Integrating Patterning Signals:
Wnt/GSK3 Regulates the Durationof the BMP/Smad1 Signal
Luis C. Fuentealba,1,2 Edward Eivers,1,2 Atsushi Ikeda,1,3 Cecilia Hurtado,1 Hiroki Kuroda,1,4 Edgar M. Pera,1,5
and Edward M. De Robertis1,*
1Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles,
CA 90095-1662, USA
2These authors contributed equally to this work.
3Present address: Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto 606-8501, Japan.
4Present address: Faculty of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka-city 422-8529, Japan.
5Present address: Lund Stem Cell Center, BMC, B13, Klinikgatan 26, S-22184 Lund, Sweden.
*Correspondence: ederobertis@mednet.ucla.edu
DOI 10.1016/j.cell.2007.09.027

SUMMARYBMP receptors determine the intensity of BMP
signals via Smad1 C-terminal phosphorylations.
Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1Cter signal was
regulated by sequential Smad1 linker region
phosphorylations at conserved MAPK and
GSK3 sites required for its polyubiquitinylationand transport to the centrosome. Proteasomal
degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK
MAPKs, as well as GSK3 inhibitors, prolonged
the duration of a pulse of BMP7. Wnt signaling
decreased pSmad1GSK3 antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos,
it was found that Wnts induce epidermis andthat this required an active BMP-Smad pathway.
Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3)
patterning gradients are integrated at the levelof Smad1 phosphorylations during embryonicpattern formation.
INTRODUCTION
Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiations is one of the
major challenges in cell and developmental biology. Embryonic patterning in Xenopus is regulated by gradientsof growth factors and their antagonists, with bone morphogenetic proteins (BMPs) controlling dorsal-ventral (D-V)
and Wnt signals anterior-posterior (A-P) cell fates (Niehrs,

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2004). This positional information must be seamlessly integrated, for when a blastula is cut in half, the embryo canself-regulate, forming perfect identical twins (De Robertis,
2006). In the ectoderm, the main cell differentiation decision is between neural and epidermal tissues, for which
excellent molecular markers exist. Neural tissue differentiates when BMP signaling is inhibited by BMP antagonists
or depletion by anti-BMP morpholino (MO) oligos, whereas
epidermis is formed at high BMP signaling levels (Harland,
2000; Reversade and De Robertis, 2005). BMP receptors(BMPR) are serine/threonine protein kinases that signal
by phosphorylating the transcription factors Smad1/5/8
at C-terminal sites (SS[PO3]VS[PO3]), causing their activation and nuclear translocation (Shi and Massagué, 2003;
Feng and Derynck, 2005).
Neural tissue can also be induced by receptor tyrosine
kinases (RTKs) such as FGF and IGF receptors via theactivation of mitogen-activated protein kinase (MAPK)(reviewed in Wilson and Edlund, 2001; De Robertis andKuroda, 2004; Stern, 2005). This neural-inducing activity
can be explained in part by an inhibitory phosphorylationin the linker (middle) region of Smad1 at four conserved
MAPK (PXS[PO3]P) sites (Pera et al., 2003; Kuroda
et al., 2005). This linker region MAPK phosphorylation
was first discovered in cultured cells treated with EGF(Kretzschmar et al., 1997) and recently reported to promote polyubiqutinylation of Smad1 by the Smurf1 E3ubiquitin ligase (Zhu et al., 1999; Sapkota et al., 2007),
a finding independently confirmed here. FGF/MAPK signals are known to oppose BMP/Smad1 in many developing organs (De Robertis and Kuroda, 2004). Remarkably,mouse phosphorylation-resistant mutations in the MAPK
sites of Smad1, introduced by homologous knock-in,
generated embryonic fibroblasts in which the transcriptional activation of a reporter gene by BMP becomes
resistant to repression by addition of FGF (Aubin et al.,
2004; Sapkota et al., 2007). Thus, the role of Smad1 as
an interface for integrating RTK and BMP signals is firmly
established.

Although less generally recognized, the Wnt signaling
pathway also influences neural induction. Wnts play multiple roles during development: at the early blastula stage,
canonical Wnt signaling provides a dorsalizing signal via
activation of xTcf3 (Harland, 2000; Heasman, 2006), andat the neurula stage, it regulates neuronal differentiation
via inhibition of NeuroD (Marcus et al., 1998). At the gastrula stage, overexpression of Wnt8 causes antineural
effects in Xenopus (Christian and Moon, 1993). Wnt antagonists such as Dickkopf-1 (Dkk1) and secreted Frizzledrelated proteins (sFRPs), promote neural differentiationin Xenopus, chick, and mouse ES cells (Glinka et al.,
1998; Wilson and Edlund, 2001; Aubert et al., 2002). The
canonical Wnt pathway signals through the inhibition of
glycogen synthase kinase 3 activity (Logan and Nusse,
2004). GSK3 is a protein kinase that usually requires prephosphorylated substrates, phosphorylating Ser/Thr residues located four amino acids upstream of sites primed
by other kinases (Cohen and Frame, 2001). Frequently,
as in the case of b-catenin, such coupled phosphorylations by two protein kinases are followed by polyubiquitination and degradation in the proteasome (Liu et al., 2002;
Cohen and Frame, 2001). This project was initiated when
we noticed conserved GSK3 sites in vertebrate Smad1/5/
8 proteins that could be phosphorylated by GSK3 after
priming by MAPK. This was exciting, because a pathway
in which
DkkxWntxGSK3xpSmad1Cter xNeural plate
could potentially explain the proneural effects of antiWnts.
In the present study, we demonstrate that phosphorylation at the GSK3 sites represses the transcriptional activityof Smad1 by enhancing proteasomal degradation of
pSmad1Cter. We found that GSK3 phosphorylation is an
essential requirement for polyubiquitination. Pulse-chaseexperiments with BMP7 revealed a novel cellular pathwaythat regulates the duration of the BMP signal. First, BMPR
causes C-terminal phosphorylation (pSmad1Cter) andnuclear translocation. Next, the pSmad1MAPK phosphorylation is mediated by MAPK enzymes (Erk, p38, and JNK)
in the nucleus. Subsequently, GSK3 recognizes the prephosphorylated linker region, generating pSmad1GSK3 atan unknown cellular location. The triply phosphorylated
protein is transported along microtubule-like structures
to the centrosome. The centrosome is the cellular location
at which proteasomes normally accumulate in manycell types (Badano et al., 2005). The GSK3 activity that
phosphorylates Smad1 was regulated by Wnt3a protein
addition in cultured cells, leading to the accumulation of
pSmad1Cter. A constitutively active low-density lipoprotein receptor-related protein 6 (CA-LRP6) caused redistribution of pSmad1GSK3 from the centrosome to cytoplasmic particles resembling the recently described LRP6
signalosomes (Bilic et al., 2007). In vivo epistatic experiments indicated that Smad1 phosphorylations by GSK3
play a key role in mediating the effects of Wnt signalingon neural development at the gastrula stage. In dissociated ectodermal cells, overexpression of Wnt8 and its
coreceptor LRP6 induced epidermal differentiation in a
Smad1/5/8-dependent manner. The results suggest thata branch of the canonical Wnt pathway signals throughthe stabilization of BMP-Smad signals.
RESULTS
GSK3 Phosphorylation Inhibits Smad1 Function
Inspection of the Smad1 sequence revealed four GSK3
sites located four amino acids upstream of the PXSP
Erk/MAPK sites in the linker region (Figure 1A). These
potential GSK3 sites were conserved in BMP-responsive
Smad1/5/8 across vertebrates and in Drosophila Mad(Figure S1 in the Supplemental Data available online).
Phosphorylation-resistant mutations (Ser/Thr to Ala) wereintroduced into a human Smad1 expression construct(Kretzschmar et al., 1997) previously characterized in Xenopus embryos (Pera et al., 2003; Kuroda et al., 2005).
These sites were mutated individually or in combination;strongest effects were found when all four GSK3 sites
were mutated (data not shown) in a construct designated
SGM (Figure 1B). The phenotypic effects of SGM werecompared to those of Smad1 wild-type (SWT) andSmad1 mutated at the MAPK sites (designated SMM).
Overexpression of mRNA encoding GSK3 or MAPK phosphorylation-resistant mutants in early Xenopus embryos
resulted in hyperactive Smad1 proteins that caused
strongly ventralized phenotypes, as indicated by transcript accumulation of the BMP-inducible marker Sizzled(Figures 1C–1F). A constitutively active phospho-mimetic
form of Smad1, in which the C-terminal SVS phosphorylation sites were mutated into EVE (designated SEVE;
Figure 1G), provided a novel reagent independent of
BMPR signaling (compare Figure 1D to Figure 1H).
GSK3 and MAPK phosphorylation-resistant mutants of
SEVE strongly induced Sizzled, not only ventrally but
also ectopically in the floor plate (Figures 1I and 1J, insets).
We conclude that the function of the putative GSK3 phosphorylations in vivo, like those of MAPK, is to downregulate Smad1 activity.
We next tested whether Smad1 was a substrate for
GSK3 that required priming by MAPK (Cohen and Frame,2001). The entire linker region of human Smad1 wastagged with six histidines, expressed in E. coli, and purified. In vitro phosphorylations were performed in twostages, and it was found that GSK3b caused the incorporation of [g-32P]ATP by Smad1 linker protein, but only after
prephosphorylation by Erk/MAPK (Figure 1K, compare
lanes 2 and 4).
Antibody reagents specific for human phosphoSmad1GSK3 (p-Serine 210) and phospho-Smad1MAPK(p-Serine 214) were developed. A synthetic peptide(SS[PO3]DPGS[PO3]PFQMPADT) proved extremely antigenic. After affinity purification on a column of the same
peptide phosphorylated only in Ser214, one rabbit produced a very high titer antibody for pSmad1MAPK, such

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Figure 1. Smad1 Is Phosphorylated and Inhibited by GSK3
(A) Smad1 contains MAPK (blue) and GSK3 (red) phosphorylation sites in its linker region. The PPAY binding site of Smurf1 is boxed and serines 210
and 214 used to raise antibodies indicated by asterisks.
(B) Smad1 constructs encoding Smad1 wild-type (SWT) or phosphorylation-resistant mutants for GSK3 (SGM) and MAPK (SMM) sites.
(C–F) Injection of SGM or SMM, but not of SWT, mRNA increased expression of the ventral marker sizzled in Xenopus embryos.
(G) A BMP-independent phospho-mimetic activated Smad1 (SEVE) in which the SVS terminus was mutated into EVE.
(H–J) Activity of SEVE is increased by phosphorylation-resistant linker mutations.
(K) GSK3 radioactively phosphorylates Smad1 in vitro, but only when primed by MAPK. The recombinant Smad1 linker substrate was about 90% purein polyacrylamide gels (data not shown).
(L and M) Phospho-specific antibodies for hSmad1 Ser214 (pSmad1MAPK) and Ser210 (pSmad1GSK3 antibody B was used).
(N) Phospho-specific antibodies (pSmad1MAPK and pSmad1GSK3-A) demonstrate that GSK3 phosphorylation of recombinant Smad1 requires MAPK
priming, in nonradioactive Western blots. Recombinant Smad1 substrate was used in the same amount as in (K).

that it detected overexpressed hSmad1-wt, but not thephosphorylation-resistant mutant S214A, at dilutions of
1:200,000 (Figure 1L). A second rabbit produced a phospho-specific antibody for pSmad1GSK3 Ser210 active at
dilutions of 1:30,000 after affinity depletion with phospho-Ser214 peptide and positive purification with phospho-Ser210 peptide (antibody A). An additional antipSmad1GSK3 Ser210 (antibody B), derived with peptide
PHSPTSS[PO3]DPGSPFQ as antigen, detected overexpressed hSmad1-wt but not the phosphorylation-resistantmutant S210A (Figure 1M). Immunoblots of nonradioactive biochemical reactions confirmed that Ser214 in

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recombinant Smad1 protein was indeed phosphorylatedby Erk/MAPK and that the phosphorylation of Ser210 by
GSK3 was strongly dependent on prephosphorylation by
MAPK and underscored the phospho-specificity of ourantibodies (see Figure 1N, lanes 2–4). We conclude that
Smad1 is phosphorylated by GSK3 in vitro in a MAPKdependent fashion.
GSK3 phosphorylations were required for the polyubiquitinylation of Smad1 (Figure 2A). The Smad ubiquitination regulatory factor 1 (Smurf1) E3 ubiquitin ligase binds
to a PPXY sequence (Zhu et al., 1999) located near the
Smad1 linker MAPK phosphorylation sites (Figure 1A).

Figure 2. GSK3 and MAPK Phosphorylation Sites Regulate Polyubiquitinylation of Smad1 and Its Transcriptional Activity(A) Polyubiquitinylation of Smad1 requires GSK3 and MAPK phosphorylation sites. Cells were cotransfected with Smad1-flag, Smurf1, andUbiquitin-HA.
(B) GSK3 inhibitors decrease polyubiquitinylation of human Smad1. SB415286 (SB4, Biomol) was used at 40 mM, and LiCl at 30 mM, for 24 hr.
(C) SGM and SMM are more active than SWT mRNA in a BRE-luciferase transcriptional reporter assay in ectodermal Xenopus explants.
(D) The GSK3 inhibitor LiCl increases BMP4-induced transcriptional activation of BRE-luciferase in 293T cells 8 hr after cotransfection. Data are represented as mean ± standard deviation, three independent experiments.

We used the assay of Zhu et al. (1999) in 293T cells, inwhich flag-tagged Smad1-WT or its GSK3 or MAPK
mutants were cotransfected with HA-tagged ubiquitin
and Smurf1, and Smad1 was immunoprecipitated via its
flag-tag. Polyubiquitinylation of GSK3 or MAPK phosphorylation-resistant Smad1 proteins (SGM and SMM) was
greatly reduced when compared to their wild-type counterpart (Figure 2A, lanes 2–4). The mono- or diubiquitinylated forms were less affected. In addition, polyubiquitinylation of SWT in 293T cells was inhibited by the GSK3
inhibitors SB415286 or lithium chloride (LiCl) (Figure 2B).
We conclude that phosphorylation of Smad1 by GSK3 isessential for its polyubiquitinylation in cultured mammalian cells.
We next tested whether the transcriptional activity of
Smad1 was regulated by GSK3 phosphorylations. Experiments were performed in Xenopus ectodermal explants,
which have physiological endogenous BMP signalinglevels sufficient for reporter gene studies (Kuroda et al.,2005). It was found that the phosphorylation-resistantmutants SGM and SMM caused significantly stronger
induction (3- to 4-fold) of a BMP reporter (BREx2-Id1-luciferase; Korchynskyi and ten Dijke, 2002) than SWT(Figure 2C). In addition, the effect of the GSK3 inhibitor
LiCl on BMP-dependent transcription was tested. Cotransfection of BMP4 and BREx2-Id1-luciferase DNAs
into 293T cells showed that LiCl treatment significantly
increased the transcriptional activity of endogenous
BMP-Smads (Figure 2D). These results were congruent
with the microinjection of SGM and SEVE-GM mRNAs in
Xenopus embryos (Figures 1E and 1I) and suggested
that GSK3 activity normally represses Smad1 signaling
by promoting proteasomal degradation.

GSK3 and MAPKs Regulate the Durationof BMP Signals
We next investigated whether the coupled inhibitory phosphorylations by MAPK and GSK3 regulated the duration of
the BMP7 signal in pulse-chase experiments (Figure 3).
The intensity of the Smad1 signal is determined by thelevels of C-terminal phosphorylation (pSmad1Cter) by
BMPR (Feng and Derynck, 2005). The phosphorylationsof endogenous Smad were analyzed in L cells, which
respond particularly well to BMP in serum-free medium(serum complicates analyses because it contains BMP
and FGFs). Note that in these and all subsequent cultured
cell experiments, endogenous Smad1 was analyzed. By
using this assay, we found that the MAPK and GSK3 phosphorylations were triggered by BMPR activity.
A strong pSmad1Cter signal was elicited at time 0 after a
15 min pulse of 5 nM BMP7 (Figure 3A, compare lanes 1
and 2), which partially decreased after 1 hr and greatly
diminished by 2.5 hr (lanes 3–5). Interestingly, pSmad1MAPK
antibodies also detected a BMP7-induced band, but witha 0.5 hr delay. The pSmad1GSK3 antibody detected BMPinduced phosphorylations 0.5 and 1 hr after treatment,
which decreased by 2.5 hr (Figure 3A, lanes 2–5). Total
Smad1 protein levels did not change during this period;
this is in agreement with observations that only a small
fraction of the total Smad1 is phosphorylated at physiological BMP levels, with a large reservoir of inactive
Smad1 persisting at most times (e.g., Kuroda et al.,2005). BMP treatment does not increase activity levelsof the three main cellular MAPKs (Erk, p38, and JNK)
(Kuroda et al., 2005; Sapkota et al., 2007; data not shown).
Experiments with chemical inhibitors (U0126 for MEK/Erk,
Calbiochem CFPD inhibitor for p38, SP600125 for JNK)

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Figure 3. The Duration of the pSmad1Cter Signal Is Regulated by MAPK and GSK3
(A) Sequential phosphorylation of Smad1 by BMPR, MAPK, and GSK3 after a pulse of BMP7.
(B) Induction of pSmad1MAPK by BMP7 is blocked by triple inhibition of MAPKs (lane 7). Inhibitors used were: MEK/Erk (10 mM U0126), p38 (10 mM
Calbiochem CFPD p38 inhibitor), and JNK (25 mM SP600125). Lane 6 contained both p38 and JNK inhibitors.
(C) The duration of pSmad1Cter signal is prolonged by inhibition of MAPKs, GSK3 (40 mM SB415286 or 30 mM LiCl), or proteasome activity (50 mM
lactacystin).

showed that the Smad1 MAPK phosphorylation triggeredby BMP7 results from the combined constitutive activity ofthese three MAPKs in cultured cells (Figure 3B; Figure S2).
We have also observed, with a phosphorylation-resistantC-terminal mutant, that MAPK and GSK3 can phosphorylate Smad1 in a BMPR-independent fashion (data notshown); this activity may help explain why neural induction
by Chordin requires an intact MAPK pathway (Pera et al.,2003). Recently, Sapkota et al. (2007) reported that BMP
induced MAPK phosphorylation, but suggested the involvement of a different and as yet unknown kinase; however, they did not test a cocktail of the three inhibitors as in
Figure 3B (lane 7). We conclude from these pulse-chase

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experiments that BMP treatment triggers three sequential
phosphorylations in Smad1: first by BMPR, second by
MAPKs, and third by GSK3.
The sequential nature of these events suggested thatthey might function in the regulation of the duration of the
BMP signal, which would be terminated by the coupledactivity of MAPK and GSK3 via degradation in the proteasome. To test this hypothesis, we performed BMP7 pulsechase experiments in the presence of the MAPK inhibitor
cocktail, the GSK3 inhibitors LiCl or SB415286, or the proteasome inhibitor lactacystin. As shown in Figure 3C, allthese treatments significantly increased the duration of
the pSmad1Cter signal. These results suggest a molecularFigure 4. MAPK and GSK3 Phosphorylations Target Smad1 and Total Polyubiquitinated Proteins to the Centrosome
in Cos7 Cells(A–C) pSmad1Cter accumulates in the centrosome (arrows) in response to proteasomal inhibition by lactacystin (24%, n = 323).
(D) The proteasomal subunit S20a5 normally
accumulates in the centrosome in Cos7 cells(arrows).
(E and F) Nuclear and centrosomal localizationof pSmad1MAPK.
(G–I) pSmad1GSK3-A (green) colocalizes withthe centrosome marker g-Tubulin (red). Notethat in (E) through (I), BMP4 was not added,
but the medium contained 10% fetal calf serum, which provides growth factors such as
BMP and FGF.
(J and K) Specificity of pSmad1MAPK and
pSmad1GSK3-A staining: phospho-peptide
competition and l-phosphatase sensitivity.
(L) pSmad1GSK3 accumulates in the centrosome 8 hr after lactacystin (50 mM) treatment(a 25-fold increase in volume).
(M–O) Treatment of Cos7 cells with lactacystin
induces accumulation of total cellular polyubiquitinylated proteins in the centrosomal region
marked by pericentrin in red. In one cell, a ringof pericentrosomal staining is seen.

pathway in which, after phosphorylation by BMPR, theduration of the pSmad1Cter signal is controlled by coupled
MAPK and GSK3 phosphorylations and protein degradation in the proteasome.
pSmad1MAPK/GSK3 Is Targeted to the Centrosome
Unexpectedly, when the subcellular localization of the various forms of phosphorylated Smad1 in cultured cells was
examined, it was found that Smad1 targeted for degradation accumulated in the centrosomal region (Figure 4). The
pSmad1Cter antigen accumulates in the nucleus of Cos7
cells after BMP4 treatment in serum-free conditions, but
we noted that in cells treated with the proteasome inhibitor

lactacystin, pSmad1Cter was also found in a cytoplasmic
region (Figures 4B and 4C) that appeared to correspondto the centrosome (when a kidney-shaped nucleus ispresent, as in Figure 4C, the centrosome is almost invariably located in its concavity; Wilson, 1928). The centrosome, the region of cytoplasm that surrounds the centrioles, is a center for ubiquitin-mediated proteolysis inwhich the proteasomal machinery becomes concentrated(Badano et al., 2005). We corroborated this by staining
Cos7 cells with proteasomal S20a5 subunit antibodies.
Proteasomes were present both in nucleus and cytoplasm, but were more concentrated in the centrosome(Figure 4D, arrows).

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Phospho-Smad1MAPK was observed both in the nucleus
and in a bright centrosome-like body in the cytoplasm (Figures 4E and 4F). The pSmad1GSK3 antibody stained thenucleus weakly and the cytoplasm strongly, along microtubule-like filaments that converged on the centrosome,which was unequivocally identified by colocalization withthe established centrosomal marker g-tubulin (Figures
4G–4I). Specificity controls showed that centrosomal
phospho-Smad1 staining was eliminated or reduced bycompetition with the corresponding phosphorylated peptide (Figure 4J), by l-phosphatase treatment (Figure 4K),
and by the MAPK inhibitor cocktail or the GSK3 inhibitor
SB415286 (Figure S2). Centrosomal localization was
greatly enhanced by treatment of Cos7 cells with the proteasomal inhibitor lactacystin (Figure 4N), supporting theview that Smad1 is degraded by proteasomes located in
the centrosome.
Finally, we investigated whether this cellular mechanism
represented a wider phenomenon that might apply toother proteins targeted for degradation. An antibody
against total polyubiquitin chains stained diffusely both
nucleus and cytoplasm (Figure 4M), but after lactacystin
treatment, polyubiquitin antigen became clearly concentrated in the pericentrosomal region overlapping, and insome cases surrounding, the Pericentrin centrosomal
marker (Figures 4N and 4O). We conclude that the cellulardegradation pathway we have identified probably is usedby many other proteins marked for destruction in addition
to Smad1, because total polyubiquitinylated proteins also
became concentrated in centrosomes when proteasome
activity was inhibited.
Smad Degradation Is Regulated by Wnt
As shown in the model in Figure 5A, the experiments presented so far suggest that an elaborate cellular regulatory
pathway is involved in the termination of BMP signaling.
Nuclear C-terminal phosphorylated Smad1 is subjected
first to MAPK phosphorylation in the linker region andthen to GSK3 phosphorylation, triggering the polyubiquitinylation of Smad1. Once targeted for degradation, Smad1
is transported, probably along microtubules, to the centrosome, where the triply phosphorylated and polyubiquitinylated Smad1 is degraded by proteasomes. A key
question, with important implications for the integrationof patterning signals, is whether GSK3 phosphorylationof Smad1 is regulated by canonical Wnt signaling.
To investigate this, the effects of treating cultured L cells(in the absence of serum and in the presence of BMP7 andFGF2) with purified Wnt3a protein were examined. The
levels of pSmad1GSK3 were decreased, but not eliminated,
1 hr after Wnt3a addition, while the levels of pSmad1Cterincreased (Figure 5B). The increase in pSmad1Cter was
due to stabilization, rather than to secondary inductionof BMP ligands, because transfection of b-catenin DNA(both in wild-type and stabilized mutant forms) did not
significantly affect pSmad1Cter phosphorylation levels in
L cells (data not shown). Wnt3a protein also affected
Smad1 phosphorylations in human embryonic stem cells

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(hESCs), causing pSmad1GSK3 to decrease moderately
and pSmad1Cter to accumulate (Figure 5C). We conclude
that the levels of pSmad1Cter are stabilized by Wnt3atreatment, in agreement with the pathway proposed in
Figure 5A.
We next investigated whether the activation of Wnt signaling caused a change in the cellular localization of thepSmad1GSK3 antigen. A recent study has demonstrated
that Wnt signals cause the colocalization of componentsof the b-Catenin degradation complex, such as GSK3
and Axin, with intracellular membrane vesicles containing
the Wnt receptor LRP6 (Bilic et al., 2007). Transfection of
CA-LRP6 DNA (Tamai et al., 2004) into Cos7 cells caused
a dispersion of the centrosomal pSmad1GSK3 antigen into
small cytoplasmic particles when compared to untransfected cells (Figures 5D and 5E). These cytoplasmic
puncta most likely correspond to LRP6 signalosomes(Bilic et al., 2007) because they colocalized with endogenous GSK3 protein (Figure 5G). In addition to dispersing
the centrosomal material into smaller particles, CA-LRP6
caused the pSmad1GSK3 antigen to accumulate in thecell nucleus (Figures 5D–5G). Perhaps transport to thecentrosome is required for nuclear export of Smad1
marked for destruction. We conclude that activationof the Wnt pathway triggers a major redistribution of
Smad1 forms targeted for degradation within the cell.
We next turned to the Xenopus embryo. An old enigma
is why overexpression of Wnt antagonists, such as Dkk1,
cause dorsalized phenotypes (expanded neural plate at
the expense of epidermis) almost indistinguishable tothose of embryos microinjected with BMP antagonists,such as Chordin protein (Figures 6A–6C). This suggested
the possibility that Wnt inhibitors might regulate BMP
signaling at gastrula through the pathway indicated in
Figure 6D. This hypothesis was tested by coinjection of
Dkk1 and Xenopus BMP4 mRNA (Figures 6E–6H). BMP4
overexpression inhibited expression of the forebrain/eye
marker Rx2a, and Dkk1 mRNA blocked this inhibition(compare Figures 6G and 6H). Dkk1 is one of the strongest
and best characterized inhibitors of canonical Wnt signaling, which functions by removing the LRP6 Wnt coreceptor from the cell surface (Glinka et al., 1998; Niehrs, 2004).
Therefore, in principle, Dkk1 should not have inhibited
BMP4 signaling.
To explore this in vivo in a Wnt loss-of-function situation,
we used a previously characterized Xenopus laevis Wnt8
MO (Lee et al., 2006). xWnt8 is the main Wnt expressed
posteriorly during gastrulation (Christian and Moon,
1993), and its knockdown causes dorsalization with expansion of brain at the expense of epidermis (marked by
Cytokeratin) (Figures 6J and 6N). Microinjection of wildtype Smad1 mRNA had little effect on the xWnt8 depletion
phenotype (Figures 6K and 6O), yet injection of the sameamount of SGM mRNA was able to abrogate the dorsalizing effects of Wnt8 MO (Figures 6L and 6P). This indicatesthat the GSK3 phosphorylation sites in microinjected
Smad1 play a crucial role in the dorsalizing effects caused
by Wnt8 depletion. The phosphorylation of Smad1 GSK3

Figure 5. Wnt3a Regulates Smad1 Phosphorylation(A) Model of biochemical and cellular signaling pathway integration at the level of Smad1 phosphorylations.
(B) Wnt3a protein (300 ng/ml, R&D Systems) inhibits Smad1 phosphorylation by GSK3 and stabilizes pSmad1Cter. L cells cultured in the absence ofserum were treated with purified Wnt3a protein for 1 hr; 0.3 nM BMP7 and 40 ng/ml FGF2 were added after 20 min of Wnt treatment.
(C) Wnt3a (300 ng/ml) stabilizes pSmad1Cter and b-Catenin in hESCs after 2 hr.
(D and E) Activation of the Wnt pathway with transfected CA-LRP6 (Tamai et al., 2004) disperses pSmad1GSK3 from the centrosome into small cytoplasmic puncta. Note that in CA-LRP6-transfected cells, the levels of pSmad1GSK3 in the nucleus are elevated.
(F and G) The cytoplasmic puncta containing pSmad1GSK3 most likely correspond to LRP6 signalosomes because they costain with GSK3 antibodies.
Transfected cells were identified by cotransfection of CFP, not shown here.

sites requires MAPK priming and, in agreement with this,phosphorylation-resistant SMM, but not SWT mRNA, also
inhibited the effects of Wnt8 MO in the embryo (Figure S3).
Dkk1 overexpression is expected to block all canonical
Wnt signaling at gastrula. Dkk1 mRNA microinjection
expanded the neural plate, and overexpressed wild-type
Smad1 was ineffective at counteracting this (Figures
6Q–6S), whereas the GSK3 phosphorylation-resistantmutant SGM was able to reverse the enlargement of theneural plate produced by Dkk1 mRNA (compare Figures
6S and 6T). These epistatic experiments support the hypothesis that the GSK3 phosphorylation sites in Smad1
are critical targets of Dkk1 and Wnt8 signaling during Xenopus development.

The Antineural Effects of Wnt/GSK3 Require
Smad1 but Not Tcf3
We next investigated the molecular pathway by which
Wnt/GSK3 regulates the decision between epidermal
and neural cell fates in vivo. In Xenopus, it is sufficient todissociate ectodermal (animal cap) cells to switch froman epidermal to a neural fate (Wilson and Hemmati-Brivanlou, 1995). This default neural differentiation is causedby sustained activation of MAPK/Erk (Kuroda et al., 2005).
Ectodermal explants normally differentiate into epidermis,
while dissociated animal cap cells form neural tissue(Figure 7A, compare lanes 2 and 3). LiCl, an inhibitor of
GSK3, inhibits neural tissue and causes epidermal differentiation (Figure 7A, lanes 3–6).

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Figure 6. The Dorsalizing Effects of Wnt
Inhibition Are Rescued by SGM(A–C) Dkk1 (n = 28) and Chordin (n = 45) protein(R&D) injections into the blastula cavity (60 nl of
2.5 mM and 0.5 mM, respectively) present considerable phenotypical similarities.
(D) Proposed molecular pathway.
(E–H) Dkk1 mRNA suppresses the antineuralphenotype of BMP4 overexpression (n foreach sample was 18, 13, 23, and 18, respectively).
(I–P) Wnt8 MO causes dorsalization, which isblocked by GSK3-resistant SGM, but not by
SWT mRNA (n for each sample was 22, 17,23, and 26, respectively).
(Q–T) The expansion of the neural plate by
Dkk1 mRNA is rescued by GSK3-resistantSGM, but not SWT mRNA (n = 30, 22, 21,
and 27, respectively).

Microinjection of pCSKA-Wnt8 and pCS2-LRP6 DNA,
which are expressed at gastrula stage (Christian and Moon,
1993), induced epidermis 8-fold in dissociated animal cap
cells (Figure 7B). Importantly, epidermal induction by Wnt
was blocked by coinjection of mRNA encoding a dominant-negative Smad5 mutant mimicking the zebrafish
somitabun mutant (mouse DN-Smad5; Beck et al., 2001)
that blocks all BMP/Smad1/5/8 signaling (Figure 7B).

988 Cell 131, 980–993, November 30, 2007 ª2007 Elsevier Inc.

The epidermal-inducing effect of Wnt8/LRP6 DNA, which
was in itself a novel observation, was not caused by anincrease of BMP4 mRNA levels, which did not change significantly when compared to uninjected cells (mean 0.9 ±0.2 standard deviation) in the same samples. This is inagreement with previous work in Xenopus showing that
xWnt8 is unable to induce BMP4 (Hoppler and Moon,
1998). In whole embryos, DN-Smad5 eliminated epidermalFigure 7. Wnt Signaling Induces Epidermis in a Smad1/5/8- and b-Catenin-Dependent, but Tcf3-Independent, Manner(A) LiCl induces epidermis (Cytokeratin) and inhibits neural differentiation (NCAM, Otx2). Radioactive RT-PCR analysis of whole embryos (WE), animalcap (AC) explants, and dissociated animal cap cells at stage 13. MyoD indicates lack of mesoderm induction and Ornithine decarboxylase (ODC)
equal loading.
(B) Quantitative PCR of dissociated animal caps injected with pCSKA-Wnt8 and pCS2-LRP6 DNA. DN-Smad5 mRNA was coinjected to block
Smad1/5/8 activity. Cytokeratin mRNA levels at stage 13 were normalized for ODC mRNA, and the standard deviation from three independent experiments is indicated.
(C and C0 ) Wnt3a protein (60 nl of 16 ng/ml) microinjected into the blastula cavity at stage 9 inhibits anterior neural plate and expands epidermis (n = 40
and 42, respectively).
(D and D0 ) DN-Smad5 converts the entire ectoderm into neural tissue and is epistatic to Wnt3a protein injection (n = 27 and 35). ADMP MO was coinjected to eliminate all traces of epidermis.
(E and E0 ) Dkk1 mRNA expands the neural plate (n = 100 and 53).
(F) DN-Tcf3 mRNA eliminates the neural plate; only a ring of Sox2 expression in ventral mesoderm remained (90%, n = 30).
(F0 ) Dkk1 mRNA rescues neural plate in the presence of DN-Tcf3 in 60% of embryos (n = 70).
(G and G0 ) The induction of neural plate by Dkk1 mRNA has a complete requirement for b-Catenin (100%, n = 17 and 55, respectively).
(H) Model in which the BMP (D-V) and Wnt (A-P) patterning pathways are integrated at the level of Smad1/5/8 phosphorylations. Black arrows indicate
direct protein-protein interactions and blue arrows transcriptional regulation by Smad1/5/8; all interactions are supported by overexpression or morpholino studies in Xenopus (Lee et al., 2006, and data not shown).

differentiation, causing ubiquitous neural differentiation of
the entire ectoderm (particularly in the presence of MO
for ADMP, a BMP expressed at low Smad1/5/8 signalinglevels; Figures 7C and 7D; Reversade and De Robertis,2005). Microinjection of Wnt3a protein at late blastula ex-panded the embryonic epidermis but was without effectin a DN-Smad5 background (Figures 7C0 and 7D0 ). Similarly, GSK3 MO, which expands epidermis and inhibits
neural tissue in the whole embryo, was unable to induce
epidermis in DN-Smad5 embryos (Figure S4). We conclude

Cell 131, 980–993, November 30, 2007 ª2007 Elsevier Inc. 989

that Wnt/GSK3 signals induce epidermis in Xenopus embryos and that this requires an active BMP/Smad1/5/8
pathway.
The canonical Wnt pathway signals by preventing thedegradation of b-Catenin, an adaptor protein containing
armadillo repeats, which accumulates in the nucleus,binds to the repressor T cell factor 3 (Tcf3), and activates
the transcription of Tcf target genes (Molenaar et al., 1996;
Logan and Nusse, 2004). This pathway controls patterningin the Xenopus embryo during cleavage stages, causing
the formation of the early embryonic dorsal axis (Harland,
2000; Heasman, 2006). At the gastrula stage, Wnts (andLiCl) have the opposite effect, promoting the formationof epidermis and ventral tissues (Figure S5B). Becauselate Wnt signaling appeared to be mediated in partthrough the regulation of Smad1 activity, we investigatedwhether the late effects of Wnt signaling in Xenopus weredependent on Tcf3 and b-Catenin. Dkk1 mRNA provides
an excellent reagent to explore this because it specificallyinhibits all gastrula-stage canonical Wnt signaling. Dkk1
greatly expands the anterior neural plate (Figure 7E0 ).
DN-Tcf3 encodes a 31 amino acid deletion lacking theb-Catenin binding site (Molenaar et al., 1996), which
once bound to DNA blocks early Wnt signaling, resulting
in embryos devoid of neural plate (Figures 7E and 7F).
Remarkably, it was found that Dkk1 mRNA was able torestore neural plate development in 60% of these embryos (Figure 7F0 ). We also investigated whether the lossof neural plate caused by depletion of b-Catenin could
be rescued by Dkk1 and found, to our surprise, a complete
requirement for b-Catenin (Figures 7G and 7G0 ). We conclude from these epistatic experiments that Wnt signaling
promotes epidermal development via a Smad-dependent
pathway and that Dkk1 promotes neural development ina Tcf-independent, yet b-Catenin-dependent, manner.
DISCUSSION
We investigated the function of the four conserved GSK3
phosphorylation sites in Smad1. During the course of thework, we generated new phospho-specific antibody reagents for pSmad1MAPK and pSmad1GSK3 that revealed
a cell biological pathway by which the duration of the
BMP signal is exquisitely modulated. Three novel observations were made. First, we found that BMP signaling
triggers three sequential Smad1 phosphorylations by
BMPR, MAPK, and GSK3. Second, we found that the
GSK3 and MAPK phosphorylations specifically regulate
the duration of the BMP signal. Finally, experiments in cultured mammalian cells and in Xenopus embryos indicatedthat Smad1 phosphorylation by GSK3 serves to integrate
the BMP and Wnt signaling pathways.
Sequential Smad1 Phosphorylations
GSK3 phosphorylation required priming by phosphorylated MAPK sites located in the linker region of Smad1.
GSK3 phosphorylation-resistant mutations generated hyperactive forms of Smad1, as in the case of MAPK-

990 Cell 131, 980–993, November 30, 2007 ª2007 Elsevier Inc.

resistant mutations (Kretzschmar et al., 1997; Pera et al.,
2003; Aubin et al., 2004; Sapkota et al., 2007). High-titer
phospho-specific antibody reagents for GSK3 (Ser210)
and MAPK (Ser214) revealed the biochemical pathway
of sequential phosphorylations summarized in Figure 5A.
C-terminal phosphorylation by BMPR triggered nuclear
translocation (Shi and Massagué, 2003) and was followed
about 30 min later by phosphorylation at MAPK sites, presumably in the nucleus. This served as priming for phosphorylation by GSK3. The intracellular site of the GSK3
phosphorylation is not known because GSK3 is foundboth in nucleus and cytoplasm (Huang et al., 2007). The
combination of both MAPK and GSK3 phosphorylations
is essential for the polyubiquitinylation of Smad1 by the
Smurf1 E3 ubiquitin ligase (Zhu et al., 1999). Recently,
an independent study showed that MAPK phosphorylation is essential for Smad1 polyubiquitinylation (Sapkota
et al., 2007), a discovery fully confirmed here. One difference between the two studies is that here we report anessential requirement for GSK3 for polyubiquitinylationin vivo, whereas Sapkota et al. (2007) found only a partial
impairment in their in vitro studies. The broad theme thatemerges from these complementary studies is that linker
phosphorylations are required for Smad1 polyubiquitinylation in a negative feedback loop (Sapkota et al., 2007;
this work).
Triply phosphorylated Smad1 is polyubiquitinylated,binds to cytoplasmic microtubule-like filaments, and istransported to the centrosome (Figure 4). The centrosome
has been described as the proteolytic center of the cell(Badano et al., 2005). Inhibition of the proteolytic activityof proteasomes with lactacystin caused a marked accumulation of phospho-Smad1 destined for degradation
specifically in the centrosome. Because, as demonstrated
here, total polyubiquitin chains also accumulate in thecentrosome after lactacystin treatment, many other proteins targeted for degradation must also use this pathway.
The study of Smad1 has led to the identification of an elaborate cellular pathway involved in the degradation of thistranscription factor.
The Duration of the Smad1 Signal Is Regulated
by a Cellular Proteolytic Pathway
Smad1 is a transcriptional activator that controls the activity of hundreds of downstream target genes (Shi and Massagué, 2003; Feng and Derynck, 2005). During signaling, it
is important to control both the intensity and the durationof the signal. The classic example is provided by PC12
cells, in which a transient stimulus of MAPK/Erk by EGF
causes cell proliferation, whereas the more sustained activation of Erk caused by NGF induces neuronal differentiation and neurite outgrowth (Marshall, 1995). Whereas theintensity of the Smad1 signal is determined by the dose of
BMP, its duration is regulated by the coupled phosphorylations mediated by MAPK and GSK3. Thus, the half-life ofphospho-Smad1Cter was prolonged in pulse-chase experiments by inhibiting the three main endogenous MAPKs(Erk, p38, and JNK), GSK3, or proteasomal degradation

(Figure 3). This novel biochemical pathway offers rich possibilities for the differential regulation of target genes by
the Smad1 signal.
Integrating the A-P and D-V Axes
via Smad1 Phosphorylations
In Xenopus, the D-V axis is regulated by a gradient of BMP
maximal in the ventral and lowest in the dorsal, and theA-P axis by a gradient of Wnt lowest in the head and highest in the posterior blastopore where xWnt8 is expressed(Niehrs, 2004). When normal patterning is challenged bytransplanting a new organizer or cutting the embryo in
half, the new pattern is seamlessly integrated (De Robertis,
2006). The model shown in Figure 7H indicates how a selfregulating patterning mechanism may integrate the A-P
and D-V axes. This model is based on recent studies on
BMP signals and their antagonists (Reversade and De
Robertis, 2005; Lee et al., 2006). Smad1/5/8 are presented
as key players that can transcriptionally activate ventral
genes (BMP4, the protease Xolloid-related, and its inhibitor
Sizzled) as well as xWnt8. Previous work has shown that
BMP4 is required for the expression of xWnt8, whereas
xWnt8 does not affect BMP4 levels in the Xenopus gastrula(Hoppler and Moon, 1998). On the dorsal side, BMP signals
inhibit the transcription of genes such as ADMP (a dorsal
BMP-like molecule), Chordin, and Dkk1. The additional
layer of regulation of Smad1/5/8 by MAPKs activated by
RTKs such as FGFR, IGFR, and EGFR was not included
for simplicity. The key new node of interaction in this modified patterning network is that Wnt8 inhibits the phosphorylation of Smad1 by GSK3, resulting in a longer BMP signal
which, in turn, is inhibited by Dkk1. In this view, the D-V axis
would be specified by the intensity of the BMP signal andthe A-P positional information by its duration.
Our model proposes that Wnt enhances the BMP/
Smad1 signal. Such a molecular pathway could have
implications for the pathogenesis of hereditary bone diseases. BMPs were isolated as potent inducers of bonemorphogenesis, yet recent genetic studies in humans
and mice have shown that the Wnt coreceptor LRP5 playsa central role in bone mineralization. Loss-of-function
mutations in LRP5 cause osteoporosis and gain-of-function mutations cause osteopetrosis (reviewed in Koay
and Brown, 2005). Conversely, heterozygosity of Dkk1,
an LRP5/6 inhibitor, causes excessive bone mass formation (Morvan et al., 2006). Like Dkk1, Sclerostin (SOST) isa Wnt antagonist that binds to LRP5/6 and leads to increased bone mass in loss-of-function mutations (Semenov et al., 2005). It would be interesting to determine
whether these osteoblast differentiation syndromes arecaused by misregulation of the duration of the Smad1/5/
8 signal through the Wnt/GSK3 pathway.
The canonical Wnt pathway can inhibit GSK3 activity
causing b-Catenin stabilization (Logan and Nusse, 2004).
Wnt3a protein moderately decreased pSmad1GSK3 levels
while increasing the activated pSmad1Cter form (Figure 5).
In cytological analyses, activation of the Wnt pathway by
a CA-LRP6 caused a striking change. The pSmad1GSK3

antigen lost its centrosomal localization and was detected
in intracellular puncta corresponding to the recently
discovered LRP6 signalosomes that contain GSK3 andother components of the Wnt-regulated protein degradation complex (Bilic et al., 2007). In addition, pSmad1GSK3levels in the nucleus increased in cells transfectedwith CA-LRP6. This redistribution from the centrosome
into putative signalosomes explains the residual levels of
pSmad1GSK3 found in Wnt-treated cells. Relocation of
GSK3 away from the cellular proteolytic center locatedin the centrosome may disrupt the flow of Smad1 targeted
for degradation and suffice to explain the stabilization of
pSmad1Cter caused by Wnt3a protein, as well as theincrease of pSmad1GSK3 levels in the nucleus. Perhaps
other features of the canonical Wnt/GSK3 pathway,
such as the stabilization of b-Catenin, are mediated by
the relocation of Wnt-regulated protein destruction complexes away from the centrosomal proteasomes (Bilicet al., 2007).
The new Wnt signaling branch identified here requires
Smad1 and is independent of Tcf3, yet requires b-Catenin(Figure 7). This requirement suggests that both b-Catenin
and Smad1 might be degraded by a common Wnt-regulated destruction complex containing GSK3, Axin, andother components (Logan and Nusse, 2004). In thisregard, we note that b-Catenin has a primary structure related to armadillo domain adaptor proteins and may help
carry Smad1, and perhaps other proteins, to their destruction. A direct interaction between Smad4 and b-Catenin
has been reported (Nishita et al., 2000). In addition, b-Catenin binds to the microtubular motor Dynein (Ligon et al.,2001) and b-Catenin can localize to microtubules (Huang
et al., 2007). An attractive working hypothesis is that thedestruction of b-Catenin could serve to drive the flow of
Smad1 targeted for degradation to the centrosome. In
Drosophila, work currently under completion supports
the view that Wingless can stabilize the Mad transcription
factor (E.E., L.C.F., J. Clemens, and E.M. De R., unpublished data). With 1500 transcription factors in the humangenome, one might predict that in future the degradationof other proteins may also be found to be stabilized by
Wnt/GSK3 signaling.

EXPERIMENTAL PROCEDURES
Mammalian Cell Culture Experiments
Mouse L fibroblasts, Cos7, and HEK293T cells were cultured in DMEM
supplemented with 10% fetal bovine serum (GIBCO) and cultured at
37 C in 5% CO2. For pulse-chase experiments, L cells were incubatedin serum-free medium (1/3 Iscove’s, 2/3 F12 DMEM, GIBCO) for 4 hr,
and 5 nM BMP7 added for 15 min. Chemical inhibitors were added 1 hrprior to the BMP7 pulse. Wnt3a protein (R&D Systems) was added at
300 ng/ml in the absence of serum. FGF-2 (Invitrogen) and BMP4
(R&D Systems) were added at 40 ng/ml and 0.3 nM, respectively.
CA-LRP6 (LRP6-DN, Tamai et al., 2004) was transfected into Cos7
cells with Fugene HD (Roche). We note that the pericentrosomal region
is best visualized in sparse cell cultures well below confluence at theG1 phase. Ubiquitination assays were performed as described by
Zhu et al. (1999).

Cell 131, 980–993, November 30, 2007 ª2007 Elsevier Inc. 991

Western Blots and Immunostainings
Western blots of endogenous proteins were as described (Kuroda
et al., 2005), with polyclonal rabbit antibodies against pSmad1MAPK(1:30,000), pSmad1GSK3-A (1:15,000), pSmad1Cter (1:1000; Cell Signaling), and total Smad1 (1:1000; Zymed). b-catenin antibody (Sigma,
1:8000), GSK3b antibody (BD Transduction Labs, 1:1000), anti-His
antibody (Santa Cruz, 1:1000), and anti-flag-HRP conjugate (Sigma,
1:1500) were also utilized. Synthetic peptide was used to immunize
two rabbits (Covance). A second pSmad1GSK3 antibody phosphorylated at site 210 (designated B) that works on Western blots was
generated. For cell immunostaining, primary antibodies were used
as follows: pSmad1MAPK (1:2500), pSmad1GSK3 (1:1500), total polyubiquitin (1:500, Biomol), proteasome 20Sa5 subunit (1:200, Abcam),g-Tubulin (1:500, Sigma), and Pericentrin (1:1000, Abcam). For
additional immunostaining methods, see Supplemental Experimental
Procedures.
In Vitro Phosphorylations
His-tagged linker region (residues 146–264) of Smad1 was cloned in
the periplasmic expression vector pET26b, expressed in E. coli BL21
cells, and purified with His Bind Resin (Novagen). The priming reaction
was performed for 40 min at 30 C in kinase buffer (8 mM MOPS
[pH 7.3], 0.2 mM EDTA, 10 mM MgCaH3PO4, and 20 mM MgCl2) containing 1 mM cold ATP and 5 U/ml recombinant p42/Erk2 (New England
Biolabs). After heat inactivation of Erk (65 C for 20 min), a second
reaction containing [g-32P]ATP (Amersham) and recombinant GSK3b(Upstate) was performed for 1 hr at 30 C.
Xenopus Microinjections
All microinjections were four times marginal (or in the animal pole for
ectodermal explants) at the 4-cell stage. Amounts injected per blastomere were as follows: Xwnt8 MO (10 ng; Lee et al., 2006), ADMP-MO(8.5 ng), b-catenin-MO (11 ng at 2-cell stage), pCSKA-Wnt8 DNA(30 pg, Christian and Moon, 1993), pCS2-LRP6 DNA (10 pg, Tamai
et al., 2004), DN-xTCF3 mRNA (200 pg, Molenaar et al., 1996),
DN-Smad5 mRNA (100 pg, Beck et al., 2001), Dkk1 mRNA (50 pg
per embryo, Glinka et al., 1998), xBMP4 mRNA (50 pg), hSmad1
mRNA or its linker mutants (375 ng), and the constitutively active phospho-mimetic hSmad1 SEVE or its linker mutants (200 pg). Luciferase
assays were as described in Kuroda et al. (2005). For quantitative
PCR, RNA was isolated with Absolutely RNA preparation kit (Stratagene), DNase treated, and reverse transcribed with random primers.
SYBR Green real-time quantitative PCR was performed in a MX3000
Cycler (Stratagene). Methods for mRNA synthesis, whole-mount insitu hybridizations, and PCR primers are available at http://www.
hhmi.ucla.edu/derobertis/index.html.
Supplemental Data
Five figures and Experimental Procedures are available at http://www.
cell.com/cgi/content/full/131/5/980/DC1/.
ACKNOWLEDGMENTS
We thank Drs. G. Thomsen, J. Massagué, D. Bohmann, X. He, R.
Moon, J. Slack, H. Clevers, P. ten Dijke, and C. Niehrs for reagents,
Y. Sun for advice, and members of our laboratory for comments on
the manuscript. This work was supported by the NIH (HD21502-21).
C.H. was a recipient of a Heart & Stroke Foundation of Canada Fellowship. E.M. De R. is an investigator of the Howard Hughes Medical
Institute.
Received: March 2, 2007
Revised: July 29, 2007
Accepted: September 13, 2007
Published online: November 30, 2007

992 Cell 131, 980–993, November 30, 2007 ª2007 Elsevier Inc.

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