M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6

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Expression of Siamois and Twin in the blastula Chordin/Nogginsignaling center is required for brain formation in Xenopus
laevis embryos
Hideyuki Ishibashia,1, Noriko Matsumuraa,1, Hiroshi Hanafusab,c, Kunihiro Matsumotob,c,E.M. De Robertisd, Hiroki Kurodaa,*aFaculty of Education (Biology), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japanc
CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya 464-8602, JapandHoward Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USAb

A R T I C L E I N F O

A B S T R A C T

Article history:

The blastula Chordin- and Noggin-expressing (BCNE) center located in the dorsal animal

Received 7 March 2007

region of the Xenopus blastula embryo contains both prospective anterior neuroectoderm

Received in revised form

and Spemann organizer precursor cells. Here we show that, contrary to previous reports,

4 October 2007

the canonical Wnt target homeobox genes, Double knockdown of these genes using anti-

Accepted 9 October 2007

sense morpholinos in Xenopus laevis blocked head formation, reduced the expression of

Available online 12 October 2007

the other BCNE center genes, upregulated Bmp4 expression, and nullified hyperdorsalization by lithium chloride. Moreover, gain- and loss-of-function experiments showed that

Keywords:

Siamois and Twin expression is repressed by the vegetal transcription factor VegT. We pro-

Cell signalingpose that VegT expression causes maternal b-Catenin signals to restrict Siamois and Twin

Spemann organizer

expression to the BCNE region. A two-step inhibition of BMP signals by Siamois andSiamoisTwin – first by transcriptional repression of Bmp4 and then by activation of the expression

Twinof the BMP inhibitors Chordin and Noggin – in the BCNE center is required for head formation.

Chordin 2007 Elsevier Ireland Ltd. All rights reserved.

NogginBMP
Wnt
Xenopus1.

Introduction

Head formation is of considerable interest in developmental
biology (reviewed in De Robertis and Kuroda, 2004; Niehrs,
2004; Stern, 2005). In vertebrate embryos, the establishmentof the anteroposterior (A-P) axis has been most fully studiedin amphibians, and embryological and molecular biologicalapproaches have led to the identification of many moleculesthat control A-P patterning. Several lines of evidence indicatethat head formation in Xenopus is controlled by a maternal
nuclear b-Catenin signal located in the dorsal side of the blastula embryo. However, at later stages of development zygotic* Corresponding author. Tel./fax: +81 54 238 4304.
E-mail address: ehkurod@ipc.shizuoka.ac.jp (H. Kuroda).
1
These authors equally contributed to this work.
0925-4773/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mod.2007.10.005

M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6

Wnt/b-Catenin signals have the opposite effect, ventralizing
the embryo (Christian and Moon, 1993). The late Wnt/b-Catenin effects are probably mediated by the increased duration of
BMP signals caused by inhibition of phosphorylation of
Smad1/5/8 by GSK3 (Fuentealba et al., 2007). The early effects,
however, are mediated by the well-known b-Catenin/Tcf3 (TCell Factor-3, a transcriptional repressor) pathway (Schneider
et al., 1996; Houston et al., 2002; De Robertis and Kuroda,
2004). In the early blastula embryo, overexpression of Wnt ligands, their downstream signaling components, or their target genes, on the ventral side of the early embryo results inaxis duplication, including perfect secondary head structures.
The nuclear localization of b-Catenin on the dorsal side
extends broadly from the bottom (vegetal) to the top (animal)pole of the blastula in Xenopus (Schneider et al., 1996). The egg
cytoplasm is heterogeneous, and when zygotic gene transcription starts at the mid-blastula transition the Nieuwkoop
signaling center is formed in the dorsal-vegetal region(Fig. 1A). Nieuwkoop center cells express Xenopus Nodal-related (Xnr1, 2, 4, 5, and 6), TGFb-superfamily factors that havepotent mesoderm-inducing activity (Agius et al., 2000; Takahashi et al., 2000). High levels of Xnrs emanating from the Nieuwkoop center induce the Spemann organizer in overlying
cells at gastrula, while the Nieuwkoop center cells themselves
go on to form anterior endoderm. The b-Catenin signal induces the expression of BMP antagonists such as Chordin
and Noggin in the BCNE (blastula Chordin- and Nogginexpressing) center, which has only limited overlap with the
Nieuwkoop center (Fig. 1A; see also Fig. 1E in Kuroda et al.,2004). Blastula expression of Chordin and Noggin in the cellular
precursors of the brain itself causes the anterior neuroectoderm to be predisposed to neural induction by the underlying
endomesoderm and explains the enigmatic phenomenon ofplanar neural induction in dorsal Keller explants in Xenopus(Ruiz i Altaba, 1993; Kuroda et al., 2004).
In addition to Chordin and Noggin, other genes are expressed in a localized fashion in the BCNE center: the homeobox genes Siamois and Twin, the BMP antagonist Xnr3, thewinged-helix gene FoxA4a/pintallavis/HNF3b, and the BMP-likegene Admp (Fig. 1A, Kuroda et al., 2004; Wessely et al., 2004;
Reversade and De Robertis, 2005). The homeobox genes Siamois and Twin are expressed immediately after the mid-blastula transition and are direct targets of the early b-Catenin/
Wnt signal with potent dorsalizing activity (Lemaire et al.,
1995; Carnac et al., 1996; Laurent et al., 1997). According totheir high similarity in amino acids sequence (88% in the
homeodomain) and perfectly overlapping expression patternthese genes may have redundant functions during Xenopus
embryogenesis or act synergistically, possibly as heterodimers, to regulate gene transcription (Laurent et al., 1997).
In the present study, we have analyzed the function of
Siamois and Twin using morpholino antisense oligomers(MO) (Heasman, 2002). The loss-of-function phenotype for
Siamois has been previously investigated using dominantnegative forms containing an engrailed repression domain(Fan and Sokol, 1997; Kessler, 1997). However, it is important
to study gene function in a true loss-of-function situation by
MO knockdown rather than in a dominant-negative approach, since microinjection of dominant-negative forms
could affect other regions where the desired genes being
investigated are not expressed, and these constructs persist
much longer time in the embryo than the wild-type products. We report here that the Siamois and Twin in combination are key regulators of all genes expressed in the BCNE
center, and are required for head formation and planar neural induction. Furthermore, we show that the vegetal expression of the maternal T-box transcription factor VegT (Zhang
et al., 1998) is the key component that restricts the expression region of Siamois and Twin to the dorsal-animal BCNE
center, thereby generating the distinction between the BCNE
and Nieuwkoop signaling centers in the animal and vegetal
hemispheres.

2.

Fig. 1 – Siamois and Twin are regulated by b-Catenin. (A) The
positioning of two dorsal signaling centers at blastula. The
BCNE center expresses Chordin (Chd), Noggin (Nog), Siamois(Sia), Twin (Twn), Xenopus nodal-related 3 (Xnr3), FoxA4a/
Pintallavis/HNF3b, Anti-dorsalizing morphogenetic protein(ADMP). D, dorsal; V, ventral. (B) The effect of b-Cat-MO,
demonstrating that BCNE center genes require b-Catenin.
(C–F) Blocking of Xnr inductive events by CerS mRNA
injection inhibits Chordin expression at gastrula but not at
blastula (when the BCNE is active), while Siamois and Twin
are independent of Xnrs. Ornithine decarboxylase (ODC) wasused as loading control.

59

Results and discussion

2.1.
Depletion of both Siamois and Twin causes anterior
truncations
The expression of Siamois, Twin, Chordin, and Noggin requires maternal b-Catenin, as shown by microinjection ofa MO that inhibits b-Catenin (b-Cat-MO) translation. b-Catenin depletion completely blocked the expression of these
BCNE genes in the stage 9 blastula (Fig. 1B). It has beenpreviously reported that Twin expression is blocked by adominant-negative form of Smad4 (Nishita et al., 2000).
We now investigated whether Siamois and Twin expression
is regulated by Nodal. The mesoderm-inducing activity ofendogenous Xenopus nodals (Xnrs) can be blocked by over-60

M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6

expression of CerS protein, the C-terminal portion of Cerberus (Bouwmeester et al., 1996; Piccolo et al., 1996; Ramiset al., 2007) (Fig. 1C–F). The expression of Chordin at gastrula stage is greatly inhibited by CerS, but the initial blastula expression is not. This indicates that the early
expression of Chordin is regulated by b-Catenin, whereas
Chordin expression at gastrula is driven by Xnrs (Fig. 1C)(Wessely et al., 2001). However, no differences were detected in Siamois and Twin expression in CerS injected embryos (Fig. 1D and E), indicating that Siamois and Twin are
independent of Xnrs and are exclusively regulated by bCatenin. In these RT-PCR analyses the house-keeping gene
Ornithine decarboxylase (ODC) was used as an mRNA loading
control (Fig. 1F). We conclude from these results that Siamois and Twin are under the control of the early b-Cateninsignals, but not of Xnrs.
Loss-of-function studies were carried out with Siamois
and Twin antisense morpholino oligomers (Sia-MO and
Twn-MO) designed to hybridize to the translation initiationsites of these genes (Fig. 2A). There are only six base differences between these MOs, yet each MO effectively blocked
only the translation of its own target gene (Fig. 2B). Thus,
Sia-MO and Twn-MO injected on their own served as internal specificity controls. Sia-MO blocked the secondary axis
inducing activity of ventrally injected Siamois mRNA
(Fig. 2C and D), but Twn-MO did not (Fig. 2E), and vice-versa(data not shown).

Fig. 2 – The function of Xenopus laevis Siamois and Twin isblocked by the antisense morpholino oligomer reagents
designed in this study. (A) The MOs for Siamois and Twintarget the translation initiation site. (B) In vitro translated
Siamois and Twin protein were blocked only by each specificMO. (C) Secondary axes induced by ventral injection SiamoismRNA (5 pg); 77.3% (17/22) of embryos have secondary axes
with formation of complete heads. (D) Secondary axisinducing activity by Siamois mRNA is blocked by Sia-MO(4 ng); 0% (0/25) of the embryos formed secondary axes. (E)Twn-MO (4 ng) did not block the effect of Siamois mRNA;
78.9% (15/19) of embryos had secondary axes with heads.

Fig. 3 – Siamois and Twin are required for head formation. (A)Twn-MO injected embryo. Small eyes were detected in 10.3%
of injected embryos (3/29). (B) Sia-MO injected embryo.
Small eyes were detected in 87.5% of embryos (42/48). (C)Twn-MO and Sia-MO co-injected embryos. 80% of embryos(28/35) lost head structures. (D) Co-injection of Twn-MO andSiamois mRNA rescued the Twn-MO phenotype to normal or
mild dorsalization (big eyes in 42.9% (15/35) of the embryos).
(E) Sia-MO and Twin mRNA coinjected embryo. Small eyes
were detected in 14.8% of embryos (4/27). (F) Control
uninjected sibling embryo for (A–E). (G) Sagittal histologicalsection of the embryo shown in C. (H) Sagittal histologicalsection of embryo shown in F. (I) Whole-mount in situ
hybridization analysis of the anterior neural marker Otx2 instage 26 embryo co-injected with Twin-MO and SiamoisMO. (J) Otx2 expression in control embryo at stage 26. fb,
forebrain; mb, midbrain; hb, hindbrain; nt, neural tube; no,notochord; he, heart.
Single Sia-MO or Twn-MO injections were without phenotypic effect, except for slight reduction in eye size
(Fig. 3A and B, compare to Fig. 3F). The Twn-MO small eye
phenotype was over-rescued by a small amount (1 pg) of
Siamois mRNA (Fig. 3D), and that of Sia-MO was rescuedby 1 pg of Twin mRNA (Fig. 3E). Importantly, when bothgenes were depleted together, a severe head defect phenotype was observed with high penetrance (Fig. 3C). MOs for
the BCNE center gene Chordin (Chd-MO) also have somehead inhibitory activity (Oelgeschläger et al., 2003), but theeffect of co-injection of Sia-MO and Twn-MO was muchstronger (Fig. 3C). In histological sections, forebrain development, but not hindbrain or spinal cord, was severely impaired (Fig. 3G and H). In in situ hybridizations, theanterior brain marker Otx2 was blocked by co-injection of
Sia-MO and Twn-MO (Fig. 3G–J), while the more posterior
neural markers krox20 and HoxB9 continued to be expressed(data not shown). Notochord and heart formation still took
place in embryos depleted for Siamois and Twin (Fig. 3G
and H). We conclude that Siamois and Twin are required
for anterior head formation in Xenopus development, in particular the forebrain.

M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6

2.2.
Siamois and Twin function as key regulators of the
BCNE center
The BCNE center is under the control of b-Catenin. Lithium
chloride (LiCl) inhibits glycogen synthase kinase-3b (GSK-3),
stabilizing b-Catenin and increasing its levels in the nucleus(Klein and Melton, 1996; Schneider et al., 1996). Embryos treated at the 32-cell stage with LiCl are dorso-anteriorized,
resulting in radial head structures lacking trunk–tail structures (Kao et al., 1986) (Fig. 4A). Siamois mRNA overexpression
in the whole embryo is sufficient to mimic the LiCl-dorsalized
phenotype (Lemaire et al., 1995). To test whether Siamois andTwin mediate the LiCl phenotype, loss-of-function experiments were performed. Co-injection of Sia-MO and Twn-MOprior to LiCl treatment completely blocked the dorsalizing effects of LiCl (Fig. 4A–C). This indicates that Siamois and Twin
constitute one of the main gene outputs acting downstreamof the b-Catenin signal.

Fig. 4 – Dorsalization by LiCl and planar neural induction
require Siamois and Twin. (A) Embryo treated with LiCl at
the 32-cell stage. The average of dorso-anterior index (DAI);
was 9.58 (Kao et al., 1986). (B) Embryo co-injected with SiaMO and Twn-MO prior to LiCl treatment. The average of DAI
was 5.38. (C) Control embryo for (A) and (B). In all cases 8 ngof MOs and 1 pg of mRNA were injected. (D) Phenotype of
dorsal-animal injection of Sia-MO (total 4 ng) and Twn-MO(total 4 ng); 76.0% (19/25) of embryos lost head structures. (E)The phenotype of dorsal-vegetal injection of Sia-MO and
Twn-MO. 7.7% (2/26) of embryos lost head structures. (F)Sibling control embryo at stage 38 for (A) and (B). (G) RT-PCR
analyses of the effects of Sia-MO and Twn-MO on whole
embryos at stage 9 and 11. (H) Neuroectoderm (NE) region of
Keller explants used for molecular studies. (I) RT-PCR
analyses of the effect of Sia-MO, Twn-MO, Chd-MO, andChordin mRNA on the NE region shown in (H).

61

The entire BCNE center region is derived from dorsal-animal blastomeres at the 8-cell stage, whereas the Nieuwkoop
center is derived from dorsal-vegetal blastomeres (Kuroda
et al., 2004). To test whether Siamois and Twin also function
as Nieuwkoop center genes, we injected both MOs into dorsal-animal or dorsal-vegetal blastomeres at the 8-cell stage
and found that Siamois and Twin were required for head formation in the animal blastomeres, but not in ventral ones
(Fig. 4D–F). At late blastula, stage 9, the expression levelsof Chordin and Noggin were significantly reduced by dorsalanimal but not by dorsal-vegetal depletion of Siamois andTwin (Fig. 4G, lanes 1–3). At mid-gastrula, stage 11, theexpression levels of Chordin and Noggin were not affectedby loss-of-function of Siamois and Twin (Fig. 4G, lanes 4–6).
These data are consistent with the view that at blastula Siamois and Twin function exclusively in the BCNE center, activating the expression of Chordin and Noggin. At gastrula
stages, however, Chordin and Noggin are regulated by Nodal-related signals emanating from Spemann organizer
mesoderm.
We reported previously that planar neural induction in
Keller dorsal gastrula explants requires Chordin (Kuroda
et al., 2004). We now asked whether the expression of Siamois
and Twin in the prospective neuroectoderm at blastula isresponsible for the ‘‘planar’’ neural induction signals. To
investigate this, we examined gene expressions in the neuroectodermal (NE) region in Keller sandwiches (Fig. 4H) (Keller
and Danilchik, 1988; Kuroda et al., 2004). The neuroectodermal region of control Keller sandwiches expressed the anterior neural marker Otx2 and the neuronal marker N-tubulin(Fig. 4I, lane 2), whereas the expression of Otx2 was blocked
by co-injection of Sia-MO and Twn-MO (Fig. 4I, lane 3), justas in the case of Chd-MO (Fig. 4I, lane 5). These results showthat the planar neural induction observed in Keller explants
requires Siamois and Twin. Microinjection of Chordin mRNA
partially rescued the anti-neural effect of Sia-MO and TwnMO, and completely the inhibition of planar induction by
Chd-MO (Fig. 4I, lanes 4 and 6). We conclude from theseexperiments that planar neural induction requires Siamois
and Twin. As described earlier, this neural induction also
has a complete requirement for the expression of their downstream target Chordin (Kuroda et al., 2004).
In addition to Siamois and Twin, the BCNE center expresses
Chordin, Noggin, Xnr3, FoxA4a/pintallavis/HNF3b, and Admp
(Wessely et al., 2004; Reversade and De Robertis, 2005). To
investigate the relationship between these other BCNE center
genes and Siamois and Twin, we examined their transcription
levels at blastula stage 9. Single MOs had very weak effects,
although Sia-MO-injected embryos had a slightly stronger
phenotype than Twin-morphants (Fig. 5A, lanes 1–3). However, all BCNE center genes, except for Siamois and Twin, were
inhibited by co-injection of Sia-MO and Twn-MO (Fig. 5A,
lanes 1 and 4). In particular, Chordin, Noggin, and FoxA4a/Pintallavis/HNF3b expression became undetectable (Fig. 5A, lane
4 and Fig. 5B and C). The Xnr3 promoter region has target
binding sequences for LEF/TCF and for homeodomain transcription factors (McKendry et al., 1997). Thus, the residual
expression observed in the case of Xnr3 and Admp might be
explained by a feed-forward loop in which these genes are directly regulated by b-Catenin, as well as by Siamois and Twin.

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M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6

Fig. 5 – Siamois and Twin are key regulators of thetranscription of BCNE center genes. (A) RT-PCR analyses ofthe effects of Sia-MO, Twn-MO, or both on whole embryos.
(B) Chordin and Noggin expression at blastula in the animal
cap and marginal zone (BCNE) of normal embryos. (C) Coinjection of Twn-MO and Sia-MO eliminates Chordin and
Noggin expression. Embryos are shown in animal view.

These results indicate that the Siamois and Twin transcriptional activators are central players in the regulation of BCNE
center gene expression. They are required for the expressionof Chordin, Noggin and FoxA4a/Pintallavis/HNF3b, but not for
their own expression, which depends entirely on the b-Catenin signal.

2.3.
Double inhibition of BMP by Siamois and Twin bytranscriptional repression and BMP antagonists
The traditional embryological view held that neural tissue
forms during gastrulation, when zygotic signals from dorsal
mesoderm antagonize BMPs in the overlying dorsal ectoderm(Kuroda et al., 2004). Recent studies, however, have shownthat the early b-Catenin is sufficient to cause neural tissue
formation in the ectoderm in the absence of underlying
mesoderm, explaining earlier observations of planar neuralinduction in Xenopus (Ruiz i Altaba, 1993). Some studies havepreviously shown that b-Catenin inhibits BMP transcription(Baker et al., 1999; Leung et al., 2003). On the other hand, we
reported that nuclear b-Catenin causes the expression of
Chordin and Noggin in the BCNE center and that these BMP
antagonists are required for neuralization (Wessely et al.,
2001; Kuroda et al., 2004). In an attempt to reconcile these
two lines of evidence, we now investigated the function of
Siamois and Twin in this process.

Fig. 6 – Two-step inhibition of BMP4 by Siamois and Twin.
(A) Experimental procedure. (B) RT-PCR analyses for theeffects of Siamois, Twin, and the BMP antagonists Chordin
and Noggin on Bmp4 and its target gene Vent1. The nodalrelated target gene Mix1 was used to indicate that CerS was
effective in blocking mesoderm and endoderm formation inthese experiments. (C) Diagram showing a two-step modelfor the regulation of BMP4 via Siamois and Twin. BMP
signaling is blocked in two steps (1) transcriptional
repression and (2) BMP antagonists such as Chordin and
Noggin which are induced by Siamois and Twin.
Small dorsal and ventral ectodermal explants were excisedfrom embryos injected with CerS mRNA to prevent mesoderm
formation (Fig. 6A). CerS, which consists of the cystine knot ofCerberus, is an excellent reagent for this type of experimentbecause it blocks specifically the activity of all mesoderminducing Nodal-related genes (Piccolo et al., 1996; Agiuset al., 2000). Double depletion of Siamois and Twin caused
Bmp4 to be expressed in the dorsal animal cap fragments(Fig. 6B, compare lanes 2 and 4), and injection of SiamoismRNA inhibited Bmp4 expression in ventral animal cap fragments (Fig. 6B, lanes 5 and 7). Depletion of Chordin and Nogginincreased expression of the BMP target Vent1 dorsally (Fig. 6B,
lanes 2 and 3), and Chordin mRNA inhibited Vent1 but did notaffect Bmp4 transcription (Fig. 6B; lanes 5 and 6). At the gastrula stage, the opposing homeobox genes Goosecoid andVent1/2 have been reported to be mutually antagonistic in dorsal–ventral patterning (Sander et al., 2007). We believe that
Siamois and Twin act on Vent1 at the blastula stage, before
the later gastrula regulatory event of the mutual repression
by goosecoid and Vent1 takes place.
These results suggest to us a model in which Siamois andTwin first downregulate endogenous Bmp4 mRNA transcription at the blastula dorsal animal region in a BMP antagonist-independent manner (as proposed by Baker et al., 1999;
Leung et al., 2003). In a second step, Chordin and Noggin produced in the BCNE center inhibit any residual BMPs. In thistwo-step model, depicted in Fig. 6C, BMP signals are clearedM E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6

out in the BCNE center by transcriptional repression and also
by BMP antagonists, both dependent on Siamois and Twin
expression.
Since Siamois and Twin are transcriptional activators(Kessler, 1997), how can they repress transcription of Bmp4?
In zebrafish, the homeobox gene bozozok induced by the
Wnt/b-Catenin signal encodes a transcriptional repressor
and is able to directly inhibit Bmp2b transcription (Leunget al., 2003). A distinct possibility is that Xenopus might havean as yet undiscovered bozozok-like transcriptional repressor
homologue induced by Siamois and Twin that negatively regulates the transcription levels of Bmp4, explaining arrow number 1 in Fig. 6C.

63

2.4.
VegT defines the border between the BCNE center andthe Nieuwkoop center
What determines the molecular differences between the
BCNE and Nieuwkoop centers? To address this, we used a
VegT-MO (Heasman et al., 2001) against the maternal vegetally-expressed T-box transcription factor VegT (Zhang et al.,1998). This morpholino does not inhibit translation of the zygotic alternatively spliced VegT transcript designated as Antipodean (Stennard et al., 1999). We noted that VegT-MO-injected
embryos always had big heads and cement glands, which are
typical signs of low-BMP (Fig. 7A and B). This is what one
might expect from an expansion of the BCNE center.
To investigate whether VegT controls BCNE center gene
expression, we injected both Wnt8 mRNA to activate b-Catenin(Christian and Moon, 1993) together with VegT mRNA. This
combination recreates dorsal-vegetal conditions, mimicking
a Nieuwkoop center in animal caps. In the conventional view
currently held by the Xenopus field, Siamois and Twin are expressed in the Nieuwkoop center (Lemaire et al., 1995; Laurent
et al., 1997). Therefore, the expectation would be that animal
caps co-injected with Wnt8 and VegT mRNAs would express
large amounts of Siamois and Twin. Surprisingly, BCNE center
genes such as Chordin, Noggin, Siamois, and Twin, which arestrongly induced by increasing doses of Wnt8 mRNA (Fig. 7C,
lanes 2-5), were not upregulated in the presence of VegT mRNA
(Fig. 7C, lanes 6-8). In contrast, the Nieuwkoop center genes
Xnr2 and Xnr6 were induced by VegT mRNA (Fig. 7C, lanes 6–9). These data show that VegT mRNA inhibits the induction of
BCNE center genes caused by Wnt8/b-Catenin (Fig. 7C).
Loss-of-function experiments support the view that VegT
serves to exclude the BCNE from the vegetal pole. The Chordin
and Siamois expressing region at the BCNE center was greatly
expanded towards the vegetal side by depletion of VegT
(Fig. 7D–G). As shown earlier, this early expression of Chordin
requires Siamois and Twin (Fig. 5A–C). This result is somewhat
conflicting with a previous report by Houston et al. (2002) inwhich the inhibition of VegT reduced the level of Chordin andbFig. 7 – The BCNE center is restricted to the dorsal-animal
region by the maternally localized VegT transcription factor.
(A) The phenotype of VegT-MO. White arrowhead indicatesenlarged cement gland. (B) Control embryo for (A). (C) RT-PCR
analyses of the effects of Wnt8 and VegT mRNA for animal
caps. 1, 4, or 16 pg of Wnt8 mRNA and 100 pg of VegT mRNA
were injected. (D) Chordin expression in control embryo atstage 9. White and black arrowheads indicate the animaland vegetal poles, respectively. (E) Siamois expression incontrol embryo. (F) Chordin expression of VegT-MO (16 ng)
injected embryo. (G) Siamois expression of VegT-MO injected
embryo. Note the expansion of Chordin and Siamois
expression towards the vegetal side upon inhibition of VegT.
(H) Real-time PCR analyses of Chordin mRNA expression
levels from stage 8 to 12. (I) Siamois mRNA expression levelsin the same samples. (J) Model of the regulation of the two
blastula signaling centers. The border between the BCNE
and Nieuwkoop centers is generated by repression by twomaternally provided transcription factors: B1-type Sox(animal; Zhang et al., 2004) and VegT (vegetal).

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M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6Goosecoid, while we now report an increase. Therefore, we
examined by real-time PCR analysis the level of expressionof the mRNAs for Chordin and Siamois over the time course
from stages 8 to 12 (Fig. 7H and I). The expression of Chordin
and Siamois in VegT-MO-injected embryos was increased toapproximately twice of control levels at stage 9. However, atstage 12 Chordin expression was greatly reduced by VegT-MO,
while Siamois expression was not changed at stage 12(Fig. 7H and I). One important experimental difference is that
Houston et al. (2002) used antisense DNA oligo depletion in oocytes, while we used an antisense morpholino injected intocleavage stage embryos. It has been observed previously thatthese two loss-of-function approaches result in similar, yet
not always identical phenotypes (Heasman et al., 2001). In
addition, the timing of the samples may have had slight differences (Fig. 7H and I). It should also be mentioned that previous
reports have shown that Chordin expression is activated by bCatenin in animal but not vegetal cells (Darras et al., 1997).
Taken together, the results presented in this section suggest that Siamois and Twin are regulated differently from Nieuwkoop center genes, since VegT represses them. Recently,
maternal B1-type Sox genes expressed in the animal cap havebeen shown to restrict Xnr expression, except for Xnr3, to thevegetal hemisphere (Zhang et al., 2004). Thus, it appears thatthe border between the BCNE and Nieuwkoop centers isestablished by the maternally encoded gene products VegT
and B1-Sox. Fig. 7J shows a model in which VegT and B1-Sox
are proposed to regulate the distinction between the BCNE
and Nieuwkoop centers within the dorsal region of stabilized
nuclear b-Catenin.

2.5.

The making of a brain

The role of the prospective neuroectoderm in amphibian neural induction has been the subject of much debate (Spemann,
1938; Holtfreter et al., 1955; Nieuwkoop and Koster, 1995; Kuroda et al., 2004). It has long been known that dorsal animal
cap explants respond much better than ventral ones to induction by dorsal organizer mesoderm and the mesoderm-inducer Activin (Sharpe et al., 1987; Sokol and Melton, 1991). In
addition, dorsal-animal blastomeres at the 8-cell stage thathave not received mesoderm induction signals, have a selfdifferentiation predisposition to become anterior neural tissue (Kinoshita et al., 1993).
The BCNE center is formed in cells that give rise to neuroectoderm, in particular brain, even in the absence of a mesodermal substratum. Extirpation and transplantation
experiments have shown that the BCNE center is requiredfor brain formation in the embryo (Kuroda et al., 2004). Recently, the requirement of Noggin expressed in the blastulaanimal cap ectoderm for neural differentiation has provided
independent support for our proposal that the BCNE in prospective neuroectoderm plays a critical role in the differentiation of the central nervous system in Xenopus (Huang et al.,2007). The results of the present study show that all known
BCNE center genes require the combined action of the homeobox genes Siamois and Twin. Thus, the process of brain induction can be traced back to the earliest stages of embryonicdevelopment, as is the case in other embryos such as the chick(reviewed in Stern, 2006; De Robertis and Kuroda, 2004). Neuralinduction is not an event that occurs at gastrula as believed
classically (Spemann, 1938), but rather starts with the corticalrotation of the 1-cell zygote, which stabilizes nuclear b-Catenin in the dorsal side of the embryo. The prospective neuroectoderm is activated by b-Catenin and repressed by VegT on thevegetal side (Fig. 7J). b-Catenin activates expression of Siamois
and Twin at mid-blastula, causing the repression of Bmp4 transcription (Baker et al., 1999; Leung et al., 2003), as well as theexpression of the Bmp-antagonists Chordin and Noggin in theprospective neuroectoderm (Kuroda et al., 2004; Huang et al.,2007). Using morpholino knockdown we have demonstrated
here that the Siamois and Twin homeobox proteins are required for the expression of BCNE center genes such as Chordin, Noggin, FoxA4a/pintallavis/HNF3b, Xnr3, and Admp, which
in turn participate in the formation of the vertebrate brain.
In addition, we showed that Siamois and Twin are also required for the dorsalizing effects of LiCl treatment of early
Xenopus embryos. In conclusion, Siamois and Twin represent
the main output of b-Catenin in the future neuroectoderm at
the blastula stage, and these homeobox genes play a key roleas determinants of brain formation in Xenopus.

3.

Experimental procedures

3.1.

Embryo manipulations

Xenopus laevis embryos were obtained by in vitro fertilization(Sive et al., 2000). Animal cap assays were performed in 1·
Steinberg’s solution (58 mM NaCl, 0.67 mM KCl, 0.83 mM
MgSO4, 0.34 mM Ca(NO3)2, 4.6 mM Tris–HCl, pH 7.4, 100 mg/L
Kanamycin) at stage 9 (7 h after fertilization at 22 C). PCR
conditions and primers, as well the protocol for whole-mountin situ hybridization, are described in http://www.kurodalab.com.

3.2.

RNA and morpholino injections

To generate synthetic mRNAs, pRN3-Siamois was linearizedwith Sfi1 and transcribed with T3 RNA polymerase (Lemaire
et al., 1995), pST64T-Twin with Sal1 and SP6 polymerase (Laurent et al., 1997), and pCS2-Chd, pCS2-Wnt8, and pCS2-VegTwith
NotI and SP6 RNA polymerase. MOs for Siamois, Twin, Chordin,
Noggin, and VegTwere ordered from Gene Tools, LLC. They consisted of the following sequences; Sia-MO: 5 0 -TTCAGCCTCATAGGTCATGTCTGTC-3 0 (this work), Twn-MO: 5 0 -TTCAGAGTCACAAGTCATGTCTGTG-3 0 (this work), b-Cat-MO: 5 0 -TTTCAAC
CGTTTCCAAAGAACCAGG-3 0 (Kuroda et al., 2004), Chd-MO1: 5 0 ACGTTGTGTGTGGTATAGTGAGGGT-3 0 and Chd-MO2: 5 0 -ACAG
GATTTTTGTGGTTGTCCCGAA-3 0 (Oelgeschläger et al., 2003),
Nog-MO: 5 0 -TCACAAGGCACTGGGAATGATCCAT-3 0 (Kuroda
et al., 2004), VegT-MO: 5 0 -CCCGACAGCAGTTTCTCATTCCAGC3 0 (Heasman et al., 2001). MOs were resuspended in sterilewater to a concentration of 1 mM each and then further diluted
to give working solutions.

3.3.

In vitro translation

Siamois and Twin proteins were in vitro translated using TnT
T3/SP6 Coupled Transcription/Translation System (Promega)M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 –6 6in the presence of Sia-MO or Twn-MO and [35S]-methionine,
according to the manufacturer’s instructions. Half of eachreaction was analyzed by 8% SDS–PAGE and visualized byautoradiography.

Acknowledgements
We thank P. Lemaire, K.W.Y. Cho, R. Moon, C. Wylie, and T.
Hayata for materials; V. Sander and members of the Kuroda
laboratory for comments on the manuscript. This work was
supported by the Research Reports of Uehara Memorial Foundation and the Sumitomo Foundation, NIH Grant HD21502-21,
and the Howard Hughes Medical Institute.

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