theconsequences of this activation differ along the animal-vegeaxis. VCC-injected animal caps acquire two properties of thead organiser: they express chordinand are able to dorsaliseventral mesoderm. In contrast, vegetal caps from UV-irradiatembryos adopt an anterior mesendoderm fate, as demonstrby the expression of cerberus.

The VCC dorsal factors act along the Wnt/ β-cateninpathwayFormation of the dorsal axis of amphibian embryos initiated by cortical rotation, which moves dorsal factorfrom the vegetal cortical region to a more equatoriposition. Microinjection of vegetal cortical cytoplasm(VCC) into the ventral equatorial region of a recipienembryo produces an ectopic dorsal axis, demonstrating tthe dorsal factors can be experimentallytransferred (Yuge et al. 1990; Fujisue et al.1993; Holowacz and Elinson 1993). Theaxis-inducing property of the cortex wasfurther demonstrated by Kageura (1997),who showed that transplantation of thedorsal vegetal cortex to ventral positions ledto the induction of a secondary axis. A clueas to the nature of the dorsal factors comesfrom microinjection of VCC into animalpole cells. In this location, the dorsal factorsenhance dorsoanterior development, withoutinducing mesoderm, suggesting that they actas competence modifiers (Holowacz andElinson 1995).

Both noggin and members of the Wnt/β-catenin pathway have the capacity to producea dorsal axis by acting as competencemodifiers, rather than as mesoderm inducers.Recent experiments using UV-irradiatedembryos suggest a connection between thedorsal activity of the VCC and the Wnt/β-catenin pathway. Cortical rotation is inhibitedby UV, and two Wnt/β-catenin dependentevents, namely nuclear translocation of β-catenin and expression of Siamois, now occurin cells at the vegetal pole rather than at theequatorial region (Schneider et al., 1996;Brannon and Kimelman 1996; Cui et al.,1996). Dorsal activity of the VCC alsoremains at the vegetal pole in UV irradiatedeggs (Fujisue et al. 1993; Holowacz andElinson 1993).

Our present results greatly strengthen thehypothesis that the VCC dorsal factors act in

Fig. 4.Dorsalisinpole cells from Umarginal zone (Vtracer rhodaminepole from UV-treembryos (D. Vegcultured until sta(B) Photographs(VMZ) with UV vexplants (bottomvegetal poles faithe presence of in the dorsal vegRLDx labelled ce

B

althe Wnt/β-catenin pathway. Microinjection of VCC intoanimal pole cells leads to the expression of Siamoisand Xnr3,both of which are downstream targets of the Wnt/β-cateninpathway and are not expressed in response to either nogginmesoderm inducers (Carnac et al., 1996; Smith et al., 199


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Culture until tadpole stagesand stain with 12/101

RLDx

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V D V D

VMZAn. C

mRNA

VMZVeg pole

St. 8.5

St. 10

St. 8.5

2-cell St.

2-cell St.

2-cell St.

Fig. 5.Dorsalising activity of animal or vegetal pole cells injectedwith Siamoisor chordinmRNA. (A) Experimental strategy. Stage 10ventral marginal cells (derived from embryos injected with RLDx)were combined either with animal cap (stage 8.5-9) derived fromembryos previously injected in the animal pole with 900 pg ofchordinmRNA or 100 pg SiamoismRNA or with vegetal capderived from embryos injected similarly in the vegetal pole. Theconjugates were cultured until stage 30-35 and immunostained wthe muscle antibody 12/101. (B) Photographs of sections throughconjugates of VMZ explants with chordinmRNA injected animalcaps (chd an) or vegetal poles (chd veg), or with SiamoismRNA-injected animal caps (Sia An) or vegetal poles (Sia veg). Whileinjection of chordinmRNA confers dorsalising activity to bothanimal and vegetal pole cells, injection of SiamoismRNA leads tothe secretion of dorsalising signals in animal caps cells only. chd,chordin; Sia, Siamois. Left panels: 12/101 staining; right panels:RLDx labelled cells.

A

B

Furthermore, our results show that the amount of dorsactivity in the VCC of one egg is sufficient to activate Siamoisand Xnr3 ectopically. This observation indicates that overexpression of Wnt/β-catenin pathway members is likely to bemimicking the natural activity found in the VCC. Consistenwith this idea, analysis by in situ hybridisation of the preciselocation of Xnr3 and Siamois transcripts in UV-irradiatedembryos demonstrate that expression of these genesstrongest in the cells of the surface layer that have inherited VCC.

Siamois and Xnr3 are activated in all embryoniccells that inherit dorsal maternal determinantsIn his elegant cortical transplantation experiments, Kageu(1997) found that transplantation of dorsal vegetal cortex to tventral marginal zone caused the development of an ectoaxis, while transplantation to more animal or vegetal positiofailed to do so. Kageura’s interpretation of these results wthat the dorsal factors are only active when they come contact with a core factor localised in the equatorial region the egg. Our results do not support this interpretation. We fithat activation of Siamois and Xnr3 by vegetal cortexcytoplasm is not restricted to the marginal zone. This activtion can occur in both animal and vegetal pole cells andtherefore not regulated by cellular context. Also, the stronactivation of Siamoisand Xnr3at the vegetal pole of UV-irra-diated embryos, which do not undergo cortical rotatioindicates that this process is not needed for the activationthe maternal dorsal determinants.

This constitutive relationship between the activation of thVCC/β-catenin pathway and the expression of Xnr3/Siamoisalso appears to hold true during normal development: durithe extensive pre-gastrula cell movement (Bauer et al., 199the broad domain of nuclear localisation of β-catenin at themid-blastula stage (Fig. 1C from Schneider et al., 1996) shifted to a more vegetal position which corresponds to tearly gastrula domain of expression of Xnr3/Siamois.

Regionalisation of the anterior territories of theorganiserBy the mid-gastrula stage, the domain of expression Xnr3/Siamoiscan be subdivided along the anteroposterior axinto 3 territories, defined by different patterns of expression the Siamoistarget genes cerberusand chordin. The anterior-most domain, located at the leading edge of migrating cellsthe anterior endomesoderm (AEM) which expresses cerberusbut not chordin. Cells in the intermediate domain, which corresponds roughly to the presumptive head mesoderm, exprboth genes, while more posterior organiser cells exprechordin but not cerberus(Bouwmeester et al., 1996). Thesethree domains differ in their inducing properties. The AEMlacks dorsalising activity, as demonstrated by the fact thEinsteck grafts of AEM do not induce ectopic axial or heastructures (Bouwmeester et al., 1996). Although the role of thdomain in Xenopuspattern formation has not been thoroughlinvestigated, in the mouse the anterior primitive endodermrequired for the formation of rostral neurectoderm (Thomaand Beddington, 1996). In Xenopus, Einsteck experiments withhead mesoderm lead to ectopic head formation. Similar expiments with the posterior domain lead to axis duplicationindicating that this territory has dorsalising activity (Lemair

ith

4284

didnges

ofe

otof


of chordinand cerberusby Siamois is context dependent. (A) Cerberusois. 4-cell embryos were injected with 100 pg of SiamoismRNA in a

cation or with 100 pg of enR-SiamRNA in a dorsal vegetal position.ted embryos were reared until stage 10 before excising ventral vegetald. Whole embryos (WE) and ventral vegetal explants from uninjected-injected embryos (VVZ+Sia) were analysed at stage 11 for cerberusordin, but not cerberus, is activated by the VCC in animal caps. Theples as in Fig. 1 were subjected to RT-PCR analysis for the expression ofrus. Abbreviations are as in Fig. 1. (C) cerberusis expressed at the

V-irradiated embryos. Control or UV-irradiated (U.V.) whole embryosxplants were analysed at stage 11 by RT-PCR. In the experiment shown

hordinexpression was undetectable in the UV-irradiated wholewn). enR-Sia: embryos injected at the vegetal pole with 100 pg of enR-

and Kodjabachian, 1996). Fate maps from 32-cell embryindicate that late gastrula AEM is mainly derived from thvegetal-most dorsal blastomeres, while the more postedomains are derived from blastomeres located in a progrsively higher position along the animal-vegetal axis (Baueral., 1994; Vodicka and Gerhart, 1995).

Our results suggest that the position along the animvegetal axis of the cleavage stage blastomeres from whichAEM and head mesoderm are derived could play a major rin the distinction between these two territo-ries. We find that inheritance of dorsal deter-minants by vegetal pole cells leads to theactivation of cerberusbut not of chordin,suggesting that these cells have acquired ananterior mesendodermal fate. Consistentwith this proposition, these cells lack dor-salising activity. In contrast, activation of theVCC/Siamoispathway in animal cells leadsto the activation of chordinbut not cerberusand to the acquisition of dorsalising activity.Although this has not been tested here, it islikely that marginal zone cells, which alsohave dorsalising activity, activate bothcerberus and chordin in response to the acti-vation of the VCC/β-catenin/Siamoispathway.

Taken together, our results suggest that theregionalisation of the anterior domain of theorganiser into AEM and presumptive headmesoderm results from the differential inter-pretation of the activation of the VCC/β-catenin/Siamoispathway along the animal-vegetal axis. In the light of these findings, analternative interpretation of the results of thecortex transplantation studies (Kageura,1997) can be proposed. Transplantation ofdorsal vegetal cortex to both equatorial andvegetal positions leads to Siamois/Xnr3expression. However, while transplantationto an equatorial position leads to thesecretion of a dorsalising factor and toectopic axis induction, transplantation to avegetal position results instead in theformation of AEM-like tissue, which lacksaxis-inducing properties.

Molecular basis for the differentialresponse to the VCC/ β-catenin/ Siamois pathwayIn an attempt to understand why vegetal polecells do not secrete dorsalising factors inresponse to Siamois, we have tested threepossibilities. Firstly, we found that theobserved difference is not due to a generalinability of these cells to secrete dorsalisingpeptides. Secondly, it has been proposed thatthe FGF pathway may be activated at a lowerlevel in vegetal cells than in marginal zonecells (Cornell et al., 1995). We thereforeexamined the effect of artificially increasingthe level of activation of the FGF pathway in

Fig. 6.Activation is a target of Siamventral vegetal loControl and injeccells when neede(VVZ) or Siamoisexpression. (B) Chsame cDNA samchordinand cerbevegetal pole of U(WE) as well as eon the left panel,cembryos (not shoSiamRNA.

ose

riores- et

al- theole

vegetal cells. Under the experimental conditions tested, this not restore the ability of vegetal cells to secrete dorsalisifactors in response to Siamois. Finally, Henry and colleagu(1996) also suggested that decreasing the level of activityan activin-like pathway could convert vegetal cells into moranimal cells. In our hands however, injection of tARmRNA didnot lead to the secretion of dorsalising polypeptides by SiamoismRNA-injected vegetal pole cells. These experiments do nexclude the possibility that a very precise level of activation


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the FGF and/or activin-like pathways is needed for embryocells to secrete dorsalisers in response to Siamois. Howeverthey suggest that the differential response to Siamoisdoes notsolely rest on the difference in the level of activation of thetwo pathways. Several maternal T-box transcription factoexpressed in the vegetal cells have recently been isola(Lustig et al., 1996; Stennard et al., 1996; Zhang and Kin1996; Horb and Thomsen, 1997). In the future, it will be interest to investigate whether these factors play a role in differential response of vegetal and sub-equatorial cellsSiamois.

We thank J. Brockes, F. Watt and the Developmental StudHybridoma bank for antibodies, A. M. Duprat, V. Ecochard, RHarland, Y. Sasai and W. C. Smith, A. Hemmati-Brivanlou and MUmbauer for DNA constructs. During the initial stages of this worS. D. was kindly hosted in the laboratory of J. C. Boucaut to whowe express our gratitude. We are also grateful to D. Caillol teaching in situ hybrisations to S. D., to A. Ribas, J. Kim and G. Téfor maintaining our frog colonies and to M. O’Reilly for criticareading of the manuscript. This work was supported by grants frthe CNRS (ATIPE) and the Ligue Départementale (Bouches Rhône) Contre le Cancer to P. L., by a Human Frontier ScieProgram (HFSP) post-doctoral fellowship to Y. M. and an MRC grato R. P. E.

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(Accepted 13 August 1997)

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