Developmental Cell, Vol. 4, 219–230, February, 2003, Copyright 2003 by Cell Press

Chordin Is Required for the Spemann Organizer
Transplantation Phenomenon in Xenopus Embryos
Michael Oelgeschläger,1 Hiroki Kuroda,
Bruno Reversade, and E.M. De Robertis*
Howard Hughes Medical Institute andDepartment of Biological Chemistry
University of California, Los Angeles
Los Angeles, California 90095

Summary
We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis
pseudoalleles with morpholino antisense oligomers(Chd-MO) markedly decreased Chordin production.
Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior
tissues, phenocopying the zebrafish chordino mutant.
A strong requirement for Chordin in dorsal development was revealed by experimental manipulations.
First, dorsalization by lithium chloride treatment wascompletely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely
blocked the induction of the central nervous system(CNS), somites, and notochord by organizer tissuetransplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin forneural plate differentiation.
Introduction
The dorsal lip, or Spemann organizer, of the amphibian
gastrula has remarkable inductive properties. When
grafted into the ventral side of a host embryo, this small
group of cells can induce a second embryonic axis—
including a central nervous system (CNS), dorsal mesoderm, and secondary gut—in neighboring host cells.
Considering these striking properties, isolating the molecules that mediate these cell-cell inductions has been
an intensive area of research (reviewed in De Robertis
and Aréchaga, 2001). A large number of candidate secreted proteins specifically expressed in the organizer
region have been isolated (Harland and Gerhart, 1997;
De Robertis et al., 2000). Surprisingly, many of thesefactors encode secreted antagonists of growth factor
signaling. These include bone morphogenetic protein(BMP) inhibitors, such as Chordin, Noggin, Follistatin,
and Xnr3; Wnt inhibitors, such as Dkk, Frzb-1, and Crescent; Nodal signaling inhibitors, such as Lefty/Antivin;and inhibitors of multiple signaling pathways, such as
Cerberus, an inhibitor of BMP, Wnt, and Nodal signals.
In overexpression studies in Xenopus, all of these molecules have potent effects on the differentiation of embryonic cells. In order to unravel the respective contributions of each antagonist to the organizer phenomenon,*Correspondence: derobert@hhmi.ucla.edu
1
Present address: Max-Planck-Institut fur Immunobiologie, Stebeweg 51, D-79108 Freiburg, Germany.

one should ablate the function of each individual genein the grafted tissue. Here we test the requirement for
Chordin in Xenopus development.
Chordin is a large secreted protein expressed specifically in the organizer, which, when overexpressed, canrecapitulate most of the activities of the organizer (Sasai
et al., 1994, 1995). Chordin protein is expressed abundantly, reaching extracellular concentrations of 6–12 nM
in the dorsal lip (Piccolo et al., 1996). Chordin directly
binds BMPs via cysteine-rich modules (called CRs), preventing BMPs from binding to their cognate receptors(De Robertis et al., 2000). In Drosophila, the Chordin
homolog Short gastrulation (Sog) is expressed in ventral
neuroectoderm and is required for CNS development
and dorsal-ventral patterning of the embryo (François
et al., 1994; Srinivasan et al., 2002). The conservation
extends to cofactors of Chordin/Sog, in particular, themetalloproteinase Xolloid/Tolloid, which cleaves Chordin/
Sog, and the BMP binding protein Twisted gastrulation(Tsg), which forms ternary complexes with Chordin/Sog
and BMPs (Piccolo et al., 1997; Marqués et al., 1997;
Oelgeschläger et al., 2000; Chang et al., 2001; Ross etal., 2001).
In zebrafish, the Chordin homolog chordino was identified as the strongest ventralized mutant resulting fromsaturation genetic screens (Hammerschmidt et al., 1996;
Schulte-Merker et al., 1997). In chordino mutants dorsal
marker gene expression is reduced and ventral markers
are expanded in the mesoderm and ectoderm at thegastrula stage. Although chordino embryos recover atlater stages, they develop with small heads and eyes,
U-shaped somites, shortened trunk, and increased ventroposterior tissues, including blood islands, before dying after three days of development. These zebrafish
studies demonstrated that Chordin is required for dorsal-ventral patterning during gastrulation. In the mouse,
the situation is different, for most chordin⫺/⫺ embryos
have a normal CNS. Mutant embryos lack anterior notochord, and the pharyngeal endoderm, which expresses
Chordin, is greatly reduced. The pharyngeal defect results in a set of head, neck, and cardiac malformations
that mimic human DiGeorge syndrome. In a small percentage of embryos, a ventralized phenotype at gastrula
was seen, in which the ventral mesoderm giving rise to
the allantois was expanded at the expense of intraembryonic mesoderm (D. Bachiller, J. Klingensmith, and
E.D.R., submitted). Although the mouse chordin mutant
behaves differently from the zebrafish with respect to
CNS development, when the BMP antagonist noggin isalso mutated, the forebrain does not develop, indicatingthat Noggin can partially compensate for the lack of
Chordin (Bachiller et al., 2000).
In the present study we downregulated the expressionof Chordin protein in Xenopus using morpholino antisense oligomers (Summerton, 1999; Heasman, 2002).
The overall phenotype resembled that of the chordino
mutation, with reduction of dorsoanterior, and expansion of ventroposterior, structures in the embryo. The

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Figure 1. Morpholinos Targeting Two Distinct chordin mRNAs Reduce Chordin Expression and Promote Ventralization(A) Sequence of the morpholinos targeting the two Chordin mRNAs.
(B) Western blot analysis of secreted Chordin protein. Two-cell embryos were injected four times with a total of 8 ng of Chd-MO-1 (lane 2)
or Chd-MO-2 (lane 3) or with a combination of the two (4 ng each; lane 4). Dorsal lips were isolated at gastrula, and explants were cultured
in Ca2⫹-Mg2⫹-free medium (CMFM) for 12 hr. The supernatant was then analyzed in western blots with an immunopurified anti-Chordin
antibody; Coomassie blue staining of the cell pellets served as loading control.
(C–E) Embryos microinjected four times at the two-cell stage with a total of 8 ng of each Chordin morpholino or a mixture of the two. The
ventralized phenotype observed by coinjection of the two morpholinos generated a highly penetrant ventralized phenotype (⬎75%) in many
independent experiments.
(F) The effect of Chd-MO(1⫹2) was rescued by microinjection of 10 pg chd mRNA lacking the 5⬘ UTR.

neural plate and resulting CNS were reduced, and embryos were more susceptible to the ventralizing influence of overexpressed xTsg. Making use of the embryological manipulations available in Xenopus, we provide
evidence that Chordin plays a central role in the dorsalization of the embryo by LiCl treatment and in the dorsalization of mesoderm by Activin protein. Transplantationof dorsal lips microinjected with morpholinos showedthat Chordin is required for the inductive activity of transplanted Spemann organizer tissue.
Results
Inhibition of Both Chordin Pseudoalleles
Causes Ventralization
To determine the loss-of-function phenotype of Chordin
in Xenopus development, we designed an antisense
morpholino directed against the 5⬘ untranslated region(UTR) of chordin mRNA (MO-1; Figure 1A). No phenotype
was observed in a range of concentrations (Figure 1C).
Xenopus laevis is an allotetraploid species, thought tohave originated from the hybridization of two different
Xenopus species in the course of evolution. Therefore,
many genes are present in two copies, called pseudoalleles, which can differ in sequence (Kobel and Du Pasquier, 1986; Tinsley and Kobel, 1996). A search of the
Xenopus database revealed a chordin EST (GenBank
accession number BG019466) that differed considerably
in sequence from the original chordin cDNA (Sasai etal., 1994). A morpholino targeting the translation initiation site of this second ortholog (Figure 1A) produced
either no phenotype (Figure 1D) or weak ventralizations
at high concentrations (data not shown). Analysis of theproteins secreted by dorsal lip explants with an affinitypurified anti-Chordin antibody (Larraı́n et al., 2001)
showed that microinjection of each morpholino on its
own only moderately reduced Chordin secretion but thatthe combination of MO-1 and MO-2 greatly downregulated production of endogenous Chordin protein (Figure 1B).
When both morpholinos, designated Chd-MO(1⫹2),
were coinjected (4 ng of each per embryo), a ventralized
phenotype was observed with high penetrance (Figure1E). The specificity of these reagents for the two formsof Chordin was highlighted by microinjection of double
the amount of MO-1 or MO-2 (8 ng per embryo), which
was without phenotypic effect (Figures 1C and 1D). Embryos developed with small heads and enlarged ventroposterior regions (Figure 1E) and were reminiscent of
the chordino phenotype in zebrafish (Hammerschmidt
et al., 1996). This distinct phenotype has been previously
observed in experimental conditions that lead to an increase of BMP signaling in early Xenopus embryos, suchas the microinjection of Xolloid, BMP1, xTsg, Smad5,
and BMP4 (Piccolo et al., 1997; Goodman et al., 1998;
Oelgeschläger et al., 2000; Beck et al., 2001; our unpublished data). Microinjection of a synthetic chordin mRNAlacking the 5⬘ flanking region (10 pg, once dorsal) rescued the ventralized phenotype of Chd-MO(1⫹2) (Figure

Chordin and the Organizer
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morpholinos interfere with endogenous Chordin production, causing Xenopus embryos to develop withsmall heads and enlarged ventroposterior regions.
Chordin Downregulation Promotes Ventral Fates
To investigate the function of Chordin in embryonic patterning, we examined a number of molecular markers
in Chd-MO(1⫹2)-injected embryos. The expression of
chd and noggin at early gastrula, as well as the formationof the dorsal lip, were not affected (Figures 2A and 2B
and data not shown). At the late gastrula stage the expression of chd, noggin, and follistatin mRNAs was not
significantly affected by Chd-MO(1⫹2), except for ashortening of the length of the axial mesoderm (future
notochord), presumably caused by a mild inhibition of
convergence-extension gastrulation movements (Figures 2D–2F). RT-PCR analysis confirmed that the levelsof chordin, noggin, or follistatin mRNAs were not significantly affected by Chd-MO(1⫹2) at early or late gastrula
stages (Figure 2G). In zebrafish, the chordino mutation
also affects the morphogenetic process of convergenceextension (Myers et al., 2002). At the neurula stage, theneural plate, marked by the pan-neural marker Sox2,
was reduced in size, particularly in the anterior (Figure2C). The neuron-specific marker N-tubulin (Richter et al.,1988) confirmed the moderate reduction in CNS tissue(Figure 3E). The overall anterior-posterior patterning was
only mildly affected, with a reduction of Otx-2 in theforebrain and HoxB9 in the spinal cord (Figure 2H). However, the ventral marker sizzled (which encodes a secreted Wnt antagonist of the Frzb family; Salic et al.,1997) revealed a considerable expansion of ventral tissues (Figure 2I). This is of interest because Chordin isexpressed in the dorsal midline. Thus, Chordin inhibition
can affect the differentiation of ventral tissue far awayfrom its source, as it is known to be the case for its
Drosophila homolog, Sog (Srinivasan et al., 2002).
Histological sections at the swimming tadpole stage
showed that the eyes, brain, and spinal cord were reduced and that the blood islands were expanded in
ventral mesoderm of Chd-MO(1⫹2)-injected embryos(Figures 2J–2K″). The pharynx was hypoplastic and had
defects in the development of peripharyngeal structures, such as the hyoid and branchial arch cartilages(Figure 2K⬘ and data not shown). These pharyngeal phenotypes are reminiscent of those of the chordin knockout in the mouse (D. Bachiller, J. Klingensmith, and
E.D.R., in preparation). The notochord was present in
Chd-MO(1⫹2)-injected embryos (Figures 2K⬘, 2K″, and3B⬘). This is unlike chordino, in which the posterior notochord is lacking, and the mouse chordin knockout, inwhich the anterior notochord regresses (Hammerschmidt et al., 1996; our unpublished data). In Xenopus
embryos injected with Chd-MO(1⫹2), the myotomes
were reduced and U shaped, replacing the normal chevron-shaped appearance (compare Figures 3A″ and 3B″).
U-shaped somites have been described in chordino mutants, as well as in a large number of mutants in the
Sonic hedgehog (Shh) signaling pathway in zebrafish,
and reflect defects in horizontal myoseptum development caused by reduced notochord signaling. We conclude that inhibition of Chordin affects patterning of the

Xenopus embryo, causing a reduction of dorsoanterior,and an expansion of ventroposterior, tissues.
Chd-MO(1⫹2) Enhances the Effect of xTsg mRNA
We next analyzed the effects of Chd-MO in Xenopus
embryos overexpressing xTsg. A single ventral injectionof xTsg mRNA caused embryos to develop with a reduced head, an expanded endodermal cavity, and aposteriorized anus, which tends to detach from the endodermal mass at the swimming tadpole stage (Changet al., 2001; Figure 3C). This phenotype was consistent
with that of a moderate increase in BMP signaling, since
it was also observed in microinjections of Smad5 andconstitutively active BMP receptor mRNAs (Beck et al.,
2001; our unpublished data). In xTsg-injected embryos
the notochord was hypoplastic, particularly in the posterior, and the myotomes were U shaped (Figures 3C⬘ and3C″). When xTsg mRNA was injected into embryos inwhich Chordin expression was reduced by the ChdMO(1⫹2), the resulting embryos were severely ventralized, with only patches of residual notochord, reduced somites, and greatly decreased numbers ofN-tubulin-positive neurons in the CNS (Figures 3D and
3E). The reduction in the amount of CNS and myotomal
muscle was confirmed by RT-PCR analysis (Figure 3F,
lane 5).
The synergy between Chd-MO(1⫹2) and xTsg mRNAmight be explained by two different mechanisms. First,
xTsg is known to enhance the cleavage of Chordin by
Tolloid-like proteases, both in vitro and in microinjected
Xenopus embryos (Scott et al., 2001; Larraı́n et al., 2001).
It is conceivable that, in our experiments, xTsg may
eliminate residual amounts of Chordin protein in ChdMO(1⫹2) embryos. This explanation seems unlikely,
considering the chordino phenotype in zebrafish andthe considerable reduction observed in Chordin protein
levels after injection of Chd-MO(1⫹2) (Figure 1B). Second, a number of additional proteins that contain BMP
binding cysteine-rich repeats similar to those seen in
Chordin exist in vertebrates (Garcia Abreu et al., 2002);
xTsg overexpresion could antagonize the activities ofthese other proteins as well as that of Chordin. We conclude from these results that reduction of Chordin by
Chd-MO(1⫹2) synergizes with xTsg mRNA, resulting ina much stronger ventralization than that observed witheither reagent on its own.
Chordin Is Required for Dorsalization by LiCl
Chordin was originally identified as a cDNA induced bylithium chloride treatment of early Xenopus embryos(Sasai et al., 1994). LiCl inhibits glycogen synthase kinase-3␤ (GSK-3), stabilizing ␤-catenin (Klein and Melton, 1996; Schneider et al., 1996). This, in turn, leads tothe induction of chordin and other organizer-specificgenes, such as noggin, follistatin, and Xnr-3 (Wessely
et al., 2001). Embryos treated with LiCl are dorsoanteriorized, resulting in radial head structures lacking trunktail structures because of the transformation of the entire
marginal zone mesoderm into Spemann’s organizer(Kao and Elinson, 1988; Figure 4B). As shown in Figure
4C, we found that microinjection of 200 pg chordinmRNA into the animal pole of four-cell Xenopus embryos
is sufficient to mimic the LiCl phenotype (Figure 4C).

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Figure 2. Inhibition of Endogenous Chordin Results in Reduction of Neuroectodermal and Expansion of Ventral Mesodermal Markers
A mixture of the two Chordin morpholinos (4 ng of each) was injected four times, radially, at the two-cell stage.
(A and B) Expression of the organizer marker genes chordin and noggin was unchanged in wild-type (wt, left) and Chd-MO(1⫹2)-injected(right) embryos at early gastrula.
(C) The neural plate was reduced in Chd-MO(1⫹2)-injected embryos, as shown by the neural marker Sox2 (stage 12).
(D, E, and F) At stage 12 the expression of chordin, noggin, and follistatin was not significantly affected by Chd-MO(1⫹2) injection. The
expression domains are shorter, indicating a mild inhibition of convergence-extension gastrulation movements.
(G) RT-PCR analysis of sibling embryos. The mRNA levels of chordin, noggin, and follistatin were not significantly changed in Chd-MO(1⫹2)injected embryos at stage 10.5 or stage 12.
(H) At tadpole stages the A-P pattern of neural organization was relatively normal, as shown by expression of Otx2 (forebrain), Krox-20(hindbrain), and HoxB9 (spinal cord).
(I) Ventral mesoderm, marked by the expression of sizzled, is expanded in Chd-MO(1⫹2)-injected embryos. The upper panel shows a lateralview, and the lower panel shows a ventral view, of the same embryos.
(J and K) At late tadpole stages (stage 42) Chd-MO(1⫹2)-injected embryos develop with smaller head structures. Histological sections (J⬘,J″, K⬘, and K″) revealed a reduction of midbrain (mb), pharynx (ph), eye (ey), and spinal cord (sc) and an expansion of ventral blood islands(bl) in Ch-MO(1⫹2)-injected embryos. No obvious changes in somites (so) or notochord (no) were observed.

To test whether Chordin is required for the LiCl phenotype, loss-of-function experiments were performed. LiCl
treatment greatly increased synthesis of Chordin protein

by dissociated Xenopus embryos, and microinjectionof Chd-MO(1⫹2) prior to LiCl treatment inhibited the
production of secreted Chordin protein (Figure 4D). As

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Figure 3. Reduction of Chordin and Overexpression of xTsg Synergize, Causing Ventralization of Xenopus Embryos(A) Uninjected wild-type embryo at stage 40, stained with the notochord-specific antibody MZ15 (A⬘) or the muscle-specific antibody 12-101(A″).
(B) Embryos microinjected with Chd-MO(1⫹2) at the two-cell stage have normal notochord staining (B⬘), but the muscles have adopted a
U-shaped phenotype (B″).
(C) Embryos injected once ventrally with 1 ng of xTsg mRNA at the four- to eight-cell stage. Note that the notochord is reduced and absent
from the posterior in strongly affected embryos (C″). Muscle development is disturbed (C″), and muscles adopt a U-shaped phenotype.
(D) Embryos coinjected with xTsg and Chd-MO(1⫹2) are strongly ventralized.
(E) N-tubulin expression in stage 25 wild-type embryos, embryos injected at the two-cell stage with 8 ng Chd-MO(1⫹2), 1 ng xTsg mRNA, or
both.
(F) RT-PCR analysis of sibling embryos; Chd-MO(1⫹2) and xTsg mRNA cause the repression of anterior neural markers Rx2a (eye), Six-3(forebrain), and engrailed (midbrain). The pan-neural marker NCAM and the dorsal mesodermal marker actin are reduced. The ventral mesodermal marker sizzled is upregulated by Chd-MO(1⫹2) injections, but repressed by xTsg mRNA injections (Chang et al., 2001).

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Figure 4. Chordin Is Required for the Dorsalization of Xenopus Embryos by LiCl Treatment(A) Stage 45 untreated tadpole.
(B) Sibling embryo treated with 120 mM LiCl at the 32-cell stage for
30 min.
(C) Stage 45 embryo microinjected into the animal pole with 200 pg
chordin mRNA at the four-cell stage.
(D) Chordin production is inhibited by Chd-MO(1⫹2), even after LiCl
treatment (compare lanes 2 and 3). Whole embryos were dechorionated and dissociated at stage 9 and cultured for 14 hr at 19⬚C in
CMFM to allow detection of Chordin protein in the supernatant.
(E and F) LiCl-treated embryos display a radial cement gland at
tadpole stages and do not develop trunk-tail structures (dorso anterior index [DAI] ⫽ 8, n ⫽ 73)(G) Embryos at the two-cell stage with 8 ng Chd-MO(1⫹2) before
LiCl treatment are resistant to LiCl (G; DAI ⫽ 6.4, n ⫽ 38).

can be seen in Figures 4F and 4G, Chordin morpholinos
blocked the effect of LiCl. In some LiCl-treated embryos
the chordino-like phenotype with small heads and big
bellies was observed (Figure 4G). Thus, although ChdMO(1⫹2) had a mild effect in the intact embryo, it had avery strong effect when the embryo was experimentally
dorsalized. We conclude that Chordin is required for
LiCl dorsalization.
Chordin Is Required for Dorsalizationof Mesoderm by Activin
At the gastrula stage the organizer secretes a “horizontal
signal” that induces somite formation in ventral mesoderm (Dale and Slack, 1987). This dorsalization of themesoderm can be mimicked by treatment of animal cap
explants with the morphogen Activin (Green et al., 1992;
Dyson and Gurdon, 1998). Activin has a dual activity on

ectodermal animal explants. First, it is able to induce
ventral mesoderm and, second, Activin generates in adose-dependent way a dorsal-ventral set of cell fates(Green et al., 1992). Since Activin is known to induce
chordin transcription (Sasai et al., 1994), we tested
whether Chordin was required for the dorsalization ofanimal cap explants by Activin. Uninjected animal caps
or those injected with MO-1 or MO-2 individually elongated after treatment with 2 ng/ml Activin protein (Figures 5A–5D). This elongation reflects the convergenceextension movements that take place concomitantlywith dorsal mesoderm (somite) formation. When MO-1
and MO-2 were injected together, however, animal caps
retained their spherical shape (Figure 5E). This effectcould be rescued by the injection of chordin mRNA lacking the 5⬘ UTR (Figure 5F).
RT-PCR analysis indicated that the treatment of caps
containing Chd-MO(1⫹2) had reduced levels of the muscle markers Myf5 and ␣-Actin and of the neural marker
NCAM (Figure 5G, lanes 3–5). These molecular markers
were rescued by coinjection of chd mRNA lacking the5⬘ UTR (Figure 5G, lane 6). The ability of Activin to induce
ventral mesoderm was not impaired by Chordin knockdown. The ventral mesoderm markers xHox3/eve1 and␣-Globin were not only not inhibited, but were increased,by microinjection of Chd-MO(1⫹2) into Activin-treatedcaps (Figure 5G, lane 5).
Immunoblot analysis confirmed that 2 ng/ml Activin
induced abundant secretion of Chordin protein in animal
caps, which was inhibited by Chd-MO(1⫹2) (Figure 5H).
Interestingly, when the cell pellets were analyzed witha phospho-Smad1-specific antibody, which provides ameasure of BMP receptor activity (Persson et al., 1998),
Activin repressed, and Chd-MO(1⫹2) restored, BMP signaling (Figure 5H). The induction of the BMP antagonists
noggin, follistatin, and cerberus by Activin treatment inanimal cap explants was not significantly affected by
Chd-MO(1⫹2) injection (Figure 5I). At the end of gastrulation, at a time by which competence to respond to
Activin is lost (Grimm and Gurdon, 2002), noggin andfollistatin transcription was only weakly reduced, whereas
the levels of chordin transcripts were clearly downregulated by Chd-MO(1⫹2) (Figure 5J, lane 4). Taken together, the results suggest that Chordin plays a criticalrole in the dorsalization of mesoderm by Activin treatment but is not required for the induction of ventral
mesoderm by this morphogen.
Chordin Is Required for Spemann
Organizer Activity
To further analyze the function of Chordin in dorsal development, we carried out two series of transplantation
experiments. Dorsal lips were isolated from early gastrulae microinjected with the lineage tracer biotin dextran
amine (BDA) with or without Chd-MO(1⫹2). The graft
tissue comprised 30⬚ of the marginal zone and extended
from the base of the blastocoel to the early dorsal lip.
In the first experimental series (Figure 6A), the dorsal
lip replaced the endogenous organizer (isotopic andisochronic transplantation). In the absence of morpholinos the graft contributed to axial structures, in particular, to the notochord and dorsal endoderm, and noninvoluted cells contributed to ventral CNS, including the floor

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Figure 5. Chordin Is Required for the Induction of Dorsal Mesoderm by Activin(A–F) Two-cell embryos were injected fourtimes into the animal pole with a total of 8 ng
of Chd-MO-1, Chd-MO-2, Chd-MO(1⫹2), orwith Chd-MO(1⫹2) and 10 pg of chordinmRNA lacking the 5⬘ UTR of the chordin
cDNA. Animal explants were isolated at stage
8, treated with 2 ng/ml Activin, and cultured
until stage 25. Three independent experiments were performed with similar results.
(G) RT-PCR analysis of samples shown in(A–F). Activin treatment induced the expression of the somite marker genes Myf5 andActin and the pan-neural marker NCAM. ChdMO(1⫹2) inhibited the expression of dorsal
markers but upregulated the expression ofventral marker genes (xHox3 and ␣-Globin).
(H) A subset of the animal caps were cultured
overnight in CMFM. The supernatant was analyzed in Western blots with an immunopurified antibody specific for the interrepeat region of Chordin (␣-I-Chd; Larraı́n et al., 2001),
and the cell pellet was analyzed with an antibody specific for phospho-Smad-1.
(I) RT-PCR analysis of Activin-treated animal
caps at stage 10.5 injected with ChdMO(1⫹2). The morpholinos did not affectmesoderm induction (Xbra) or the expressionof other BMP antagonists (cerberus, chordin,
noggin, and follistatin).
(J) RT-PCR analysis of Activin-treated animal
caps at stage 12. The microinjection of ChdMO(1⫹2) changed noggin and follistatinmRNA levels only slightly, inhibited chordin
expression, and upregulated the ventral
mesoderm marker xHox3.

plate (Figure 6B). Transplanted organizers microinjectedwith Chd-MO(1⫹2) involuted and contributed normallyto dorsal endoderm and notochord, but, in addition,labeled cells were found in somites (Figure 6C). The cells
in the somites were found in the medial region adjacentto the notochord (Figure 6C) and indicate a modest ventralization of the mesoderm.

Unexpectedly, we noted that, in Chd-MO(1⫹2)injected organizers, the noninvoluted cells no longer
contributed to the CNS or floor plate but, rather, were
found in the epidermis, particularly in the posterior region of the embryo (Figure 6C). This phenotype was
fully penetrant, with all serially sectioned morpholinoinjected transplants showing staining in the epidermis,

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Figure 6. Chordin Is Required for Spemann’s Organizer Activity(A) Experimental design of the isotopic and isochronic transplantation experiment. Embryos were injected twice into dorsal blastomeres withBDA (biotin dextran amine lineage tracer) alone or with Chd-MO(1⫹2). Dorsal lip explants were isolated from these donor embryos andtransplanted at gastrula into unlabeled host embryos. After the embryos were sectioned in paraffin wax, BDA was detected with streptavidinconjugated alkaline phosphatase and BM purple.
(B) Transplantation of an organizer injected with lineage tracer alone allowed normal development in most cases, but some embryos developed
shortened axes (12%, n ⫽ 33). Histological sections showed that BDA staining was detected in the notochord (no) and floor plate (fp) throughout
the embryo and in anterior dorsal endoderm (de). Seven embryos were serially sectioned, stained with Streptavidin-AP, and developed with
BMP purple.
(C) Embryos that received Chd-MO(1⫹2)-injected organizer transplants developed with shortened body axes (100%, n ⫽ 15) and small eyes
(47%, n ⫽ 15) or no eyes (20%, n ⫽ 15). Lineage label was detected in the notochord, anterior dorsal endoderm, and medial somite (so).
Surprisingly, transplanted cells stained the epidermis of the host embryo (epi) in the posterior and were excluded from neural tissue. Six
embryos were serially sectioned.
(D) Dorsal lip explants transplanted into the ventral side of a host embryo recapitulate the classical Spemann-Mangold experiment.
(E) The transplantation of a wild-type organizer induced the formation of secondary axes (100%, n ⫽ 17), of which most (82%) were complete
axes containing heads and eyes. In the 11 embryos serially sectioned, BDA could be detected in the notochord, floor plate, and dorsal
endoderm of the secondary axes.
(F) Transplantation of a Chd-MO(1⫹2)-injected organizer blocked the axis-inducing activity of the explant. A few embryos formed protrusions(31%, n ⫽ 16), the strongest of which is shown here. In the seven embryos serially sectioned, BDA staining was detected in the ventral
mesoderm (vm), ventral endoderm (ve), and epidermis (epi). Note that Chd-MO(1⫹2)-injected grafts did not induce the CNS, somites, or
notochord.

whereas all sectioned control grafts showed labelingin the involuted endomesoderm and CNS, but not in
epidermis. This surprising result suggests that Chordin
is required in a cell-autonomous way in the ectoderm
for differentiation of ectodermal cells into CNS. The embryos transplanted in this way do form a ventral CNS but,when given a choice in this chimeric situation between
Chordin-expressing and -nonexpressing ectodermal
cells, prefer the Chordin-expressing cells. At the blastula
stage chordin is expressed in the preorganizer region
located in the dorsal animal cap, which might contributeto the future neural plate (Wessely et al., 2001).
In the second series of experiments, the dorsal lip
was transplanted ventrally, as in the classical SpemannMangold experiment (Figure 6D). In the controls, secondary axes were induced at high frequency (n ⫽ 17)

and, in most cases, were complete and included eyes,with the grafted cells contributing to notochord, floor
plate, and dorsal endoderm (Figure 6E). Microinjectionof Chd-MO(1⫹2) had a striking effect on the graft, abolishing secondary axis induction (n ⫽ 16). In some embryos weak ectodermal protrusions were seen, and thestrongest one is shown in Figure 6F. Chd-MO(1⫹2)treated organizers were completely unable to induce
CNS, somites, notochord, or secondary gut. The grafted
cells contributed mostly to ventral mesesoderm, endoderm, and epidermis. In none of the serially sectioned
control Spemann grafts was epidermal staining observed, whereas all Chd-MO(1⫹2) grafts analyzed hadlabeled cells in the epidermis.
Figure 7 shows the differentiation of dorsal lips cultured for three days in saline solution (stage 38). Dorsal

Chordin and the Organizer
227

Figure 7. The Mesodermal Specification of Dorsal Lip Explants Is Not Changed by Chd-MO(1⫹2) Microinjection(A) Explanted dorsal lips (n ⫽ 25) developed into anterior neural tissue (eye, ne), notochord (no), endoderm (en), and cement gland (cg) atstage 38.
(B) Chd-MO(1⫹2)-injected dorsal lip explants (n ⫽ 27) developed into notochord (no), muscle (mu), posterior neural tissue (ne), otic vesicle(ov), and epidermis (epi).
(C and D) Uninjected and Chd-MO(1⫹2)-injected dorsal lip explants were positive for the notochord-specific antibody MZ15 (n ⫽ 5).
(E and F) Uninjected and Chd-MO(1⫹2)-injected dorsal lip explants were stained by the muscle-specific antibody 12-101.
(G) RT-PCR analysis of dorsal lip explants at stage 10.5 showing that organizer gene expression (chd, noggin, follistatin, and cerberus) was
not affected by Chd-MO(1⫹2) at the time of the transplantations.
(H) RT-PCR analysis of Chd-MO(1⫹2)-injected dorsal lip explants at stage 26. In Chd-MO(1⫹2)-microinjected embryos the anterior neuralmarkers Six-3 and Rx2a were inhibited, the pan-neural marker NCAM and the cement gland marker XAG were reduced, and the notochordmarker Xnot and the somite marker gene actin were not affected.

lip explants identical to those used for the grafting experiments differentiate into anterior brain (containing
eye tissue), notochord, cement gland, and some muscle(Figure 7A; n ⫽ 25). The organizer retained its specification as dorsal mesoderm in Chd-MO(1⫹2)-injected explants, forming notochord and muscle (Figure 7B; n ⫽27). Antibody markers confirmed that notochord (Figure
7D; 5 out of 6 explants) and muscle (Figure 7F; 5 outof 5 explants) were present in Chd-MO(1⫹2)-injecteddorsal lips. The expression of organizer-specific BMP
antagonists was not affected by Chd-MO(1⫹2) in dorsal
lips analyzed by RT-PCR at the time of transplantation

Developmental Cell
228

(Figure 7G). Whereas the specification of mesoderm was
not changed, the neural tissue was reduced and had amore posterior character in Chd-MO(1⫹2)-injected dorsal lips (Figure 7B). Uninjected dorsal lip explants contain cement glands but, as is well known, are not surrounded by epidermis (Holtfreter, 1938; Figure 7A).
Morpholino-injected dorsal lips had smaller cement
glands and were covered by epidermis in nonneuralregions (Figure 7B), indicating ectoderm of a more ventral character. Molecular analyses of dorsal lips cultured
until tailbud stages (Figure 7H) confirmed that dorsal
mesoderm specification was maintained. Since thespecification of the gastrula mesodermal organizer isunchanged, these transplantation experiments indicatethat Chordin protein is required for the inductive properties of Spemann organizer tissue transplanted into theventral side of Xenopus embryos.
Discussion
Spemann’s organizer has been studied intensively inAmphibia, but loss-of-function studies have not beenpossible. Using antisense morpholino oligomers we now
show that the phenotype of Chordin knockdown inwhole Xenopus embryos resembles that of chordino
mutants in zebrafish. Considering the similarities between Xenopus and zebrafish development, this result
is not remarkable in itself. However, when embryonicdevelopment was challenged in a number of experimental situations, an unexpectedly strong requirement for
Chordin was revealed. In particular, we found that dorsalization by LiCl, Activin, and Spemann organizer grafts
was dependent on Chordin function. The loss of one
gene product may be compensated by redundant molecular mechanisms in the context of the whole embryo.
In experimental conditions, compensatory mechanisms
may be insufficient to mask the effects of the loss of
Chordin protein. The results illustrate the importance ofbeing able to manipulate the embryo in order to understand molecular mechanisms of embryonic induction invertebrates.
Chordin knockdown phenocopies the chordino mutation in zebrafish (Hammerschmidt et al., 1996; SchulteMerker et al., 1997). The Xenopus embryos displayed amoderately ventralized phenotype, with reduced headand expanded ventroposterior structures. Overall, theeffect in intact embryos was relatively mild, and neuraltissue was induced, although the neural plate was somewhat reduced (Figure 2). This weak ventralized phenotype was greatly enhanced in embryos injected withxTsg mRNA (Figure 3). Vertebrates have many Chordinlike CR-containing proteins, and xTsg can interact withseveral of them (Garcia Abreu et al., 2002; our unpublished data). It is possible that these or other redundant
molecular mechanisms, such as those of the BMP antagonists Noggin, Follistatin, Xnr3, or Cerberus, might
compensate for the lack of Chordin in whole embryos.
However, this compensation does not take place at thetranscriptional level, at least for noggin and follistatin,
in Chd-MO(1⫹2)-injected embryos (Figure 2G). In themouse the noggin and chordin gene products can compensate for each other, since their requirement for forebrain development is only seen in the double mutant(Bachiller et al., 2000). In Xenopus, an embryo that canbe easily manipulated, it is possible to uncover the requirement of Chordin for embryonic induction with avariety of experimental treatments.
Spemann’s organizer is induced by an early nuclear
␤-catenin signal on the dorsal side of the embryo (reviewed in Harland and Gerhart, 1997; De Robertis et al.,2000). Treatment of embryos with LiCl at the 32-cellstage stabilizes ␤-catenin, leading to the formation of aradial organizer spanning the entire marginal zone (Kaoand Elinson, 1988). LiCl embryos develop as radial headstructures lacking trunk-tail structures, which can bephenocopied by overexpression of Chordin in the animalpole (Figure 4C). Chordin is greatly upregulated by LiCl
treatment (Wessely et al., 2001), and, remarkably, thephenotypic effects of LiCl can be completely blocked
by Chordin morpholinos (Figures 4F and 4G). Rescueof LiCl dorsalization has been previously obtained bytreatments that increase BMP signaling, such as microinjection of BMP4, Xolloid, or xTsg mRNAs (Fainsod etal. 1994; Larraı́n et al., 2001). The present results indicatethat the transcriptional upregulation of the BMP antagonist Chordin is an essential downstream component mediating the effect of LiCl on embryonic patterning.
Another agent that induces Chordin is the TGF-␤ superfamily member Activin. In animal cap explants strong
induction of Chordin protein by Activin was observed
and was blocked by Chd-MO(1⫹2). Activin treatment
reduces Smad1 phosphorylation (a readout of BMP signaling), which was restored in the presence of ChdMO(1⫹2) (Figure 5H). Animal cap elongation, muscle
differentiation, and neural differentiation were inhibitedby Chd-MO(1⫹2). Thus, Chordin appears to be required
for the dorsalization of ventral mesoderm by Activin, amuch-studied morphogen (Green et al., 1992; Dyson
and Gurdon, 1998). However, the induction of ventral
mesoderm by Activin is not inhibited by Chd-MO(1⫹2).
The transcription of noggin and follistatin was not significantly affected in these experiments. In the future it will
be interesting to analyze the effects of Chordin protein
level reduction on the dose-dependent threshold effectsof Activin and endogenous Xnr signals on mesoderm
patterning.
Overexpression of BMP4 antagonizes the inductionof dorsal mesoderm by Activin (Jones et al., 1992). In
previous work, Goodman et al. (1998) showed that themetalloproteinase Xolloid or BMP1 blocked muscle formation in Activin-treated animal caps. These enzymes
cleave Chordin as well as other substrates, such as
probiglycan, procollagen, and prolysyl oxidase (Scott etal., 2000; Uzel et al., 2001); the present work demonstrates that Chordin is the component required for the
Xolloid/BMP1 effect.
In addition to the dorsalization of ventral mesoderm
by LiCl and Activin, Chordin is required for the inductive
activity of dorsal lips transplanted into the ventral sideof a host gastrula. Chd-MO(1⫹2)-injected dorsal lips
were completely unable to induce CNS, somites, or notochord, and they themselves differentiated into ventral
endoderm, mesoderm, and ectoderm (Figure 6F). This
result is in sharp contrast to the phenotype seen in intact
embryos, in which dorsal tissues continue to differentiate after microinjection of Chd-MO(1⫹2). It seems possible that, in whole embryos, other BMP antagonists canChordin and the Organizer
229

compensate for the lack of Chordin, but not when asmall group of cells is introduced into ventral tissue andpresented with the challenge of transforming ventral
cells expressing high BMP levels into dorsal tissue atthe gastrula stage. In Spemann grafts the lack of a single
BMP antagonist, Chordin, has a profound effect on neural-inducing activity and on the self-differentiation of thegraft. Interestingly, mouse node transplants have beenshown to be impaired in their neural-inducing activity
after transplantation into chick hosts when the organizer
gene goosecoid is mutated, even though neural induction is normal in intact gsc⫺/⫺ mouse embryos (Zhu etal., 1999). One possibility is that, since BMP expressionis under positive feedback (Hammerschmidt et al.,1996), the grafted tissue is overrun by BMP from surrounding ventral tissue when Chordin is lacking. Anotherpossibility is that the mild phenotype observed in whole
embryos may result from organizer-independent dorsalization signals, such as, for example, FGFs or Xnr’s.
The present experiments address only the loss-of-function of Chordin. Other BMP antagonists, such as Noggin,
Follistatin, and Cerberus, could also be required for the
Spemann organizer phenomenon. In the future it will beinteresting to test these individually.
An intriguing result noted in our transplantation experiments was that, in organizers transplanted into the dorsal side (Figure 6A), ectodermal cells were unable tocontribute to the CNS and floor plate when injected with
Chd-MO(1⫹2), forming only epidermis. Thus, it appears
that Chordin is required in a cell-autonomous way in theectoderm for the differentiation of neural plate in thesetransplantation experiments. Recently, Baker et al.
(1999) showed that ␤-catenin overexpressed in the ectoderm greatly expands the neural plate, and Wessely etal. (2001) described a ␤-catenin-dependent region ofchordin expression in the preorganizer region locatedin the dorsal side of the animal cap shortly after midblastula. Future studies will address whether the early expression of chordin in the blastula preorganizer is required for neural induction.
Experimental Procedures

explants for each sample in 50 ␮l CMFM at room temperature. The
supernatant was directly used for Western blot analysis with anantibody specific for the interrepeat region of Chordin (␣-I-Chd;
Piccolo et al., 1997; Larraı́n et al., 2001) that had been blot affinity
purified (Larraı́n et al., 2001). For the analysis of phospho-Smad1
levels in Xenopus explants, cells were homogenized in lysis buffer(Faure et al., 2000). Anti-phospho-hSmad1 antibody (Persson et al.,1998) was used at a 1:6000 dilution for Western blots.
Acknowledgments
We thank Drs. I. Dawid, F. Watt, and C.H. Heldin for materials, C.
Coffinier, E. Pera, O. Wessely, and J. Larraı́n for comments on the
manuscript, and U. Tran and A. Cuellar for technical assistance.
M.O. was an HFSPO postdoctoral fellow. E.M.D.R. is an Investigatorof the Howard Hughes Medical Institute. This work was supportedby the National Institutes of Health (R37 HD21502-16).
Received: August 2, 2002
Revised: October 7, 2002
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