Antagonistic regulation of convergent extension movements in Xenopus by Wnt/β-catenin and Wnt/Ca2+ signaling

Antagonistic regulation of convergent extension movements in Xenopusby Wnt/b-catenin and Wnt/Ca21 signaling

Michael KuÈhla,*, Karin Geisb, Laird C. Sheldahlc, Tobias Pukropb,Randall T. Moonc, Doris Wedlichb,d

aAbteilung Entwicklungsbiochemie, Junior Group SFB 271, UniversitaÈt GoÈttingen, Humboldtallee 23, D-37073 GoÈttingen, GermanybAbteilung Biochemie, UniversitaÈt Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany

cHoward Hughes Medical Institute, Department of Pharmacology, and Center for Developmental Biology, University of Washington School of Medicine,

Seattle, WA 98195-7530, USAdUniversitaÈt Karlsruhe, Zoologisches Institut II, Kaiserstraûe 12, D-76131 Karlsruhe, Germany

Received 8 March 2001; received in revised form 26 April 2001; accepted 3 May 2001

Abstract

Convergent extension movements are the main driving force of Xenopus gastrulation. A ®ne-tuned regulation of cadherin-mediated cell±

cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to

modulate cadherin-mediated cell±cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous

Wnt/b-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on

cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1

functions in this context in the Wnt/b-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca21 pathway blocks convergent

extension movements, with potential regulation of the Wnt/b-catenin pathway at two different levels. PKC, activated by the Wnt/Ca21

pathway, blocks the Wnt/b-catenin pathway upstream of b-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca21

pathway, inhibits the Wnt/b-catenin signaling cascade downstream of b-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling

cascades regulates convergent extension movements in Xenopus. q 2001 Elsevier Science Ireland Ltd. All rights reserved.

1. Introduction

Gastrulation in vertebrates is a complex merging of

temporally and spatially coordinated cell and tissue move-

ments that leads to a reorganized embryo composed of three

germ layers. In Xenopus, gastrulation is governed by

region-speci®c movements: (i) epiboly of the animal cap,

(ii) migration of head mesoderm on the blastocoel roof, (iii)

convergent extension of the involuting marginal zone

(Keller, 1986), and (iv) vegetal rotation (Winklbauer and

SchuÈrfeld, 1999). Migration of head mesoderm requires

cell-substrate adhesion to ®bronectin deposited on the blas-

tocoel roof (Winklbauer, 1994). For epiboly and convergent

extension that are based on radial and lateral cell intercala-

tions, regulation of cell±cell adhesion is essential. Cell±cell

adhesion in the early embryo is mainly mediated by cadher-

ins, calcium-dependent cell±cell adhesion molecules

(reviewed in KuÈhl and Wedlich, 1996). Misexpression of

cadherin mutants affects cell adhesion and gastrulation

movements (Lee and Gumbiner, 1995; Broders and Thiery,

1995; KuÈhl et al., 1996; KuÈhl and Wedlich, 1996; Torres et

al., 1996). Several attempts were made to decipher how

signals involved in mesoderm induction and patterning

also control cell movements. All of these studies end up

with the observation that different types of cell movements

are differentially regulated by growth factors (Howard and

Smith, 1993). However, the detailed molecular basis for

these control mechanisms has remained obscure.

Several lines of evidence suggest that members of the

Wnt family are involved in regulating convergent exten-

sion. Wnt genes encode for glycoproteins that are involved

in cell fate decisions and cell differentiation. A role for

Wnt proteins has also been shown in regulating cadherin-

mediated cell adhesion and cytoskeletal reorganization

(Wodarz and Nusse, 1998). In PC-12 cells, transfection

of Wnt-1 enhances cell adhesion by increasing the steady

state levels of b-catenin, a known cytoplasmic interactor of

cadherins (Bradley et al., 1993). Xwnt-5A, instead, leads to

a decrease in cell adhesion when overexpressed in Xenopus

Mechanisms of Development 106 (2001) 61±76

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* Corresponding author. Tel.: 149-551-399857; fax: 149-551-3912198.

E-mail address: [email protected] (M. KuÈhl).

embryos (Torres et al., 1996). Together with other observa-

tions this bias in Wnt activity led to the model of two

functional distinct Wnt classes. Members of the Wnt-1/

Wg class (Wnt-1, -3a, -8a, and -8b) are able to induce

secondary axes formation in Xenopus embryos when

misexpressed on the ventral side. They transform

mammary C57mg cells and they increase cell adhesion.

On the other hand, members of the Wnt-5A class (Wnt-

4, -5A, and -11) do not induce secondary axes, they antag-

onize the function of the Wnt-1/Wg class and they decrease

cell adhesion (Torres et al., 1996). Members of both classes

also differentially affect convergent extension movements

during Xenopus gastrulation. Whereas members of the

Wnt-1/Wg family can enhance convergent extension

movements in the arti®cial animal cap assay (Sokol,

1993), members of the Wnt-5A class negatively in¯uence

convergent extension (Du et al., 1995; Torres et al., 1996;

Slusarski et al., 1997).

Recent observations suggest that the different classes of

Wnt activities activate different cytoplasmic signaling path-

ways. Members of the Wnt-1/Wg class stabilize cytoplas-

mic b-catenin that subsequently can enter the nucleus. Here

b-catenin interacts with transcription factors of the TCF/

LEF family and the heterodimer that functions as a tran-

scriptional activator (KuÈhl and Wedlich, 1997). The so-

called Wnt/b-catenin pathway also involves the phospho-

protein dishevelled and the serine-threonine kinase GSK-

3b, both of which function upstream of b-catenin. Members

of the Wnt-5A class can trigger intracellular Ca21-release

(Slusarski et al., 1997) and activate PKC and CamKII (Shel-

dahl et al., 1999; KuÈhl et al., 2000a,b). Further downstream

components of the Wnt/Ca21 pathway are not known. In

addition, Wnt-11 has been implicated in regulating conver-

gent extension movements through activating jun-N-term-

inal kinase (JNK) (Heisenberg et al., 2000; Tada and Smith,

2000; Wallingford et al., 2000). This Wnt/JNK pathway is

thought to resemble or be part of the planar cell polarity

pathway in Drosophila melanogaster (Shulman et al.,

1998). However, it is not clear whether there is a relation-

ship between the Wnt/Ca21 and planar cell polarity path-

ways.

The multiple proteins involved in Wnt signaling raise the

question of how Wnts regulate convergent extension move-

ments. In the present study we use in vitro `open face Keller

explants', which mimic the dorsal cell movements of

normal gastrulation, to investigate the role of the Wnt/b-

catenin and Wnt/Ca21 pathways in regulating convergent

extension movements. Our data indicate that the Wnt/b-

catenin pathway is absolutely required for proper conver-

gent extension movements due to regulation of Xnr-3

expression as a direct target gene of TCF/LEF factors.

The Wnt/Ca21 pathway inhibits convergent extension

movements via negative regulation of the Wnt/b-catenin

pathway at different levels. Thus, a ®ne-tuned balance of

opposing Wnt signaling effects serves to control convergent

extension movements during Xenopus gastrulation.

2. Results

2.1. Endogenous Wnt/b -catenin signaling is required for

convergent extension

We ®rst con®rmed reports that the Wnt/b-catenin pathway

enhances convergent extension movements in activin-treated

animal caps (Sokol, 1993) and that Wnt-5A blocks these

movements (Torres et al., 1996) (Fig. 1A). We then turned

to the Keller open face explant assay for molecular analyses,

since this assay is preferable to the animal cap assay in terms

of mimicking the normal movements of gastrulation.

Furthermore, Keller open face explants represent a very

sensitive method to study convergent extension movements.

Initial experiments using Keller explants revealed that over-

expressing Xwnt-8 RNA on the dorsal side of the embryo

does not block convergent extension movements whereas

injection of Xwnt-5A RNA does, as previously shown

(Torres et al., 1996; data not shown). Based on these obser-

vations we hypothesized that Xwnt-8-triggered Wnt/b-cate-

nin signaling might be required as a positive regulator of

convergent extension whereas Xwnt-5A-mediated Wnt/

Ca21 signaling might negatively regulate convergent exten-

sion. We therefore analyzed the effects of both Wnt signaling

pathways on convergent extension in greater detail by using

the Keller open face explant assay.

In order to quantify these experiments we established a

system that allows ef®cient scoring of open face Keller

explants even in large numbers, similar to the system used

by Tada and Smith (2000) for scoring the elongation of

animal caps. For this purpose, explants were con®ned to

one of the following groups after elongation process: (0)

no elongation and no constriction, (1) slight constriction

but no elongation, and (2) strong constriction and elongation

(as illustrated in Fig. 1B). In order to compare the results of

differently treated explants we present our data in two ways.

First, we give the data for each injected RNA by histograms

showing the distribution of each class given in percentages

(see Fig. 1C as an example for Wnt-injected explants). In

addition, we calculated the ratio of those explants showing

strong elongation (class 2) to those that show neither

constriction nor elongation (class 0). We found that these

convergent elongation coef®cients of different experiments

fall into four classes if at least 30 explants were performed

per injected RNA. Values of 1, 3, and 10, respectively, were

used to group the results of the Keller explant experiments

(see Fig. 1D). Based on this we considered that a convergent

extension coef®cient of between 3 and 10 de®nes normal

elongation behavior, whereas a value below 1 represents a

strong inhibition of convergent extension.

We ®rst analyzed the requirement of cytoplasmic b-cate-

nin for convergent extension movements. Overexpression of

XBDe349, an extracellular deleted version of XB/U-

cadherin that binds b-catenin with its intracellular domain,

results in a block of convergent extension movements (Fig.

2A). Similarly, injection of LEFDHMG that binds b-catenin

M. KuÈhl et al. / Mechanisms of Development 106 (2001) 61±7662

but that is not suf®cient to activate downstream events of

Wnt/b-catenin signaling also interferes with convergent

extension (Fig. 2A). We could reverse this effect by coex-

pressing b-catenin but not XB/U-cadherin RNA (Fig. 2B).

The latter argues that the effect of LEFDHMG on conver-

gent extension is not due to a competition of this mutant

with endogenous cadherins for cytoplasmic b-catenin. To

analyze this effect in more detail we injected RNA encoding

for conductin, which is able to trigger b-catenin degradation

(Behrens et al., 1998). Consistent with the aforementioned

results, conductin disturbs convergent extension and again

this effect could be rescued by overexpressing b-catenin but

M. KuÈhl et al. / Mechanisms of Development 106 (2001) 61±76 63

Fig. 1. Assay systems to judge convergent extension movements. (A) In animal caps of Xenopus laevis, addition of activin induces elongation of the caps. This

effect is blocked by injecting Xwnt-5A RNA into the animal pole of two-cell stage embryos. In contrast, injection of Xwnt-8 enhances elongation. A

representative experiment is shown. (B) Elongation of open face Keller explants is shown. For quantitative analyses, explants were grouped into different

classes. Normal constriction and strong elongation of the explant was classi®ed as `class 2'. Explants that just show constriction but no elongation were

grouped as `class 1', whereas explants that neither constrict nor elongate were of `class 0'. (C) Evaluation of prolactin-injected explants (control), Xwnt-5A- or

Xwnt-8-injected explants according to classes exempli®ed in (B). Xwnt-5A blocks convergent extension, while Xwnt-8 does not. Classi®cation of explants was

as given in Fig. 1C. Detailed results are given in Table 1. Calculated elongation coef®cients are also indicated. (D) Dividing class 2 (%) through class 0 (%) of a

given experiment results in the elongation coef®cient that allows rapid scoring and comparison of experiments. Values for the elongation coef®cient fall into

different groups, allowing a comparative judgment of different experiments. Each square in (D) represents an elongation coef®cient as given in Table 1. The y-

axis indicates the number n of times a given elongation coef®cient was found in the experiments presented here.

not XB/U-cadherin (Fig. 2C). We next asked whether this

effect of conductin is primarily due to its degrading effect of

cytoplasmic or cadherin-bound b-catenin. To answer this

question we prepared lysates of conductin or control-

injected embryos and separated membrane-bound and cyto-

plasmic b-catenin. Whereas conductin decreases the overall

amount of b-catenin it mainly affects cytoplasmic rather

than cadherin-associated b-catenin (Fig. 2D). In summary,

these data indicate that the positive effect and the require-

ment of Wnt/b-catenin signaling for convergent extension is

due to its stabilizing effect on cytoplasmic b-catenin.

2.2. XLEF-1 and Xnr-3 are required for convergent

extension

As b-catenin interacts with transcription factors of the

LEF/TCF family to regulate target gene expression, this

raises the possibility that nuclear events of Wnt/b-catenin

signaling are involved in regulating convergent extension.

This hypothesis is supported by our injecting an LEF-1

mutant, LEFDbBD, that still binds to DNA but does not

recruit the transcriptional activator b-catenin and thus

behaves as a dominant negative mutant with respect to


Fig. 2. Blocking the Wnt/b-catenin pathway disturbs convergent extension movements. (A) Titration of cytoplasmic b-catenin by either a cadherin mutant

(XB-cadDe349) or an LEF-1 mutant (LEF-1DHMG) leads to a block of convergent extension movements. (B) The effect of LEF-1DHMG on convergent

extension movements can be rescued by coinjecting b-catenin but not XB/U-cadherin RNA. (C) Injection of conductin RNA blocks convergent extension

movements. This can be rescued by coinjecting b-catenin but not XB/U-cadherin RNA. Classi®cation of explants was as given in Fig. 1. Detailed results are

given in Table 1. Calculated elongation coef®cients are also indicated above the bar graphs. (D) Western blot analysis to monitor b-catenin expression (arrow

head) in conductin-injected embryos. Total lysates, glycoprotein fractions, or soluble fractions of control or conductin-injected embryos were loaded as

indicated. The equivalent of two embryos per lane was used.

TCF/LEF factors (Behrens et al., 1996; Gradl et al., 1999).

This mutant clearly blocks convergent extension (Fig. 3A),

an effect that cannot be rescued by coinjections of b-catenin

(Table 1). However, the LEFDbBD mutant does not discri-

minate between TCF or LEF members of the HMG-box

transcription factor family, and XLEF-1 (Molenaar et al.,

1998) and XTCF-3 (Molenaar et al., 1996) are both

expressed during gastrulation. Thus the function of either

might be blocked by LEFDbBD. Importantly, XLEF-1 as

well as murine LEF-1 are activators of transcription whereas

XTCF-3 acts as a transcriptional repressor (Pukrop et al.,

2001). Thus it is important to determine whether XLEF-1

and XTCF-3 have distinct roles in regulating convergent

extension. Coinjection of mLEF-1 RNA was able to rescue

the inhibiting effect of LEFDbBD, whereas XTCF-3 was

not, indicating that both transcription factors differ in their

ability to regulate convergent extension movements (Fig.

3A). Consistent with this observation is the result that a

single injection of LEF-1 does not interfere with convergent

extension whereas XTCF-3 is a strong inhibitor of this

process (Fig. 3B).

Three target genes of the Wnt/b-catenin pathway have

been described in the early Xenopus embryo: the TGF-bfamily member Xnr-3 and the closely related and functional

redundant homeobox transcription factors siamois and twin

(Brannon et al., 1997; Laurent et al., 1997; McKendry et al.,

1997). We therefore asked which of these target genes might

be suf®cient to restore proper convergent extension in open

face explants de®cient in Wnt/b-catenin signaling. We

found that Xnr-3, but not siamois, was able to rescue conver-

gent extension movements in LEFDbBD- or conductin-

injected explants (Fig. 3C,D). These data indicate that

LEF-1 but not XTCF-3 is required for regulation of conver-

gent extension movements through Wnt/b-catenin signaling

and that Xnr-3 is likely a key target gene for the regulation

of convergent extension.

2.3. The ability of Xnr-3 to rescue convergent extension

movements is not due to a dorsalizing activity

As inhibition of Wnt/b-catenin signaling results in ventra-

lized embryos (Behrens et al., 1996) and as Xnr-3 has been

shown to have a modest dorsalizing activity (Smith et al.,

1995) we next analyzed whether the ability of Xnr-3 to rescue

Wnt/b-catenin signaling de®cient explants just re¯ects

different migration behavior of dorsal versus ventral meso-

derm. As expected, injection of conductin resulted in ventra-

lized embryos (Fig. 3E) and in a down-regulation of several

dorsal marker genes (Fig. 3F). Coinjection of siamois led to a

rescue of dorsal marker gene expression (Fig. 3F) although

siamois was unable to restore proper cell migration behavior

in Keller explants (Fig. 3D). Conversely, Xnr-3 was unable to

rescue dorsal marker genes in conductin-injected embryos

(Fig. 3F) despite its ability to rescue convergent extension

(Fig. 3D). The panmesodermal marker Xbra remained unaf-

fected in these experiments (Fig. 3F). This differential ability

of Xnr-3 and siamois to rescue conductin ventralized

embryos is also re¯ected phenotypically (Fig. 3E). Consis-

tent with an inability to rescue dorsal gene expression, coin-

jection of Xnr-3 with conductin failed to rescue dorsal

structures. In contrast, coinjection of siamois with conductin

rescued dorso-anterior structures like cement glands (Fig.

3E). Some of these embryos exhibited a phenotype consistent

with gastrulation defects. Speci®cally, we found that siamois

is unable to rescue Xnr-3 expression in Wnt/b-catenin signal-

ing defective embryos and vice versa. In summary, the Wnt/

b-catenin pathway is required for both dorsal mesoderm

speci®cation and for convergent extension movements. Our

®ndings indicate that these two properties of the Wnt/b-cate-

nin pathways are governed by two different target genes,

siamois and Xnr-3, respectively.

2.4. The effect of Wnt-5A on convergent extension is due to

interference with Wnt/b-catenin signaling

We next con®rmed (Fig. 4A) that injection of Xwnt-5A

RNA into the dorsal marginal zone blocks elongation of

Keller open face explants as previous described (Torres et

al., 1996). Two downstream effectors of Xwnt-5A were

recently described in the early Xenopus embryo that are

activated by intracellular release of calcium ions, protein

kinase C (PKC) and Ca/calmodulin-dependent kinase II

(CamKII) (Sheldahl et al., 1999; KuÈhl et al., 2000a,b). We

®rst asked whether one or both of them were able to pheno-

copy the effect of Xwnt-5A on Keller explants. Treatment of

Keller explants with the PKC activator PMA resulted in an

inhibition of convergent extension movements (Fig. 4A). In

addition, injection of a constitutively active mutant of

CamKII, CamKII T286D, also blocks convergent extension

movements. Supporting a role for CamKII in regulating the

effects of Xwnt-5A on convergent extension, a kinase-dead

version of this kinase, CamKII K42M, was able to partially

reverse the inhibitory effects of Xwnt-5A on Keller explants

(Fig. 4A). These experiments demonstrate that both PKC

and CamKII are likely involved in mediating the Wnt/

Ca21 signal in regulating convergent extension.

Torres et al. (1996) reported that the effects of Xwnt-5A

on convergent extension can be rescued by coexpression of

full length N-cadherin. We therefore asked whether the

effect of Xwnt-5A or its downstream components can be

rescued by overexpressing XB/U-cadherin (MuÈller et al.,

1994) or Xcadherin-11 (Hadeball et al., 1998). We focused

on these cadherins rather than on the neural-speci®c N-

cadherin since both of them are expressed in the involuting

mesoderm during convergent extension. In addition, a role

for these cadherins in convergent extension movements has

been described (KuÈhl et al., 1996; KuÈhl and Wedlich, 1996;

Broders and Thiery, 1995; Lee and Gumbiner, 1995; Hade-

ball et al., 1998). Surprisingly, overexpression of neither

XB/U-cadherin nor Xcadherin 11 was suf®cient to rescue

the block of convergent extension by either Xwnt-5A or

constitutively active CamKII (Fig. 4B).



Fig. 3. Differential ability of downstream factors of the Wnt/b-catenin pathway to rescue b-catenin targeted embryos. (A) LEF-1 but not XTCF-3 was able to

rescue LEF-1DbBD-injected embryos. (B) XTCF-3 inhibits convergent extension while injection of LEF-1 RNA has no effect on convergent extension by

itself. (C) Xnr-3 but not siamois was able to completely rescue LEF-1DbBD-injected open face explants. (D) Xnr-3 but not siamois was able to fully rescue

conductin-injected open face explants. Classi®cation of explants was as given in Fig. 1. Detailed results are given in Table 1. Calculated elongation coef®cients

are also indicated above the bar graphs in panels (A)±(D). (E) Injection of conductin RNA results in ventralized embryos. This effect can be rescued by

injection of siamois but not Xnr-3 RNA. The DAI index indicates the degree of ventralization with 5 representing wild-type embryos and 0 representing

completely ventralized embryos. (F) Marker gene analysis of embryos injected as in Fig. 3E. Conductin down-regulates dorsal marker genes like goosecoid

(gsc), chordin or XFD-1/pintalavis. This effect can be reversed by injecting siamois but not Xnr-3 RNA. Note that Xnr-3 does not upregulate siamois and vice

versa. Signals indicated by a star arise from injected RNAs.

This raises the question of whether the effect of Xwnt-5A

on convergent extension is due to an interaction with the

Wnt/b-catenin pathway rather than its direct interference

with the cadherin-mediated cell adhesion system. To test

this hypothesis we coinjected b-catenin with Xwnt-5A

into dorsal marginal zones and found that these explants

showed a slight rescue in elongation behavior (Fig. 4C).

Xnr-3, the putative downstream target of b-catenin, was

much stronger than b-catenin in restoring convergent exten-

sion when coinjected with Wnt-5A RNA (Fig. 4C). We next

examined whether this rescuing activity of Xnr-3 might be

due to its dorsalizing activity. We ®rst needed to con®rm

that the effect of Xwnt-5A on convergent extension in

Keller explants and in whole embryos (Fig. 4D) is indepen-

dent of cell speci®cation. Xwnt-5A did not change expres-

sion of two dorsal marker genes, goosecoid and chordin

(Fig. 4E). We then found that dorsal injection of Xnr-3

did not change dorsal marker gene expression, either

alone or in combination with Xwnt-5A (Fig. 4E). We

conclude that the ability of Xnr-3 to rescue convergent

extension movements in Xwnt-5A-injected Keller explants

is independent of cell speci®cation and re¯ects the ability of

Xnr-3 to modulate cell migration.

In search for molecular mechanisms underlying this


Table 1

Effect of overexpressing different RNA constructs on convergent extension: detailed dataa

Probe Experiments Explants Classi®cation of explants Coef®cient Judgment

0 1 2

Nic 32 236 26 85 125 4.81 Normal

PPL 3 30 4 1 25 6.25 Normal

Xwnt-8 3 30 4 9 17 4.25 Normal

XBDe349 5 48 23 15 10 0.43 Inhibited

LEFDbBD 10 83 38 23 22 0.58 Inhibited

LEFDbBD 1 b-catenin 3 42 17 13 12 0.71 Inhibited

LEFDbBD 1 LEF 4 53 10 18 25 2.5 Slight rescue

LEFDbBD 1 XTCF-3 3 46 19 21 5 0.26 Inhibited

LEFDbBD 1 siamois 4 49 12 22 15 1.25 Slight rescue

LEFDbBD 1 Xnr-3 5 59 5 17 37 7.4 Rescue

LEFDHMG 11 92 36 36 20 0.56 Inhibited

LEFDHMG 1 b-catenin 4 46 10 20 16 1.6 Slight rescue

LEFDHMG 1 XB/U-cadherin 4 46 25 13 7 0.28 Inhibited

cdt 7 102 59 17 26 0.44 Inhibited

cdt 1 XB/U-cadherin 3 29 20 7 2 0.41 Inhibited

cdt 1 b-catenin 4 46 8 20 18 2.25 Slight rescue

cdt 1 siamois 4 49 18 19 12 0.67 Inhibited

cdt 1 Xnr-3 3 32 1 15 16 16 Rescue

LEF-1 5 27 3 7 17 5.67 Normal

TCF-3 3 43 24 17 2 0.08 Inhibited

Xwnt-5A 9 93 50 25 18 0.36 Inhibited

Xwnt-5A 1 XB/U-cadherin 3 44 23 15 6 0.26 Inhibited

Xwnt-5A 1 b-catenin 3 45 8 17 20 2.5 Slight rescue

Xwnt-5A 1 Xnr-3 3 28 1 13 14 14 Rescue

Xwnt-5A 1 Xcad11 3 30 18 8 4 0.2 Inhibited

Xwnt-5A 1 K42M 5 55 12 16 27 2.3 Slight rescue

CamKII T286D 8 96 54 20 22 0.41 Inhibited

T286D 1 XB/U-cadherin 4 55 30 18 7 0.23 Inhibited

T286D 1 b-catenin 3 40 19 12 9 0.47 Inhibited

T286D 1 Xnr-3 5 59 10 22 29 2.9 Slight rescue

T286D 1 LEF-1 4 51 10 20 21 2.1 Slight rescue

PMA 9 155 60 58 37 0.7 Inhibited

PMA 1 b-catenin 4 39 10 9 20 2.0 Slight rescue

PMA 1 dnGSK 3 62 9 29 23 2.56 Slight rescue

a Number of experiments indicates different batches of embryos that were used to cut explants. Number of explants represents the total number of explants

cut for this study. Only embryo batches that showed proper elongation in uninjected explants were used as control. Classi®cation of explants according to the

rules as presented in Fig. 1 as well as the elongation coef®cient are given. Abbreviations: nic, non-injected control; PPL, preprolactin; XBDe349, extracellular

deleted mutant of XB/U-cadherin; LEFDbBD, N-terminal deleted mutant of LEF lacking the b-catenin binding domain; LEFDHMG, mutant of LEF-1 lacking

the DNA binding domain; cdt, conductin; Xnr-3, Xenopus nodal-related gene 3; Xcad-11, Xenopus cadherin-11; K42M, kinase-dead mutant of CamKII;

CamKII T286D, constitutively active mutant of CamKII; PMA, phorpbol-12-myristate-13 acetate; dnGSK, dominant negative mutant of GSK-3b.


Fig. 4. The Wnt/Ca21 pathway blocks convergent extension movements. (A) Both PMA and constitutively active CamKII (CamKII T286D) can phenocopy the

inhibitory effects on convergent extension of Xwnt-5A. The inhibitory effect of Xwnt-5A can be partially blocked by a kinase-dead mutant of CamKII (CamKII

K42M). (B) The inhibitory effect of Xwnt-5A on convergent extension movements cannot be blocked by overexpressing Xcadherin-11 or XB/U-cadherin. (C)

The inhibitory effect of Xwnt-5A is blocked by coinjecting b-catenin or Xnr-3 RNA. (D) Phenotype of Xwnt-5A-injected embryos in comparison to control

embryos. (E) This phenotype of Xwnt-5A is caused by defects in gastrulation movements as dorsal marker genes are not affected as analyzed by RT-PCR. In

these experiments, Xnr-3 has no dorsalizing activity excluding the possibility that the rescue of Xnwt-5A-injected explants by Xnr-3 is due to respeci®cation of

mesodermal cells. (F) RT-PCR analyses of Xwnt-5A-injected embryos. Injection of Xwnt-5A RNA leads to a reduction of Xnr-3 expression but not of siamois

expression. The inhibitory effect of Wnt-5A on Xnr-3 expression is partially blocked by either b-catenin or kinase-dead CamKII.

cross-talk of different Wnt pathways we asked whether

Xwnt-5A alters expression of Wnt/b-catenin target genes.

Most strikingly, Xnr-3 was down-regulated by Xwnt-5A,

while siamois expression was unaffected (Fig. 4F, left

panel). Xnr-3 down-regulation could be rescued by coinject-

ing either kinase-dead CamKII or b-catenin RNA together

with Xwnt-5A (Fig. 4F, right panel). These data strongly

suggest that the inhibitory effect of Wnt/Ca21 signaling is

due to an interaction of this pathway with the Wnt/b-catenin

pathway, which results in a speci®c down-regulation of Xnr-

3, the target gene important for convergent extension.

2.5. Wnt/Ca21 signaling blocks Wnt/b -catenin signaling at

multiple levels

We next questioned whether both downstream effectors of

the Wnt/Ca21 pathway, PKC and CamKII, interact at the

same level with Wnt/b-catenin signaling. Surprisingly, we

could reverse the effect of activated PKC on convergent

extension by coinjection of either dnGSK or b-catenin

RNA (Fig. 5A). In the same experiment the effects of consti-

tutively active CamKII could not be rescued by b-catenin

(Fig. 5B). This raises the possibility that PKC interacts

with the Wnt/b-catenin pathway upstream of b-catenin and

GSK-3 whereas CamKII might do so downstream of b-cate-

nin. As the phosphoprotein dishevelled (Dsh) gets depho-

sphorylated if Wnt-11 signaling is blocked by a dominant

negative Wnt-11 (Tada and Smith, 2000) we reasoned that

Dsh might be a good candidate to be phosphorylated by PKC.

To test this, we injected myc-tagged PKCa and HA-tagged

Dsh RNA into Xenopus embryos, immunoprecipitated both

proteins, and tested for the ability of PKC to phosphorylate

Dsh. Indeed we found Dsh to be phosphorylated by PKC in

vitro in a manner sensitive to a PKC inhibitor (Fig. 6A).

If CamKII interacts with the Wnt/b-catenin pathway

downstream of b-catenin, the effect of constitutively active

CamKII on convergent extension should be rescued by more

downstream components. Experiments revealed that LEF-1

and Xnr-3 were able to rescue convergent extension move-

ments in explants that had been injected with constitutively

active CamKII (Fig. 5B). We then investigated whether

CamKII is able to directly phosphorylate XLEF-1. We puri-

®ed XLEF-1 and XTCF-3 His-tagged fusion proteins

(Pukrop et al., 2001) and incubated these proteins in the


Fig. 5. The Wnt/Ca21 pathway interacts with the Wnt/b-catenin pathway at two different levels. (A) Treatment of explants with PMA blocks convergent

extension movements which can be rescued by injection of RNA encoding for b-catenin or dnGSK-3b. (B) Constitutively active CamKII (CamKII T286D)

blocks convergent extension movements which can be rescued by injection of LEF-1 or Xnr-3 RNA but not b-catenin RNA. Classi®cation of explants was as

given in Fig. 1. Detailed results are given in Table 1. Calculated elongation coef®cients are also indicated above bar graphs.

presence of radioactively labeled ATP with commercially

available CamKII. These experiments revealed that XLEF-1

but not XTCF-3 is phosphorylated by CamKII (Fig. 6B).

In summary, our data suggest that the Wnt/Ca21 pathway

interacts with the Wnt/b-catenin signaling pathway at least

at two different levels. Whereas activated PKC inhibits the

Wnt/b-catenin pathway upstream of GSK3, activated

CamKII does so downstream of b-catenin at the level of

the transcription factor complex.

3. Discussion

Gastrulation is one of the most important steps in verte-

brate development leading to the formation of three germ

layers. Here we analyzed the role of two different Wnt

signaling pathways for proper cell migration during conver-

gent extension movements during Xenopus gastrulation.

Our data indicate that the Wnt/b-catenin pathway is

required for convergent extension by activating the TGF-b


Fig. 6. Putative molecular mechanisms of Wnt pathway interactions. (A) Phosphorylation of Dsh by PKC in vitro. Embryos were single injected with either

XPKCa-myc or Dsh-HA. After mixing immunoprecipitates, a kinase reaction was performed in the presence or absence of PKC inhibitor as indicated. A signal

for phosphorylated Dsh can be observed only when PKC and Dsh are mixed in the absence of PKC inhibitor. The control panels show equal input of Dsh-HA

for each reaction as indicated. (B) Phosphorylation of XLEF-1 but not XTCF-3 by CamKII. Coomassie stain of puri®ed fusion proteins of XTCF-3 and XLEF-1

(Pukrop et al., 2001) (left panel). Equal amounts of fusion proteins as judged by SDS-PAGE and Bradford assay were incubated with CamKII and radioactively

labeled 32P-g-ATP. Only XLEF-1 was phosphorylated by CamKII but not XTCF-3 (right panel).

homolog Xnr-3. In contrast, activation of the Wnt/Ca21

pathway has a negative in¯uence on convergent extension

due to its inhibitory function on the Wnt/b-catenin pathway.

Thus, two different Wnt signaling pathways regulate

convergent extension movements in an opposing manner.

3.1. A role for the Wnt/b -catenin pathway for cell migration

Recent publications have suggested a role for Wnts in

regulating convergent extension. Overexpression of a

secreted form of the Wnt receptor Xfz-8, or the endogenous

Wnt inhibitor, FrzA, results in a block of convergent exten-

sion in vivo (Deardorff et al., 1998; Xu et al., 1998).

However, these publications did not investigate the down-

stream signaling pathways affected by these reagents. Here

we show unequivocally that a block of Wnt/b-catenin signal-

ing leads to inhibition of convergent extension movements.

This statement is based on several independent molecular

approaches. Injection of constructs that either directly tether

cytoplasmic b-catenin or lead to a degradation of cytoplas-

mic b-catenin results in disturbed morphogenetic move-

ments. We were able to show the speci®city of these effects

by coinjecting the more downstream component of the Wnt/

b-catenin pathway LEF-1. Although one might argue that the

inhibition of convergent extension in response to blocking

the Wnt/b-catenin pathway merely re¯ects ventralization of

explants we were able to show by rescue experiments that

Wnt/b-catenin signaling is required for both dorsal meso-

derm speci®cation and for convergent extension movements.

Speci®cally, we demonstrated that while siamois is able to

rescue dorsal marker gene expression in Wnt/b-catenin

signaling de®cient embryos, it does not restore proper cell

migration. On the other hand, Xnr-3 fails to rescue conductin

ventralized embryos but it fully rescues convergent exten-

sion movements. Thus, Wnt/b-catenin signaling is required

for convergent extension to occur due to its effect on Xnr-3

expression. This requirement of Wnt/b-catenin signaling for

proper gastrulation has recently also been shown in Droso-

phila (McEwen et al., 2000), and the C. elegans TCF homo-

log POP-1 has been linked to migration processes (Herman,

2001). In summary, our data and those of others indicate that

the Wnt/b-catenin pathway has to be active for proper cell

movements to occur.

Xnr-3 has been shown to be a direct target of Wnt signal-

ing (McKendry et al., 1997). Xnr-3 has also been shown to

alter cell behavior (Smith et al., 1995). Overexpressing Xnr-

3 in embryos results in tube-like extrusions and it has been

speculated that these protrusions might re¯ect an activity of

Xnr-3 in morphogenetic movements (Smith et al., 1995).

Here we showed that overexpressing Wnt-5A on the dorsal

side of the embryo results in a down-regulation of Xnr-3

expression whereas expression of siamois remained unaf-

fected in our hands. Thus, two target genes of the Wnt/b-

catenin pathway are differentially affected by the Wnt/Ca21

pathway. On ®rst sight this seems to be contradictory;

however, this ®nding clearly indicates that at least one

branch of the Wnt/Ca21 pathway exerts its function at the

level of transcription factor complexes. As these complexes

might differ between different target genes of a common

signaling pathway (Nishita et al., 2000) the effect of an

activated Wnt/Ca21 pathway on gene regulation might be

variable as here shown for Xnr-3 and siamois, respectively.

Finally, this observation also explains why Wnt-5A does not

ventralize Xenopus embryos since siamois expression

remains unaffected whereas gastrulation movements are

impaired as Xnr-3 expression is reduced. Our data that

Xnr-3 is involved in regulating convergent extension are

further supported by the observation that embryos that

have been injected with a dominant negative Xfz-7

construct also display problems with convergent extension

(Medina et al., 2000; Djiane et al., 2000; Sumanas et al.,

2000) and these embryos show reduced levels of Xnr-3

expression (Sumanas et al., 2000).

Finally, it is important to note here that our data are also

consistent with recent publications that made the statement

that Wnt/b-catenin signaling might not be required for

convergent extension movements (Wallingford et al.,

2000; Heisenberg et al., 2000). However, those experiments

were done in embryos injected with either a dishevelled

mutant or in silberblick (Wnt-11) mutant zebra®sh embryos

and thus in a situation in which a putative planar cell polar-

ity pathway (Wnt/JNK pathway) was blocked. Thus, the

Wnt/b-catenin pathway is not suf®cient to rescue conver-

gent extension movements if the Wnt/JNK pathway is inac-

tivated.

3.2. Interaction between Wnt/b -catenin and Wnt/Ca21

pathways

With respect to Wnt/Ca21 signaling we found that the

inhibitory effects of Wnt-5A on convergent extension

movements can be phenocopied by either activation of

PKC or overexpressing a constitutively active CamKII

mutant. These effects could be rescued by coinjecting single

components of the Wnt/b-catenin pathway. Consistent with

this antagonism of both the Wnt pathways is the ®nding that

expression of the b-catenin target gene Xnr-3 is reduced in

Xwnt-5A overexpressing embryos. Again, these data argue

for an important role for the Wnt/b-catenin pathway in

regulating convergent extension movements.

Furthermore, our data suggest that the two known cyto-

plasmic mediators of the Wnt/Ca21 signaling pathway, PKC

and CamKII, interact with the Wnt/b-catenin pathway at

two different levels. One candidate for phosphorylation by

PKC is Dsh, since the convergent extension effect of the

PKC activator PMA is reversed by b-catenin and by

dnGSK3 and both function downstream of Dsh in Wnt/b-

catenin signaling. Furthermore Dsh contains a DEP domain,

which recently has been suggested to interact with PKC

(Ponting and Bork, 1996). This hypothesis is also in agree-

ment with the observation that Xwnt-5A can antagonize the

axis-inducing ability of Xwnt-8 but not that of dnGSK


(Torres et al., 1996). Furthermore, Dsh is a phosphoprotein

that gets dephosphorylated in case of overexpressing

dnWnt-11 (Tada and Smith, 2000), which makes it a good

candidate for a PKC target. Finally, we here show that

PKCa is able to directly phosphorylate Dsh in vitro. Further

experiments will have to evaluate whether this also occurs

in vivo. If so, it is tempting to speculate that PKC also

in¯uences the signaling behavior of Dsh with respect to

Wnt/b-catenin and Wnt/JNK signaling.

In Xenopus, the effect of CamKII on Wnt/b-catenin

signaling might be mediated by direct phosphorylation of

XLEF-1 as shown here. However, we cannot exclude the

possibility that other components of the LEF-mediated tran-

scription factor complex are substrates for CamKII phos-

phorylation. There also remains the possibility that the

effect of CamKII on TCF/LEF factors might be indirectly

mediated by other factors regulated by CamKII. Further

biochemical analyses will have to analyze phosphorylation

of distinct proteins of this complex and the effect of these

phosphorylations on transcription factor assembly, target

gene activation, and, ®nally, convergent extension move-

ments. These experiments will also have to analyze whether

XLEF-1 is also phosphorylated in vivo and whether this

phosphorylation is linked to spatial differences in CamKII

signaling. As there is a ventral to dorsal gradient of CamKII

activity in the Xenopus embryo (KuÈhl et al., 2000a,b) this

implies that CamKII might be able to inactivate XLEF-1 on

the ventral side, while XLEF-1 is more active on the dorsal


Fig. 7. Speculative model of how different Wnt signaling pathways ensure proper convergent extension movements. The Wnt/b-catenin pathway is activated

maternally and requires LEF-1 to activate Xnr-3. Activation of this pathway might be through the maternal Xwnt-8b (Cui et al., 1995) or directly through dorsal

accumulation of Dsh (Miller et al., 1999). Activation of the Wnt/Ca21 pathway by maternal Xwnt-11 or Xwnt-5A is also required for convergent extension

movements. The Wnt/Ca21 pathway activates PKC and CamKII that interact with the Wnt/b-catenin pathway at two different levels. PKC inhibits the Wnt/b-

catenin pathway upstream of b-catenin and GSK-3b probably by phosphorylation of Dsh. As Dsh acts in two different Wnt pathways, phosphorylation of Dsh

might function as a molecular switch (S) in this context. CamKII regulates the Wnt/b-catenin pathway downstream of b-catenin at the level of the LEF-1-

mediated transcription factor complex.

side. With respect to the target gene Xnr-3, the phosphor-

ylation of XLEF-1 together with the dorsal enrichment of b-

catenin thus might contribute to the dorsoventral gradient of

Xnr-3 expression. This dorsoventral gradient of Xnr-3

expression due to dorsal b-catenin and ventral CamKII

signaling may also explain why dorsal cells show a more

active migration behavior in convergent extension move-

ments than ventral cells.

3.3. Is there a correlation between Wnt/Ca21 and Wnt/JNK

signaling?

Recent publications suggest that Xwnt-11 is able to acti-

vate JNK in a Dsh-dependent manner (Wallingford et al.,

2000; Heisenberg et al., 2000; Tada and Smith, 2000). As

Wnt-11 is also able to activate PKC and CamKII (Sheldahl et

al., 1999; KuÈhl et al., 2000a) this raises the question about the

relationship between these two pathways. Activation of JNK

by Frizzled has been shown to depend on Dsh and small

GTPases of the rho family (Strutt et al., 1997; Boutros et

al., 1998) although activation of JNK by Dsh seems to be

independent of the small GTPases (Li et al., 1999). It seems

noteworthy that in cell culture systems both Wnt-1 and Wnt-

5A have been able to activate JNK (Li et al., 1999), and JNK

has also been demonstrated to be Ca-sensitive in calmodulin-

dependent and -independent manners (Enslen et al., 1996;

Zohn et al., 1995). Our observations that Dsh can be modi®ed

by PKCa in vitro are consistent with the possibility that PKC

may be involved in regulation of Dsh with respect to JNK

signaling. Thus, several results in different systems are

certainly consistent with the possibility that Wnt/Ca21 and

Wnt/JNK signaling pathways show overlapping properties.

3.4. A model of how Wnts in¯uence convergent extension

One contradiction to be answered is the observation that

the gain of function of Wnt-11/Wnt-5A (Torres et al., 1996)

as well as the prospective loss of function of Wnt-11/Wnt-

5A by expression of dnWnt-11 leads to an inhibition of

convergent extension movements in Xenopus embryos. As

both Wnt/b-catenin signaling (this study) and Wnt/JNK

signaling (Wallingford et al., 2000; Heisenberg et al.,

2000; Tada and Smith, 2000) are required for convergent

extension movements, this contradiction might be explained

by the hypothesis that Wnt/Ca21 signaling serves as a

balancer between these pathways (Fig. 7).

In the wild-type situation, all the different Wnt pathways

are active. The caveat is that the Wnt/Ca21 pathway and the

Wnt/JNK pathway may be the same, or overlapping. The

Wnt/b-catenin pathway is required to ensure expression of

Xnr-3, the Wnt/JNK pathway may be necessary to confer

proper cell polarity or behavior. In a situation characterized

by a Wnt-5A/Wnt-11 gain of function, the Wnt/Ca21 path-

way is strongly activated (Slusarski et al., 1997; Sheldahl et

al., 1999; KuÈhl et al., 2000a,b) and we speculate that PKC

phosphorylates Dsh, leading to preferential signaling

through JNK. In addition, activated CamKII inhibits the

function of XLEF-1. This leads to a loss of Wnt/b-catenin

signaling activity, resulting in an inhibition of convergent

extension movement (this paper). The Wnt/JNK pathway is

still active when Wnt-5A/Wnt-11 is active, thereby provid-

ing polarity information. Thus, elevating the Wnt/Ca21

signaling activity shifts the balance of Wnt/b-catenin and

Wnt/JNK signaling toward the Wnt/JNK side. On the other

hand, overexpressing the dnXwnt-11 construct inhibits acti-

vation of PKC and CamKII (KuÈhl et al., 2000a,b). Thus Dsh

might not be phosporylated by PKC and therefore might

preferentially signal through b-catenin, and in addition

CamKII is not able to inhibit the function of the LEF-1-

mediated transcription factor complex. In other words, over-

expressing the dnWnt-11 construct shifts the balance of the

Wnt/b-catenin and Wnt/JNK pathways toward the Wnt/b-

catenin pathway. The loss of JNK activity then leads to a

perturbation of gastrulation movements (Heisenberg et al.,

2000; Wallingford et al., 2000) and this cannot be rescued

by an overactivation of the Wnt/b-catenin pathway. In the

future it will be important to test whether Dsh is a substrate

for PKC in vivo as suggested by our in vitro data. If it is, this

will allow further testing of the model in which Dsh is a key

control point determining which response to Wnt signaling

will predominate. Indeed, the dishevelled-associated kinase

PAR-1 is an activator of the b-catenin pathway and an

inhibitor of JNK activation (Sun et al., 2001) whereas the

dishevelled interactor naked functions in the opposite direc-

tion (Yan et al., 2001). These ®ndings support the notion

that multiple Wnt signaling pathways are linked via Dishev-

elled in vertebrates.

4. Materials and methods

4.1. Microinjections of embryos

Mature eggs were obtained by injecting females with 500

units of human chorion gonadotropin (HCG, Sigma). One

hour after in vitro fertilization, the jelly coat was removed

by treating the embryos with 2% cysteine hydrochloride in 1/

10 MBS, pH 8.2 (1£ MBSH: 10 mM HEPES, 88 mM NaCl,

1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM

MgSO4, 2.4 mM NaHCO3, pH 7.4). Injection of embryos was

performed in 3% ®coll in 1/10 MBS. At the four-cell stage

both dorsal blastomeres were injected with 5 nl RNA solu-

tion. After injection the embryos were kept for a further 1±2 h

in 3% ®coll in 1/10 MBS to prevent leakage of the embryos

until they were transferred to 1/10 MBS.

4.2. In vitro transcription and RNA constructs

All constructs used in this study were either in pSPT64 or

pCS21 in order to generate in vitro transcribed RNA by use

of mMESSAGE mMACHINE Kit (Ambion). Amounts of

RNA injected were: Xwnt-8, 0.1 ng; XBDe348, 0.5 ng; XB/

U-cadherin, 1.0 ng; Xcadherin-11, 0.5 ng; b-catenin,

0.1 ng; LEF-1, 0.5 ng; XTCF-3, 0.5 ng; siamois, 20±


50 pg; Xnr-3, 0.5 ng; LEFDbBD, 0.5 ng; LEFDHMG,

0.5 ng; conductin, 0.5 ng; Xwnt-5A, 0.5 ng; CamKII

T286D, 1.0 ng; CamKII K42M, 1 ng; dnGSK-3b, 0.5 ng.

4.3. Keller explants

Explants of the dorsal upper blastopore lip were cut at

stage 10.5 (Nieuwkoop and Faber, 1975). Squares of

approximately 0.25 mm2 were cut from control and injected

embryos and cultivated at 188C for 20 h under a piece of

cover glass in modi®ed Barth solution (1 £ MBS). Petri

dishes and cover glasses were coated with BSA. Explants

were scored as described in Section 2. For PMA treatment,

explants were kept in MBS supplemented with 100 nM

PMA.

4.4. Western blot and ConA sepharose puri®cation

Ten embryos were lysed in 100 ml NOP buffer (PBS

supplemented with 1% Triton X-100, 1% NP40 and 2 mM

CaCl2) and cleared by centrifugation and extraction with

freon. The supernatant was treated with 40 ml ConA-sephar-

ose suspension (Sigma) for 90 min. After centrifugation the

supernatant contains soluble, cytoplasmic proteins, whereas

glyosylated membrane proteins and their interactors were

bound to the sepharose. After washing three times with

RIPA buffer (10 mM Tris, pH 7.5, 2 mM CaCl2, 0.15 M

NaCl, 1% Triton X-100, 1% Na-desoxycholat, 0.1%

SDS), once with high-salt buffer (0.5 M NaCl, 10 mM

Tris, pH 7.5, 2 mM CaCl2) and once with low-salt buffer

(10 mM Tris, pH 7.5, 2 mM CaCl2), proteins were eluted

with protein sample buffer, loaded onto a standard protein

SDS acrylamide gel and analyzed by standard Western blot

procedure. The antibody used to detect b-catenin was from

Transduction Laboratories.

4.5. Expression of fusion proteins and protein

phosphorylation

Cloning, expression, and puri®cation of bacterial fusion

proteins were as described in Pukrop et al. (2001). In short,

puri®cation was as follows: transformed BL21 (DE3)

bacteria were induced by 1 mM IPTG for 2 h at room

temperature. After centrifugation, bacterial pellets were

lysed in lysis buffer (50 mM NaH2PO4, 300±1000 mM

NaCl, 10 mM Imidazole, pH 8.0) including protease inhibi-

tors (complete, Roche) and the cleared supernatent was

loaded onto Ni-NTA resins for 1 h at 48C in a batch proce-

dure. After washing with 50 mM NaH2PO4, 300±1000 mM

NaCl, 10±30 mM Imidazole, pH 8.0, the protein was eluted

10 times with 1 ml of 50 mM NaH2PO4, 300±1000 mM

NaCl, 100±300 mM Imidazole, pH 8.0. Each fraction was

analyzed by SDS-PAGE followed by Coomassie staining

using standard procedures. Phosphorylation of XELF-1 by

CamKII was performed using commercially available

CamKII (NEB) at 308C for 5 min according to the manufac-

turer's instructions.

4.6. RT-PCR analyses

Total RNA was isolated from embryos using Purescript

RNA Isolation Kit (Biozym). First strand cDNAs were

synthesized according to the Gibco protocol using Super-

script II reverse transcriptase. The PCR reaction mixtures

for investigating the expression of marker genes were

prepared using PCR Supermix (Gibco) and PCR was

performed under standard conditions. The sequences of

the used oligonucleotide primers are the following:

Sia F, 5 0-CTCCAGCCACCAGTACCAGATC-3 0

Sia R, 5 0-GGGGAGAGTGGAAAGTGGTTG-3 0

Xnr-3 F, 5 0-CGAGTGCAAGAAGGTGGACA-3 0

Xnr-3 R, 5 0-ATCTTCATGGGGACACAGGA-3 0

Goosecoid F, 5 0-ACAACTGGAAGCACTGGA-3 0

Goosecoid R, 5 0-TCTTATTCCAGAGGAACC-3 0

Chordin F, 5 0-CTGTACCAACCCAATCCGTGCC-3 0

Chordin R, 5 0-CTTGGTGCAACATCTGTCCCGC-3 0

XFD/pintalavis F,

5 0-CCTTAGCCCTACATCTGAACAA-3 0

XFD/pintalavis R,

5 0-TAAAGGGAGCTGAGGATAGGTC-3 0

Xbra F, 5 0-GGATCGTTATCACCTCTG-3 0

Xbra R, 5 0-GTGTAGTCTGTAGCAGCA-3 0

H4 F, 5 0-CGGGATAACATTCAGGGTATCACT-3 0

H4 R, 5 0-ATCCATGGCGGTAACTGTCTTCCT-3 0

Ampli®ed products were analyzed on 1.2% agarose gels.

4.7. PKC phosporylation assay

Xenopus embryos were injected with either XPKCa-myc

or XDsh-HA RNA and lysates were prepared at blastula

stages. PKC and Dsh constructs were immunoprecipitated

separately with antibodies against the tags. Kinase reaction

of mixed immunoprecipitates was for 5 or 10 min as indi-

cated using 32P-g-ATP as cosubstrate and phosphatidyl-l-

serine as a cofactor. The PKC inhibitor used was

GF109203X.

Acknowledgements

We thank Nazan OÈ lmez for technical assistance. The LEF

mutants and the conductin construct were a kind gift of W.

Birchmeier, Berlin. R.T.M. is an Investigator of the Howard

Hughes Medical Institute. This work was supported by DFG

grants to D.W. and M.K.

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Antagonistic regulation of convergent extension movements in Xenopus by Wnt/β-catenin and Wnt/Ca2+ signaling

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