Research article

3381

Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals
induces massive brain formation in Xenopus embryos
Bruno Reversade, Hiroki Kuroda, Hojoon Lee, Ashley Mays and Edward M. De Robertis*
Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662,
USA*Author for correspondence (e-mail: ederobertis@mednet.ucla.edu)

Accepted 12 May 2005
Development 132, 3381-3392
Published by The Company of Biologists 2005
doi:10.1242/dev.01901

Development

Summary
To address the patterning function of the Bmp2, Bmp4 andBmp7 growth factors, we designed antisense morpholino
oligomers (MO) that block their activity in Xenopus laevis.
Bmp4 knockdown was sufficient to rescue the ventralizing
effects caused by loss of Chordin activity. Double Bmp4
and Bmp7 knockdown inhibited tail development. Triple
Bmp2/Bmp4/Bmp7 depletion further compromised trunk
development but did not eliminate dorsoventral patterning.
Unexpectedly, we found that blocking Spemann organizer
formation by UV treatment or β-Catenin depletion caused
BMP inhibition to have much more potent effects,

abolishing all ventral development and resulting inembryos having radial central nervous system (CNS)
structures. Surprisingly, dorsal signaling molecules such as
Chordin, Noggin, Xnr6 and Cerberus were not reexpressed in these embryos. We conclude that BMP
inhibition is sufficient for neural induction in vivo, and thatin the absence of ventral BMPs, Spemann organizer signals
are not required for brain formation.

Introduction

patterning in Xenopus. These are Bmp2 and Bmp4, which canreplace the function of the DPP morphogen in Drosophila(Padgett et al., 1993), and Bmp7, which when mutated inzebrafish results in dorsalized phenotypes (Hammerschmidt
and Mullins, 2002). During gastrulation in Xenopus, Bmp4 isexpressed in a ventral domain diametrically opposed to thedorsal Spemann organizer. A number of secreted proteins areco-expressed with Bmp4 in this region, which has been
designated the ventral gastrula center (De Robertis and Kuroda,
2004). Extracellular proteins expressed in the ventral center
include Twisted Gastrulation (Tsg) (a co-factor of both Bmp4
and Chordin), the metalloproteinase Xolloid-related (whichcleaves Chordin), the Chordin-related protein Crossveinless-2,
the Bmp inhibitory pseudoreceptor Bambi, and the secreted
Frizzled-related protein Sizzled (reviewed by De Robertis andKuroda, 2004). Expression of ventral center genes coincides
with high levels of BMP signaling, which can be monitored
using phospho-specific antibodies directed against the
carboxy-terminal region of the transcription factor Smad1,
which is phosphorylated by BMP receptors (Faure et al., 2000;
Kurata et al., 2001).
At the opposite pole of the gastrula lies the Spemannorganizer, a source of secreted BMP antagonists and otherfactors that interact extracellularly with ventral center geneproducts to generate a BMP morphogenetic gradient (Harland
and Gerhart, 1997; De Robertis and Kuroda, 2004).
Traditionally, two main biological activities of the Spemann
Organizer have been distinguished (Spemann, 1938; Niehrs,
2004). The head organizer located in the early blastopore lip
induces head and trunk structures after transplantation,
whereas the trunk-tail organizer located in the late dorsal lip

Bone Morphogenetic Proteins (BMPs) are growth factors
identified by virtue of their ability to induce ectopic boneformation when introduced subcutaneously in mammals (Urist
et al., 1979). BMPs are thought to be involved in multiple
aspects of cell signaling and homeostasis, including germ cell
formation, stem cell maintenance and cell differentiation(Hogan, 1996; Fujiwara et al., 2001; Ying et al., 2003). In
particular, during early development BMPs are central players
in establishing the dorsoventral (DV) pattern of the embryo.
Many zygotic mutations affecting DV patterning have been
isolated in Drosophila and zebrafish, the majority of which
affect the BMP signaling pathway (Lall and Patel, 2001;
Hammerschmidt and Mullins, 2002; De Robertis and Kuroda,
2004). Research in Xenopus has also provided important
insights into the role of BMP signaling in embryonicpatterning. This is illustrated by the discovery that Chordin and
Noggin encode secreted BMP antagonists produced by the
Spemann organizer (Piccolo et al., 1996; Zimmerman et al.,1996). The use of dominant-negative BMP receptors (dnBMPR) or cleavage-mutant BMPs (cm-BMPs, precursor BMP
proteins in which proteolytic processing sites are impaired) has
provided valuable information on the requirements of BMP
signaling for DV patterning (Hawley et al., 1995; Yamamoto
et al., 2001; Munoz-Sanjuan and Brivanlou, 2002). However,these constructs do not allow the respective activities ofindividual BMP ligands to be distinguished, as they inhibit theactivity of multiple BMPs. Therefore, gene-specific loss-offunction reagents are greatly needed in the field.
Three BMPs stand out as candidates for regulating DV

Key words: BMP, Chordin, Sizzled, Morpholino, Spemann organizer,
Brain induction, Dorsoal-ventral patterning

Development

3382 Development 132 (15)induces tail structures. Molecular investigations using Wnt
pathway inhibitors or the multivalent inhibitor Cerberus havesuggested that head formation requires the double inhibition of
Wnt and BMP (Glinka et al., 1997), or the triple inhibition of
Nodal-related, Wnt and BMP signaling (Piccolo et al., 1999;
Niehrs, 2004). Inhibition of just the BMP pathway (by dnBMPR, cm-BMPs or individual BMP antagonists) in wild-type
embryos results in the development of partial secondary axes
with trunk/tail structures that lack head and forebrain tissues.
Recent work has shown that at earlier stages, during blastula,
two distinct dorsal centers are formed. On the dorsal animaland marginal zone, a blastula Chordin- and Noggin-expressing(BCNE) center is formed in cells that will later on give rise to
the anterior CNS (Kuroda et al., 2004). In more vegetal dorsal
cells, the Nieuwkoop center releases mesoderm-inducing
Xenopus Nodal-related (Xnrs) growth factors and theantagonist Cerberus (Kuroda et al., 2004). Dorsal β-Catenin
accumulation is triggered by sperm entry and can be blockedby irradiation with ultraviolet (UV) light during the first cycle(De Robertis et al., 2000; Weaver and Kimelman, 2004). Thus,
the Xenopus DV pattern is currently thought to arise from aseries of cell-cell interaction events involving four signaling
centers, two at blastula and two at gastrula stage, that generate
a gradient of BMP signaling (reviewed by De Robertis andKuroda, 2004). Inhibition of BMP signaling is required for the
initial neural induction in Xenopus (Harland, 2000; Stern,2005). A second layer of regulation is provided by further
inhibition of Smad1 activity by MAPK phosphorylation, which
may mediate the neural-inducing effects of FGFs and IGFs
(Pera et al., 2003; Sater et al., 2003; Kuroda et al., 2005).
In the present study, we developed BMP-specific antisense
morpholino oligomers (MO) and used them to investigate theindividual roles of Bmp2, Bmp4 and Bmp7 in early Xenopus
development. In embryonic explants, inhibition of Bmp4 andBmp7 signals caused neural differentiation in ectoderm, anddifferentiation of dorsal fates in mesoderm. Triple knockdownof Bmp2, Bmp4 and Bmp7 compromised trunk and taildevelopment, giving rise to embryos with enlarged dorsal
structures. However, these embryos retained a considerable
degree of DV polarity. Unexpectedly, we found thatknockdown of BMPs caused the formation of extensive headand brain structures in embryos in which dorsal development
had been prevented by UV treatment. Similarly, striking radial
brain structures were obtained in embryos ventralized byinjection of β-Catenin MO. Formation of the Spemannorganizer at gastrula stage, or of Nieuwkoop or BCNE centersat blastula stage, was not restored by BMP MOs in dorsalized
embryos. Thus, in the absence of the dorsal organizing centers,
inhibition of BMP signaling is sufficient to cause extensive
head and CNS induction in embryos that would otherwise
develop without any neural tissue at all.

Materials and methods
Morpholino oligomers and RNA injections
The 25-bp morpholino antisense oligomers for Bmp2, Bmp4 andBmp7 were obtained from Gene Tools and consisted of the following
sequences:
Bmp2 MO, 5′-GATCCCAGCGACCATTGTCAACCTG-3′;
Bmp4 MO, 5′-CAGCATTCGGTTACCAGGAATCATG-3′;
Bmp7 MO, 5′-TTACTGTCAAAGCATTCATTTTGTC-3′.

Research article
Morpholinos were resuspended in sterile water to a concentrationof 1 mM, which was then further diluted to give a working solutionof 0.25 mM. When co-injected with β-Catenin MO (Heasman et al.,2000) or Chordin MO [a mixture of two MOs targeting both
pseudoalleles of Chordin (Oelgeschläger et al., 2003)], a mixture wasprepared and embryos were injected four times radially at the two- tofour-cell stage with 4 nl (3 ng MO/injection). For rescue experiments
mouse Bmp4 mRNA was microinjected at 25 pg in each blastomere
at the four-cell stage.
Embryological methods
Microinjections and mRNA synthesis were performed as described(Kuroda et al., 2004). RT-PCR conditions and primers, as well as the
protocol for whole-mount in situ hybridization, are described at
http://www.hhmi.ucla.edu/derobertis/index.html, except for themodification that after hybridization all embryos (pigmented or
albino) were bleached overnight (in 10% H2O2, 20% H2O, 80%
methanol) under intense neon light to enhance contrast. Digital
photographs were taken with a Leica DC500 digital camera. For eachembryo, at least three pictures were taken in successive focal planes
and merged with Photoshop® CS.
Protein injections and western blots
Injections of recombinant mouse Fgf8b protein (R&D Systems) was
carried out as described (Pera et al., 2003). Western blot analysis of
endogenous levels of phospho-Smad1 in Xenopus whole embryos(Faure et al., 2000) used an anti-phospho-hSmad1 antibody at a 1 in
500 dilution (Persson et al., 1998).

Results
Generation of BMP-specific morpholino reagents
We generated MO reagents (Heasman et al., 2000) for Bmp2,
Bmp4 and Bmp7. To overcome the pseudotetraploid natureof the Xenopus laevis genome (Oelgeschläger et al., 2003),
a search of the EST database was performed and two
pseudoalleles were identified for each of the three genes.
Antisense MOs were designed to block translation of all BMP
pseudoalleles by targeting 100% conserved 25-bp stretches,
including the AUG initiation codon (Fig. 1A). In vitro, each
MO specifically and efficiently blocked translation of its target
mRNA (Fig. 1B). MOs were microinjected into the marginal
region of each blastomere at the four-cell stage in order to test
their effect on development (Fig. 1C).
Embryos depleted of Bmp4 exhibited the most severe
phenotypes; at early tailbud stage, enlarged brain structures(marked by Six3 in forebrain and eye, and Krox20 in hindbrain)
were observed (compare Fig. 1E and 1F). Bmp4 MO embryos
appear larger and longer than controls because of an expanded
archenteron cavity, presumably caused by increased
convergence-extension (Myers et al., 2002). This dorsalized
phenotype could be rescued by microinjection of mouse Bmp4mRNA (Fig. 1G), which has a different nucleotide sequence inthe region targeted by Xenopus Bmp4 MO. At the swimming
tadpole stage, a posteriorization of the proctodeum (anus) anda severe loss of ventral fin tissues could be observed (Fig. 1I,J).
These tail phenotypes are consistent with a decrease of BMP
signaling in Xenopus (see Fig. S1 in the supplementarymaterial) and zebrafish (Wagner and Mullins, 2002). The onsetof the Bmp4 depletion phenotype could be traced back toalterations in gene expression patterns at gastrula, the stage at
which Bmp4 expression is maximal in Xenopus (Dale et al.,1992). As shown in Fig. S2 (see supplementary material),

Neural induction in Xenopus 3383

Development

Fig. 1. Antisense MOs against Bmp2,
Bmp4 and Bmp7 inhibit endogenous
Smad1 phosphorylation and cause
dorsalization and posterior truncationsof Xenopus embryos. (A) Design of
Bmp4, Bmp7 and Bmp2 antisense MOsthat target both pseudoalleles expressedin the subtetraploid species X. laevis.
(B) In vitro transcription/translation of
Bmp4, Bmp7 and Bmp2 is specificallyinhibited by the respective MOs.
(C) MOs for Bmp2, Bmp4 and Bmp7
(at 12 ng each) were injected either
alone or in combinations at the four-cellstage radially in each blastomere.
(D) Endogenous carboxy-terminal
Smad1 phosphorylation in stage 11
embryos is decreased by co-injection ofmultiple Bmp MOs. (E-H) Bmp4 MOinjected embryos (F) are dorsalizedwith enlarged heads (compare with
control embryos, E). Red arrowheads
delineate the spinal cord marker Hoxb9(>85%, n=60). (G) Bmp4 MOdorsalized phenotype is rescued by
microinjection of 100 pg of mouse
Bmp4 mRNA. (H) Microinjection ofmouse Bmp4 mRNA alone (100 pg)
results in ventralized embryos withsmall heads, no eyes and reduced spinal
cord structures (red arrowheads).
(I-L) Bmp4-depleted embryos (J)develop into swimming tadpoles withno ventral fins and slightly larger heads
than control embryos (I). Note theposition of the anus, which is displaced(posteriorized) to the tip of the tail(white arrowhead; >72%, n=61).
(K) Bmp7-depleted tadpoles develop
with a partial loss of ventral fin and aposteriorized anus (white arrowhead;>79%, n=51). (L) Double knockdownof Bmp4 and Bmp7 results in tadpoles
lacking tail structures (>90%, n=39).

knockdown of Bmp4 decreased expression of Bmp4,
eliminated the ventral center marker Sizzled, anddownregulated the BMP downstream target genes Mix1 andVent1. The neural plate, visualized through the pan-neural Sox2
and the forebrain/midbrain Otx2 markers, was expanded.
Embryos injected with the Bmp7 MO displayed similar but
less severe phenotypes at the tadpole stage, with a partial lossof ventral fin tissue accompanied by posteriorization of theanus (Fig. 1K, arrowhead). At early tailbud, a dorsalized
phenotype, less marked than that of Bmp4 MO, was observed(Fig. 2C,D). Bmp2 MO-injected embryos were also slightly
dorsalized, but were less affected than Bmp7 MO-injected
embryos (Fig. 2B). The expression domain of Sizzled in theventral-most region at tailbud stage (Collavin and Kirschner,
2003) was found to be a particularly good reporter of BMP
signaling. The anterior portion of this ventral domain wasdependent on Bmp2 (Fig. 2A′,B′), whereas the posterior
Sizzled expression domain required Bmp4 and Bmp7 signals(Fig. 2C′,D′). Taken together, these knockdown experiments

are consistent with the notion that BMPs promote ventral
development during Xenopus gastrulation.
Dorsalization in double and triple BMP knockdowns
To further characterize the requirements for Bmp2, Bmp4 andBmp7, double and triple knockdowns were performed in all
possible combinations and analyzed by in situ hybridization(Fig. 2). In addition, western blot analyses showed that
phosphorylation of endogenous Smad1 at gastrula stage wasincreasingly inhibited by combined depletion of Bmp2, Bmp4
or Bmp7 activities (Fig. 1D). This suggested that BMP
signaling in the Xenopus embryo results from the integrationof multiple BMP signals that act in concert to promote ventral
development. In agreement with this, more severe phenotypes
were consistently observed in double or triple depletions thanin single MO injections. For example, doubly depleted
Bmp4/Bmp7 tadpoles were unable to develop tail structures(Fig. 1L).
Combinations of Bmp2, Bmp4 and Bmp7 MOs were3384 Development 132 (15)Research article

Development

Fig. 2. Triple depletion of Bmp2, Bmp4 andBmp7 results in greater dorsalization of theembryo than any single or double BMP
knockdown. Embryos were fixed at tailbud
stage and expression domains of four markers(Otx2, Krox20, Myod and Sizzled) werecompared with control siblings (n=15 per
experimental set). (A-H) Lateral view, anterior
to the left. (A′-H′) Ventral view, anterior to thetop. (H′′) Dorsal view, anterior to the top.
(A-D′) Uninjected controls and single Bmp2,
Bmp4 and Bmp7 knockdowns. (E-G′) Double
BMP knockdowns (2/4, 2/7 and 4/7). Note thedorsalized phenotype with increased Otx2
expression, radial expression of Myod in theposterior, and ventral loss of Sizzled.
(H-H′′) Triple knockdown of Bmp2, Bmp4
and Bmp7 produces further dorsalization.
Expression of Krox20 in rhombomere 5 wasseen over the entire circumference of theembryo down to the tip of the tail (whitearrowheads).

injected and their effects on the expression domains of four
markers genes, Otx2, Krox20, MyoD and Sizzled, analyzed at
tailbud (Fig. 2). Sizzled expression in the entire ventral region
was eliminated by Bmp2 MO in combination with either Bmp4
or Bmp7 MO (Fig. 2E′,F′). MyoD, a paraxial mesoderm
marker, enveloped the tailbud ventrally, forming a ring in
Bmp4 MO-injected embryos (Fig. 2C,C′) or any of the double
BMP-depleted embryos (Fig. 2E-G′), indicating an expansionof paraxial tissue into ventroposterior mesoderm. Krox20 was
unaffected or modestly increased in any single or double

depletion, but rhombomere 5 expression
was radially expanded in triple
Bmp2/Bmp4/Bmp7 MO-injected embryos(arrowheads in Fig. 2H,H′′). Thus, triple
Bmp2/Bmp4/Bmp7-depleted
embryos
were dorsoanteriorized and consisted
mainly of tissues anterior to rhombomere
5. Surprisingly, the forebrain/midbrain marker Otx2 was only
moderately enhanced in Bmp-depleted embryos at tailbud
stage, even in triple-knockdown embryos (Fig. 2H).
Differentiation of ectopic neurons marked by N-Tubulin wasobserved in the posterior ectoderm of Bmp4, Bmp2/Bmp4 andBmp4/Bmp7 MO-injected embryos (Fig. 3). These neurons,
which probably correspond to Rohon-Beard sensory neurons,
followed the pattern of the underlying paraxial mesoderm (Fig.
2) (Mullins et al., 1996; Myers et al., 2002). Expansion of theRohon-Beard neuron population has been reported in zebrafish
mutant combinations that affect the BMP activity gradient in
ectoderm (Barth et al., 1999; Nguyen et al., 2000). Fgf8 is
known to decrease the activity of the BMP signal transducer
Smad1 through an inhibitory phosphorylation of its linker
region (Pera et al., 2003). Neuronal differentiation by lowering
BMP levels strongly synergized with microinjection of theFgf8 protein into the blastocoele cavity (Fig. 3E,F). This
Fig. 3. BMP knockdown induces neuronal differentiation in posterior
epidermis and cooperates with Fgf8 signaling. (A) The differentiatedneuronal marker N-Tubulin is expressed in the CNS of uninjected
control embryos at tailbud stage (inset: dorsal view, anterior to thetop). (B-D) Inhibition of endogenous Bmp4, Bmp2/Bmp4 andBmp4/Bmp7 activities results in ectopic neuronal differentiation in
the posterior ectoderm (inset: dorsal view, anterior to the top).
Comparable results were obtained with Bmp2/Bmp7 and triple
Bmp2/Bmp4/Bmp7 MO injections (data not shown). (E,F) Control
embryos or Bmp4 MO embryos were injected at the blastula stage
with 1.5 ng of recombinant mouse Fgf8 protein into the blastocoele
cavity and analyzed at tadpole stage for N-Tubulin expression. Fgf8
protein injection in wild-type embryos resulted in minor ectopic
neural differentiation, which greatly synergized with Bmp4
depletion.

Neural induction in Xenopus 3385

Development

indicates that inhibition of BMP signaling can cooperate with
FGF signals during neural induction.
We conclude from these results that the dorsalized
phenotype observed in single Bmp2, Bmp4 or Bmp7
knockdowns is increased in double and triple MO injections.
Although development of the tail is lost, even triple
Bmp2/Bmp4/Bmp7 knockdowns retain a significant amount of
DV and anteroposterior (AP) pattern.
BMPs are required for histotypic differentiation
We next analyzed cell differentiation in Bmp4/Bmp7-depleted
ventral marginal zone (VMZ) and animal cap (AC) explants(Fig. 4A-H). RT-PCR analyses showed that VMZs depleted of
Bmp4 or Bmp7 acquired dorsal fates, expressing muscle
markers such as Myf5 and neural markers such as Ncam, NTubulin and Otx2 (Fig. 4A, lanes 2 to 4). In double
Bmp4/Bmp7 knockdowns, midline mesodermal markers suchas Sonic Hedgehog (Shh) and Xnot were induced, indicatingthat the VMZs differentiated into dorsal marginal zone (DMZ)
tissues (Fig. 4A, lanes 5 and 6). Histological analyses revealed
the formation of a large amount of neural and cement glandtissue accompanied by greatly increased cell numbers (Fig. 4,
compare B with C). The ectodermal AC explant systemprovides the gold standard for neural induction studies in
Xenopus. We found that although Bmp4 or Bmp7 MOs were
sufficient to cause some neural induction alone, their combined
knockdown was much more potent, activating the expression
Fig. 4. BMP depletion causes ventral
mesoderm to acquire dorsal fates, animal caps
to become neural, and primordial germ celllosses. (A) RT-PCR analysis of Bmp4/Bmp7depleted VMZ explants. Microinjections of
Bmp4/7 MOs cause VMZ to adopt a DMZ
fate with upregulation of neural markers, suchas Ncam, N-Tubulin, Otx2, and dorsal midline
markers, like Shh and Xnot.
(B,C) Histological sections of control VMZcompared with Bmp4/Bmp7-depleted VMZ.
Absence of Bmp4/Bmp7 signaling in VMZ
induces massive cell proliferationaccompanied by muscle differentiation (mu),and neural tissue (ne) and cement gland (cg)formation. Insets show external views of the
explants. en, endoderm; epi, epidermis; m,mesothelium; c, coelomic cavity; lpm, lateralplate mesoderm. (D-G) Animal caps depletedof Bmp4, Bmp7, or both, contain neural tissue
as shown by the expression of N-Tubulin, amarker of differentiated neurons (n=12 perexperimental set). (H) RT-PCR analysis ofanimal cap (AC) explants. Inhibition of
Bmp4/Bmp7 results in neural differentiationin animal caps as shown by the expression ofNcam and Otx2 in the absence of mesodermalmarker α-Actin. ODC provides a loading
control and Xag is a cement gland marker.
(I-L) Germ cells, marked by the probe Xpat
(small dots in the endodermal region), fail todevelop in embryos injected with
Bmp2/Bmp4/Bmp7 MOs. Primordial germ
cells still form, albeit in reduced numbers, in
Bmp4 or Bmp4/Bmp7 MO-injected embryos(n=8 per experimental set).

of neural markers such as N-Tubulin (Fig. 4D-G), Ncam andOtx2 (Fig. 4H) in ectoderm.
We also found that, consistent with the involvement of
BMPs in the specification of primordial germ cells (PGCs) inmice (Fujiwara et al., 2001), expression of Xpat, a marker for
PGCs in Xenopus (Hudson and Woodland, 1998), waseliminated in embryos triply depleted of Bmp2, Bmp4 andBmp7 (Fig. 4I-L). Knockdown of individual BMPs, and all
double combinations, still retained at least some PGCs (Fig.
4J,K and data not shown). These loss-of-function experiments
support the view that BMP signaling plays a key role in the
differentiation of neural tissue, dorsal mesoderm and PGCs in
Xenopus.
Bmp4 is epistatic to Chordin
In Xenopus, Chordin knockdown causes a ventralized
phenotype that may result from excess BMP signaling(Oelgeschläger et al., 2003). To investigate this, we compared
Bmp4 MO- or Chordin MO-injected embryos with double
Bmp4/Chd MO-injected embryos (Fig. 5A-L). Sizzled
expression, which provides an excellent readout for BMP
signaling and ventral center formation, was greatly expanded
in Chordin morphants at gastrula (Fig. 5G). Bmp4 MO- anddouble Bmp4/Chd MO-injected embryos were virtually devoidof Sizzled expression (Fig. 5D,J). At tadpole stages, a similar
result was obtained: double Bmp4/Chd MO-injected embryos
had the same dorsalized phenotype as Bmp4 MO alone (Fig.

Development

3386 Development 132 (15)Fig. 5. Bmp4 antagonizes Chordin activity in the embryo. Four-cellstage embryos were injected, into each blastomere, with Bmp4 MOs(12 ng) or Chordin MOs (7 ng), or co-injected with a mixture of thetwo (n=15 per experimental set). (A-C) Sizzled is expressed in theventral center at stage 12 (A) and on the ventral side (B,C) at tailbud
stage. (D-F) Depletion of Bmp4 results in the lack of Sizzled
expression at late gastrula stage (D) and in significant reductions at
tadpole stage (E,F). Expression of Six3, an eye marker, is expanded.
(G-I) Chordin depletion gives rise to a phenotype opposite to thatseen in Bmp4 MO embryos, with increased Sizzled expression andreduced Six3. (J-L) Bmp4 is epistatic to Chordin in co-injection
experiments.

5, compare H with K). We conclude that Xenopus Bmp4 is
epistatic to Chordin. Xenopus Chordin, as is the case inzebrafish (Hammerschmidt and Mullins, 2002), serves as a
dedicated BMP antagonist.
Radial CNS in embryos lacking β-Catenin and BMP
signals
While studying the effects of Bmp4/Bmp7 MOs in embryos
ventralized with UV light, we made an unexpected discovery.
UV treatment blocks the early nuclear β-Catenin signal,causing the development of ventralized embryos devoid of all
axial and CNS structures (Fig. 6A,C) (reviewed by De Robertis
et al., 2000). Inhibition of the early β-Catenin signal was
accompanied by increased levels of endogenous Smad1
phosphorylation, indicating high BMP signaling levels (Fig.
6H, lane 3). Because UV-ventralized embryos overexpress
Bmp4 and its target gene Sizzled (see Fig. S3 in the
supplementary material), we expected that the knockdown of
Bmp4 and Bmp7 would be less efficient or, at most, equally
efficient as in wild-type embryos (Fig. 6B). Instead, we
found that in the UV background, Bmp4/Bmp7 or
Bmp2/Bmp4/Bmp7 knockdown had a much greater effect,causing the formation of dorsalized embryos consisting almost

Research article

Fig. 6. Inhibition of Bmp2/Bmp4/Bmp7 signaling in UV-treatedembryos or β-Catenin-depleted embryos results in radial brain
formation. Bmp2/Bmp4/Bmp7 MOs were injected four times
radially (12 ng each) into two-cell-stage UV-irradiated embryos, orwere co-injected with β-Catenin MO (7 ng total) (n=45 or more perexperimental set). White arrowheads point to the blastopore/anus.
(A,B) Depletion of Bmp4/Bmp7 in wild-type embryos leads to taildefects, but leaves the head and trunk regions mostly unaffected[Dorso Anterior Index, DAI=6.6 (Kao and Elinson, 1988)]. (C) UVtreated embryos are radially ventralized and develop into ventral‘belly pieces’ lacking all CNS and head structures (DAI=0.7). They
are devoid of neural tissue, as shown by the lack of Sox2 expression
at stage 20 (inset). (D) UV-treated embryos injected with
Bmp4/Bmp7 MOs are radially dorsalized, and lack trunk and tailstructures (DAI=8.1). At stage 20, half of the ectoderm of UV-treated
Bmp2/Bmp4/Bmp7 morphants expresses the pan-neural marker Sox2
(inset). (E) β-Catenin MO-injected embryos, like UV embryos,
develop into radially ventralized belly pieces (DAI=0.3). They arealso devoid of neural tissue, as shown by the lack of Sox2 expression
at stage 20 (inset). (F) Depletion of Bmp4/Bmp7 activity in βCatenin MO-injected embryos leads to dramatically hyperdorsalized
embryos (DAI=9.5) with a radial cement gland (compare with B). At
stage 20, half of the ectoderm of β-Catenin/Bmp2/Bmp4/Bmp7
morphants expresses the pan-neural marker Sox2 (inset).
(G) Embryos dorsalized by LiCl treatment (DAI=8.6). (H) Western
blot analysis of endogenous phosphorylation of Smad1 at gastrula
stage 11. Embryos ventralized by β-Catenin MOs have high levels of
Smad1 phosphorylation, which require Bmp4/Bmp7 signals.

exclusively of dorsoanterior head structures and deficient inventroposterior tissues (compare Fig. 6B with 6D). Similarly,
in embryos ventralized with β-Catenin MO (Heasman et al.,2000), Bmp4/Bmp7 MOs induced massive head-like structures

Neural induction in Xenopus 3387

Development

Fig. 7. Depletion of Bmp2/Bmp4/Bmp7 causes theformation of head-like structures with a radiallypatterned CNS in embryos ventralized by β-CateninMOs. Wild-type, β-Catenin or triple βCatenin/Bmp4/Bmp7 MO-injected embryos were
tested for the expression of CNS markers (n=9 ormore per experimental set). (A-C) Expression of
Sox2, demarcating the CNS at stage 15 in a control
embryo, is abrogated in β-Catenin MO-injected
embryos, and greatly expanded in embryos depletedof β-Catenin and Bmp4/Bmp7. (D-F) Expression of
N-Tubulin in differentiated neurons in the posterior
CNS is abolished in β-Catenin MO-injected
embryos, but can be seen radially around theblastopore in embryos lacking β-Catenin and
Bmp4/Bmp7. (G-I) Gbx2 expression, which marks
rhombomere 1 and otic vesicles (Von Bubnoff et al.,1996), is undetectable in β-Catenin MO-injected
embryos but can be visualized in two concentricrings in embryos depleted of β-Catenin and
Bmp4/Bmp7. (J-L) Engrailed-2 expression, whichmarks the midbrain-hindbrain boundary, is notpresent in β-Catenin MO embryos but is expressed
as a ring in embryos depleted for β-Catenin and
Bmp4/Bmp7. (M-O) Expression of theforebrain/midbrain marker Otx2 is absent in embryos
injected with β-Catenin MOs but is expressed
circumferentially in embryos depleted of β-Catenin
and Bmp4/Bmp7. (P) Histological section through aβ-Catenin MO-ventralized embryo. ae, atypicalectoderm; en, endodermal tissue; m, mesothelium.
(Q) Co-injection of β-Catenin and Bmp4/Bmp7 MOs
revealed enhanced cell proliferation in ectoderm
with neural tissue (ne) differentiation and radial
cement gland formation (cg). (R) Schematic
depiction of the anteroposterior polarity of the radial
CNS formed in β-Catenin and Bmp4/Bmp7-deficientembryos deduced from in situ hybridization studies.

along the radius of the embryo, including the formation of aconspicuous radial cement gland (Fig. 6E,F). These head-likestructures were much more extensive than those seen inembryos dorsalized by Lithium Chloride (LiCl) in a wild-type
background (Fig. 6G). When Bmp2/Bmp4/Bmp7 and βCatenin MOs were co-injected, extrusions containing theblastopore at their tip were also observed (see insets in Fig.
6D,F). The total amount of Sox2-positive neural tissue in
β-Catenin/Bmp4/Bmp7- or β-Catenin/Bmp2/Bmp4/Bmp7depleted embryos was comparable (Fig. 6D,F).
Using the pan-neural marker Sox2, massive neuralinductions were observed in embryos co-injected with βCatenin, Bmp4 and Bmp7 MOs, in striking contrast to thecomplete absence of the CNS in embryos in which only βCatenin was inhibited (compare Fig. 7B with 7C). At least halfof the ectoderm was transformed into histotypic neural tissue
and cement gland (Fig. 7P,Q). Although this CNS was radiallysymmetric and lacked all DV pattern, it still expressed AP
markers. Thus, BMP depletion has a potent neural-inducing
effect in embryos ventralized by UV treatment or β-Cateninknockdown. The posterior CNS, marked by the neuronalmarker N-Tubulin at this stage, was located next to the radial
blastopore or future anus (Fig. 7D-F), whereas theforebrain/midbrain marker Otx2 was found distal to it, adjacentto the radial cement gland (Fig. 7M-O). Hindbrain (Gbx2) andmidbrain-hindbrain border (En2) markers were expressed atintermediate positions (Fig. 7G-L). These AP neural marker
results are summarized in Fig. 7R. We conclude that BMP
depletion has much greater effects on neural induction inventralized embryos than in wild-type embryos. It appears thatsignals regulated by the early β-Catenin signal can partiallycompensate for the loss of Bmp4/Bmp7 in wild-type embryos.
Inhibition of Bmp4/Bmp7 does not induce
expression of dorsal genes
Because ventralized embryos were so strongly affected by
Bmp4/Bmp7 MOs, our expectation was that expression of
dorsal genes, which is lost in β-Catenin MO and UV embryos,
would be hyper-rescued. Surprisingly, we found that at theblastula stage neither Nieuwkoop center genes (such as
Xnr6, Fig. 8A-D) nor BCNE center markers (such as
Pintallavis/Hnf3β and Chordin, Fig. 8E-L) were induced inembryos depleted for β-Catenin and Bmp4/Bmp7. RT-PCR
analysis confirmed that, as was the case for the single βCatenin knockdown, all dorsal genes tested (Chordin, Noggin,
Xnr3, Siamois, Xtwn, Pintallavis/Hnf3β, and Cerberus) were
downregulated in β-Catenin/Bmp4/Bmp7 MO co-injections at
the blastula stage (Fig. 8Q, lane 4). Even at gastrula, Spemann3388 Development 132 (15)centers, in addition to providing dorsalizing growth factor
antagonists, may be the source of additional ventralizing
signals that are lacking in UV-treated or β-Catenin-depleted
embryos, as explained below.

Discussion
In the present study, we have generated three new morpholinos
that allow the requirements for Bmp2, Bmp4 and Bmp7 in X.
laevis to be analysed. An important advantage of MO reagents

Development

organizer expression of Chordin (Fig. 8M-P) or Goosecoid (notshown) was not restored by Bmp4/Bmp7 MOs in embryos
depleted of β-Catenin.
These findings show that the induction of radial CNS
structures caused by depletion of Bmp2, Bmp4 and Bmp7 inventralized embryos is independent of Nieuwkoop or BCNE
center formation, and ultimately of Spemann organizer
function. As the extent of neural induction caused by βCatenin/Bmp2/Bmp4/Bmp7 MOs is much greater than in wildtype embryos, the results suggest that the dorsal organizing

Research article

Fig. 8. Expression of Nieuwkoop, BCNE or Spemann organizer genes is not restored in β-Catenin/Bmp4/Bmp7-depleted embryos.
(A-D) Expression of Xnr6 in the vegetal dorsal side at blastula stage 9 in the Nieuwkoop center requires β-Catenin, is unaffected by knockdownof Bmp4/Bmp7, and is not re-expressed in β-Catenin/Bmp4/Bmp7 triple knockdowns. (E-L) Expression of the BCNE center genes
Pintallavis/Hnf3β and Chordin at blastula stage 9 requires β-Catenin, is not affected by the depletion of Bmp4/Bmp7 and fails to be rescued in
β-Catenin/Bmp4/Bmp7 triple knockdowns. (M-P) Chordin expression in the Spemann organizer at gastrula stage 10 is eliminated by β-Catenin
depletion but is unchanged in Bmp4/Bmp7-depleted embryos. Note that embryos shown in panels D,H,L and P do not express any of the dorsal
genes tested, yet still give rise to hyperdorsalized embryos with large head structures. (Q) RT-PCR analysis at stage 9. Dorsal markers are notexpressed in embryos lacking β-Catenin or β-Catenin/Bmp4/Bmp7. (R-V) A simplified model highlighting the interactions taking place at
blastula stage between BMP signals and the dorsal BCNE and Nieuwkoop centers.

Neural induction in Xenopus 3389

Development

is that simple co-injection experiments allow one to perform
double or triple loss-of-function experiments that would be
difficult to carry out by genetic means. These specific reagentsare expected to be very useful in the analysis of development
and were used here to demonstrate that BMPs play a crucialrole in dorsoventral patterning and neural induction.
Unexpectedly, we found that BMP knockdown had a muchstronger effect in ventralized embryos devoid of Spemannorganizer, in which it induced the formation of extensive headstructures, including large amounts of brain tissue.
BMP loss-of-function in Xenopus
Individual knockdowns of Bmp2, Bmp4 and Bmp7 resulted inmild dorsalizations, characterized by an increase of
dorsoanterior structures and a reduction of ventral andposterior markers. In particular, Bmp4 seems to play a crucialrole in Xenopus development, as it does in mouse (Winnier etal., 1995; Hogan, 1996; Zhao, 2003), because its inhibition
produced the strongest dorsalized phenotypes. All three BMPs
had to be knocked down to obtain PGCs loss in Xenopus.
Ventral fin tissues were very sensitive to decreased BMP
signaling levels. Both Bmp4 and Bmp7 MO-injected embryos
had varying degrees of ventral fin truncations and aposteriorization of the anus (Fig. 1I-K), indicating that theposterior ventral-most region of the embryo requires maximal
BMP signaling. This is in accordance with the co-expressionof Bmp4 and Bmp7 on the ventral side of the closing blastopore(Hawley et al., 1995). In zebrafish, it is well established thatthe ventral fin requires high levels of BMP signaling (Mullins
et al., 1996; Wagner and Mullins, 2002). Interestingly, Tucker
and Slack (Tucker and Slack, 2004) have recently found that
ventral fin cells derive from the ventral blastopore in Xenopus.
In the mouse, defects in BMP signaling can cause siren(mermaid-like) phenotypes, in which ventral-posterior
mesoderm development is impaired (Zakin et al., 2005).
Given that the transplantation of the dorsal organizer has
potent effects but ventral grafts do not, ventral development has
been viewed as subordinate to dorsal Spemann organizer
signals. However, more recent studies indicate thatdiametrically opposite to the Spemann organizer, a ventral
center, which expresses a number of secreted molecules, isformed at gastrula stage (De Robertis and Kuroda, 2004). The
present loss-of-function study shows that Bmp4 is a key
positive regulator of the expression of ventral center genes,
such as Sizzled and Bmp4 itself. Furthermore, epistatic studies
showed that the ventralization caused by knockdown of
Chordin can be compensated by loss of Bmp4 function(Fig. 5). In zebrafish, the strongest dorsalized phenotypes arecaused by Bmp2b (swirl) and Bmp7 (snailhouse) mutations(Hammerschmidt and Mullins, 2002). Double mutations in
bmp2b and bmp7 in zebrafish do not increase the phenotype(Schmid et al., 2000), whereas in Xenopus double and triple
knockouts show increasing degrees of dorsalization (Fig. 2).
This could be caused by a difference in transcriptional
regulation, as zebrafish bmp2b or bmp7 single mutants display
greatly decreased bmp2b, bmp4 and bmp7 expression (Schmid
et al., 2000). In Xenopus, however, Bmp2 MO does no affectBmp4/Bmp7 transcription (data not shown).
BMP inhibition and CNS formation
Does BMP inhibition play a role in neural induction in

Xenopus? The early β-Catenin signal induces the BMP
antagonists Chordin and Noggin in the BCNE center, and theirtranscripts are required for neural tissue formation in theabsence of mesoderm (Kuroda et al., 2004). In addition, theearly β-Catenin signal represses Bmp4 transcription on thedorsal side of the embryo (Baker et al., 1999). We show that
double Bmp4/Bmp7 depletion induced ectopic neurons in theposterior of the embryo, in the region in which paraxial
mesoderm surrounds the tailbud. However, triple Bmp2, Bmp4
and Bmp7 knockdowns, although substantially dorsalized, did
not have a significant increase in forebrain/midbrain neuraltissues when compared with those of wild-type embryos attailbud. By examining just these results, one would conclude
that BMP levels are not involved in neural induction in
Xenopus, as indeed has been proposed in other species (Stern,2005). Even in Xenopus, the inhibitor Smad6 failed to induce
neural tissue when injected into ventral ectoderm (Linker andStern, 2004; Delaune et al., 2005). However, other experimentswith an inducible construct of the inhibitor Smad7, indicatethat ectopic neural tissue can be obtained by inhibiting BMP
at pre-gastrula stages, but not at later stages of development(Wawersik et al., 2005). It has also been found that the use ofmultiple dominant-negative BMPRs greatly enhances
secondary axis formation in the whole embryo (Yamamoto etal., 2001). Importantly, it has been shown that triple inhibitionof Chordin, Noggin and Follistatin causes a catastrophic lossof dorsal structures, including the CNS (Khokha et al., 2005).
Neural induction can also be caused by Receptor Tyrosine
Kinase (RTK) ligands, such as FGFs and IGFs, which canincrease BMP signaling inhibition through inhibitory
phosphorylation, via Ras/MAPK, of the linker region of Smad1(Massague, 2003; Pera et al., 2003). This inhibitory
phosphorylation is particularly importantly in dissociatedanimal caps, which undergo a sustained activation ofendogenous Ras/MAPK signals (Kuroda et al., 2005). FGF
signals may also induce neural tissue in Smad-independent
ways (Delaune et al., 2005). Although inhibition of
Bmp4/Bmp7 by itself does not induce much neural tissue inwhole embryos, it greatly synergizes with Fgf8 protein
microinjection, causing patches of neuronal differentiation
throughout the ectoderm (Fig. 3). The loss-of-function results
provided here for animal cap and VMZ explants confirm that
decreasing endogenous BMP ligands promotes neuraldifferentiation (Munoz-Sanjuan and Brivanlou, 2002).
Importantly, our results indicate that, in the intact embryo,regulatory mechanisms that limit the effects of depleting
Bmp2/Bmp4/Bmp7 on neural differentiation must exist. When
dorsal development is inhibited by ventralization by UVtreatment or by β-Catenin MOs, a massive induction of brain
structures is triggered by reduced BMP levels (Fig. 7B,C). In
this experimental situation, the effects of BMP inhibition
become obvious, so that an embryo that completely lacked a
CNS would now develop radial neural structures that cover half
the ectoderm. These results support a key role for BMP
inhibition in CNS induction in the intact Xenopus embryo.
Brain induction in the absence of Spemannorganizer
The most striking result presented here is that extensive CNS
structures are generated by the knockdown of BMP signals inembryos ventralized by UV irradiation or β-Catenin depletion.

Development

3390 Development 132 (15)In amphibians, head and trunk induction are thought to be two
separate processes, stemming from head and trunk-tailorganizing centers at early and late gastrula, respectively(Spemann, 1938; Niehrs, 2004). It has been proposed that
double inhibition of Wnt and BMP (Glinka et al., 1997), or
triple inhibition of the Wnt, BMP and Nodal pathways (Piccolo
et al., 1999), is required to form heads, whereas trunk
formation only requires BMP antagonism. While these models
are concerned with the role played by the organizer once it hasformed, we now find that the Spemann organizer is dispensable
for brain induction in embryos lacking both Spemann organizer
and BMP signals (Figs 6, 7 and 8).
The diagrams shown in Fig. 8R-V attempt to explain thesefindings in terms of the cell-cell signaling thought to take placeat blastula stage (before the actual formation of the Spemannorganizer). We propose that, in Xenopus, brain formation
results from the regulated antagonism between BMP signals
and the two dorsal blastula signaling centers (the Nieuwkoop
center and the BCNE center) that form under the influence of
nuclear β-Catenin on the dorsal side (Fig. 8R) (De Robertis
and Kuroda, 2004). Blocking early β-Catenin signals by UV
irradiation or β-Catenin depletion (Fig. 8S) prevents theformation of all dorsal centers (the Nieuwkoop center, BCNE
center and, ultimately, the Spemann organizer) and theexpression of BMP antagonists. This leads to the unopposed
activity of ventral BMPs, causing the development ofventralized embryos (also called belly-pieces) lacking all
dorsal tissues, including CNS (Fig. 8S). Knockdown of BMP
activity (Fig. 8T) in wild-type embryos by MOs principally
affects development of the posterior ventral-most structures ofthe embryo, which arise from ventral mesoderm, giving rise to
tail-less tadpoles.
Remarkably, simultaneous knockdown of both dorsal andventral signals by co-injection of β-Catenin and Bmp4/Bmp7
or Bmp2/Bmp4/Bmp7 MOs results in radially symmetricembryos containing massive CNS structures (Fig. 8U). Thesehyperneuralized embryos retained AP pattern and in future it
will be interesting to determine whether this is caused byposteriorizing inputs such as FGF, Wnt or retinoic acid. Radialhead structures have also been reported in zebrafish triple
mutants deficient for bozozok, chordino and bmp2b (Gonzalez
et al., 2000). In Xenopus, radially dorsalized embryos can beobtained by LiCl treatment, but through a different molecularmechanism. LiCl-treated embryos have radial expression of
BMP antagonists such as Chordin (Oelgeschläger et al., 2003),
which inhibit ventralizing BMP signals and result in expanded
dorsal structures (Fig. 8V). We note, however, that the radial
CNS structures formed by LiCl treatment are much smaller
than in those seen in β-Catenin/Bmp4/Bmp7-depleted
embryos.
Lineage-tracing studies have shown that brain cellprogenitors in Xenopus can be traced back to cells of the BCNE
center (Kuroda et al., 2004). These cells transiently secrete
Chordin and Noggin at blastula, forming an early domain
devoid of BMP signaling, which, if cultured in isolation, canself-differentiate into anterior CNS tissue (Kuroda et al., 2004).
In β-Catenin/Bmp2/Bmp4/Bmp7 morphants, lower BMP
levels are attained throughout the embryo leading tohyperneuralization. Foley et al. (Foley et al., 2000) haveproposed that in the chick, as gastrulation proceeds, an
important function of the organizer, located in the anterior

Research article
primitive streak in the chick embryo, would be to posteriorize
forebrain progenitors located in the epiblast. These chickforebrain precursors (which are equivalent to the Xenopus
BCNE cells) transiently express Chordin and at all stages
migrate ahead of the organizer (Streit et al., 1998; Foley et al.,2000), and, consequently, are protected from its caudalizing
signals. The posteriorizing signal produced by the primitive
streak of the chick would correspond to the trunk/tail organizer
in Xenopus. The results presented here are consistent with theproposal that neural induction precedes Spemann organizer
formation. It appears that a principal role of the Spemannorganizer of the gastrula is the maintenance and caudalizationof neural tissue, rather than its initial induction. However, ourresults also differ from the current view of chick neuralinduction (Stern, 2004), as we find a crucial role for the
inhibition of BMP activity in the initial induction of neuraltissue in Xenopus.
Yet, one surprising finding is difficult to accommodate inthis model: Bmp2/Bmp4/Bmp7-depleted embryos were
dorsalized but still retained much of their DV polarity, whereas
embryos without a Spemann organizer (β-Catenin depleted or
UV-treated), which normally develop without a trace of a CNS,
developed massive CNS structures in Bmp2/Bmp4/Bmp7
morphants. This indicates that the Spemann organizer iscapable of partially compensating for the phenotypic effects
associated with Bmp2/Bmp4/Bmp7 depletion, and thatsomehow it can mediate ventral development whenBmp2/Bmp4/Bmp7 signals are eliminated. If the organizer
were responsible only for posteriorizing neural tissue, then its
absence should mainly affect the AP pattern of the pre-existing
CNS, but not trigger the massive formation of radial brain
structures observed experimentally. We think the solution tothis conundrum lies in the possibility that the Spemannorganizer serves as a source of ventralizing signals, in addition
to dorsalizing growth factor antagonists. Our ongoing research
centers on the molecular identification of this ventralizing
activity of the Spemann organizer.
We thank Drs W. Knöchel, H. R. Woodland and C. H. Heldin formaterials, members of our laboratory for comments on the
manuscript, and D. Greenberg for technical assistance. B.R. is a fellowof the Pasteur Institute. This work was supported by the NIH (R37
HD21502-19). E.M.D.R. is an Investigator of the Howard Hughes
Medical Institute.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/15/3381/DC1

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