PLoS BIOLOGYNeural Induction in Xenopus: Requirement
for Ectodermal and Endomesodermal Signals
via Chordin, Noggin, b-Catenin, and Cerberus
Hiroki Kuroda, Oliver Wessely, E. M. De Robertis*
Department of Biological Chemistry, Howard Hughes Medical Institute, University of California, Los Angeles, California, United States of America

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing questionin vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at theblastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop
center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula
Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective
neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early b-Catenin signal.
Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the
Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap
cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive
neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE
region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Celllineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior
regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos(MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short[CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator b-Catenin. When mesoderm
involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived
from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into
prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cellstage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in
Xenopus in which an early blastula b-Catenin signal predisposes the prospective neuroectoderm to neural induction by
endomesodermal signals emanating from Spemann’s organizer.

Introduction
Vertebrate development results from a series of cell–cellinteractions in which groups of cells induce their neighbors
to acquire new cell differentiation fates. This process, known
as embryonic induction, was ﬁrst reported for the inductionof the lens in surface ectoderm by the optic vesicles
originating from the brain (Spemann 1901; Lewis 1904).
Subsequent work showed that the surface ectoderm itself also
plays an important role (reviewed by Grainger 1992). From
the analysis of lens induction, Spemann (1938) proposed thata double assurance mechanism (doppelte Sicherung) could
provide a way of explaining the robustness of vertebrate
development via reciprocal interactions between two layersof cells. Lens induction is an example of a secondary
embryonic induction. Most experimental embryologists concentrated their research on the induction of the neural plate,which is considered the primary embryonic induction(Spemann 1938; Saxén and Toivonen 1962; Harland 2000;
Gilbert 2001; Stern 2002). In the classical organizer transplantation experiment, Spemann and Mangold (1924) demonstrated that dorsal lip mesoderm is sufﬁcient to induce thedifferentiation of a complete central nervous system (CNS) in
responding ectoderm. Spemann devoted an entire chapter ofhis book to the discussion of whether a double assurance
mechanism existed in the case of neural plate induction(Chapter 8 in Spemann 1938) and concluded that the
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evidence supported a role for the underlying mesoderm,
but not for the prospective neuroectoderm.
A role for the gastrula ectoderm in neural plate formation
had been proposed on the basis of experiments in which themesoderm or the ectoderm had been damaged (Goerttler
1925) and received some subsequent support (Lehmann1928). However, further consideration of the possible roleof ectoderm in neural plate formation was hampered by ahighly inﬂuential exogastrulation experiment performed inaxolotl embryos (Holtfreter 1933), in which endomesoderm
Received November 28, 2003; Accepted January 29, 2004; Published May 11,
2004
DOI: 10.1371/journal.pbio.0020092
Copyright: Ó 2004 Kuroda et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use of, distribution, and reproduction in any medium, provided
the original work is properly cited.
Abbreviations: b-cat-MO, b-catenin morpholino oligos; BCNE, blastula Chordin- andNoggin-expressing region, BDA, biotin-dextran amine; BMP, bone morphogenetic
protein; Cer-MO, Cerberus morpholino oligos; CerS, Cerberus-Short; Chd-MO, Chordin
morpholino oligos; CNS, central nervous system; FGF, fibroblast growth factor;
GSK3, glycogen synthase kinase-3; IGF, insulin-like growth factor; LiCl, lithium
chloride; MO, antisense morpholino oligo; MAPK, mitogen-activated protein kinase;
NCAM, neural cell adhesion molecule; Nog-MO, Noggin morpholino oligos; ODC,
ornithine decarboxylase; SS, stock solution; Xnr, Xenopus Nodal-related
Academic Editor: Christof Niehrs, Deutsches Krebsforschungszentrum*To whom correspondence should be addressed. E-mail: derobert@hhmi.ucla.edu

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involution was prevented and the entire ectoderm differentiated into epidermis. Since there was no trace of CNS
tissue in these embryos, this experiment was interpreted as ademonstration that the underlying endomesoderm had theessential role in neural plate induction and that theprospective neuroectoderm had none (Holtfreter 1933;
Spemann 1938). The debate concerning whether the ectoderm itself has a role in neural plate formation has continued
to this day. In dorsal marginal zone explants (Keller and
Danilchik 1988; Keller 1991), CNS differentiation can takeplace in the absence of underlying mesoderm. It has been
proposed that in these Keller explants neural tissue induction
results from a ‘‘planar’’ signal that diffuses in the plane of theectoderm from the mesodermal organizer at gastrula (Ruiz i
Altaba 1992, 1993; Doniach et al. 1992; Poznanski and Keller1997) (see Figure 6A). However, the existence of planar neuralinduction signals has been disputed, with neural induction in
Keller explants proposed to result from ‘‘vertical’’ signals
resulting from a brief contact between ectoderm andmesoderm at early gastrula (Nieuwkoop and Koster 1995).
Therefore, a central question remains unanswered despite
many decades of research in amphibian neural induction:
What is the differentiation potential of the presumptive
neural plate material in the absence of a mesodermal
substratum? This is the problem addressed here.
Two recent technical advances led us to reinvestigate
neural induction in Xenopus. First, it is now possible tocompletely inhibit mesoderm formation by microinjecting
Cerberus-short (CerS) mRNA, a secreted antagonist speciﬁc for
Nodal-related mesoderm inducers (Agius et al. 2000). Interestingly, Xenopus embryos lacking mesoderm still developed a
CNS, including a cyclopic eye (Wessely et al. 2001). This was
surprising, because such mesoderm-less embryos did not
express multiple Spemann organizer markers such as Chordin(Chd), Noggin (Nog), and Goosecoid in dorsal endomesoderm at
the gastrula stage. Second, a technical revolution has takenplace with the availability of antisense morpholino oligos(MO) that permit loss-of-function studies in Xenopus (Heasman et al. 2000). It is now possible to combine the tools ofamphibian experimental embryology with investigations on
the role of individual genes, such as the secreted bonemorphogenetic protein (BMP) antagonist Chd (Oelgeschläger
et al. 2003) or its upstream regulator b-Catenin (Heasman etal. 2000), in experimentally manipulated embryos.
In whole embryos injected with Chd-MO, a CNS, althoughof reduced size, still develops. However, Spemann organizers
depleted for Chd lose all neural-inducing activity whengrafted to the ventral side of a host embryo (Oelgeschläger
et al. 2003). Surprisingly, when similar Chd-depleted grafts
are placed on the dorsal side, ectodermal cells lose the ability
to contribute to neural plate (Oelgeschläger et al. 2003). Thissuggested that a cell-autonomous requirement of Chd forneural plate formation might exist in the ectoderm itself. At
the blastula stage, the BMP antagonists Chd and Nog are
expressed in the dorsal animal cap and marginal zone, in aregion we had originally designated as the ‘‘preorganizer
center’’ (Wessely et al. 2001). This group of cells constitutes a
blastula Chordin- and Noggin-expressing (BCNE) region that
contains both prospective neuroectoderm cells and Spemannorganizer precursors. The BCNE region also expresses
Xenopus Nodal-related 3 (Xnr3), a secreted factor withneural-inducing properties that is expressed at high levels
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in early Xenopus embryos (Haramoto et al. 2004; Wessely et al.
2004). The early phase of expression of Chd and Nog in BCNE
cells is regulated by the dorsal accumulation of b-Catenin,
whereas later expression of the same genes in Spemannorganizer endomesoderm requires in addition Nodal-related
signals that can be blocked by CerS (Wessely et al. 2001).
In this study we analyze the mechanism of neural inductionin Xenopus by means of embryological cut-and-paste andmolecular loss-of-function experiments. We ﬁnd that theBCNE center contains much of the presumptive anterior
CNS. Loss-of-function studies show that gene products
expressed at blastula—such as Chd, Nog, and b-Catenin—
are required for neural induction in the absence of underlying endomesoderm. Cell-lineage studies show that theBCNE center itself gives rise to brain, notochord, and ﬂoor
plate. Transplantation experiments show that the BCNE
center is required for brain formation in Xenopus embryos.
Microinjection experiments at the 8-cell stage, in which ChdMO was injected into dorsal-animal and Cer-MO into dorsalvegetal blastomeres, conﬁrmed that secreted signals fromboth prospective neuroectoderm and underlying endomesoderm are required for anterior CNS development. The results
support a double assurance mechanism for brain formationof the type proposed by Spemann (1938) for lens induction.

Results
The BCNE Center Is Distinct from the Nieuwkoop Center
The initial asymmetry in Xenopus development is caused bya cortical rotation triggered by sperm entry, thought toredistribute ‘‘dorsal determinants’’ that in turn stabilize bCatenin protein on the dorsal side of the embryo (Figure 1A)(reviewed in Gerhart et al. 1991; Harland and Gerhart 1997;
De Robertis et al. 2000). At the blastula stage, the Nieuwkoop
center located in dorsal-vegetal cells secretes mesoderminducing signals such as Xnr1, Xnr2, Xnr4, Xnr5, and Xnr6 thatinduce formation of the gastrula Spemann organizer inoverlying mesoderm cells (Agius et al. 2000; Takahashi et al.
2000). The Nieuwkoop center has also been called the‘‘blastula organizer’’ in the early literature (Gerhart et al.
1991). The BCNE region develops in the dorsal animal andmarginal region. In situ hybridization analyses at the blastula
stage (7 h after fertilization) showed that the neural-inducing
secreted factors Chd, Nog, and Xnr3 are expressed in the
animal cap, in a region that includes about 45o of arc above
the ﬂoor of the blastocoel, as well as in the dorsal marginalzone (Figure 1B–1D). At the gastrula stage, the same genes are
expressed in more vegetal regions, in the Spemann organizer
located in the dorsal endomesoderm of the marginal zone
(e.g., Figure 2E).
The question arises as to whether two distinct signaling
centers coexist in the Xenopus blastula. To address this, earlyblastulae with strong dorsoventral polarity (Klein 1987) weredissected into six fragments, as shown in Figure 1E. The
results showed that, although some overlap existed, theregion expressing Chd and Nog included the animal cap,
whereas the Nieuwkoop center region that expresses Xnr2
and Xnr6 had a more vegetal location (see Figure 1E). Xnr3
expression was observed in fragments 3 and 4, indicating ahigher degree of overlap (see Figure 1E). In addition, theresults showed that the homeobox gene Siamois was expressedin the BCNE region, even though its expression has been
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Neural Induction in Xenopus
Figure 1. Two Signaling Centers Coexist in
the Xenopus Blastula(A) Diagram of early events between 1cell stage and early blastula.
(B–D) Expression of Chd, Nog, and Xnr3
transcripts just after midblastula transition (7 h postfertilization). Embryos were
hybridized as whole mounts, stored inmethanol for 1 mo at room temperature
to improve contrast, and sectioned witha razor blade.
(E) RT-PCR analysis of gene markers at
midblastula, early stage 9. Six samples
were prepared by dissections of blastula
regions as shown in the diagram.
(F) Summary of gene expression atblastula. The BCNE center expresses
Chd, Nog, Siamois, and Xnr3, while the
Nieuwkoop center expresses Xnr2, Xnr6,
and Cer.
DOI: 10.1371/journal.pbio.0020092.g001

reported to be more vegetal at later stages of development(Lemaire et al. 1995). We also found that Cerberus (Cer), a gene
expressed in anterior endoderm at gastrula (Bouwmeester etal. 1996), was a component of the Nieuwkoop center. We
conclude that two distinct signaling centers are present atblastula (see Figure 1F). The more animal BCNE center
expresses Chd, Nog, Xnr3, and Siamois, whereas the Nieuwkoop
center expresses Xnr2, Xnr6, and Cer.

Cell Lineage of the BCNE Region
To map the fate of the blastula Chd- and Nog-expressing
cells during normal development, we transplanted lineagelabeled BCNE regions isotopically into host blastulae at earlystage 9 (Figure 2A). These grafts containing the lineage tracer
biotin-dextran amine (BDA) marked the Chd-expressing
region at blastula (compare Figure 2B and 2C). A few hours
later, at early gastrula (stage 10.5), dorsal animal cap
descendants were found both in organizer endomesoderm
and in prospective neuroectoderm (Figure 2D). We note thatby early gastrula stage Chd mRNA was expressed in organizer
endomesoderm, but was no longer detectable in prospective
neuroectoderm (Figure 2E). At midgastrula, stage 11, thetransplanted tissue elongated in organizer endomesoderm
and prospective neuroectoderm, with both layers remaining
in close apposition (Figure 2F). At neural plate stages, stage
14, BCNE center descendants were found in a wide region in
the anterior neural plate and, more posteriorly, in a narrow
stripe in the midline (Figure 2G). Double staining using
nuclear lacZ mRNA as lineage tracer in combination with an
epidermal cytokeratin marker conﬁrmed that BCNE cells giverise to anterior neural plate (Figure 2H). The midline staining
in the trunk region corresponded to ﬂoor plate andnotochord in histological sections (Figure 2I). At tadpole
stage (stage 40), BCNE descendants contributed to a largepart of the brain and retina (but not lens and otic vesicles)
and to dorsal midline structures of the trunk-tail region(Figure 2J–2L). This lineage could be traced up to feeding
tadpole stages (Figure 2M and 2N) using dorsal animal cap
grafts from cytomegalovirus–green ﬂuorescent protein transgenic embryos (Marsh-Armstrong et al. 1999). These results
indicate that blastula Chd-expressing cells give rise to much of
the brain and to the ﬂoor plate and notochord in the trunk
region of the Xenopus embryo.
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The Dorsal Animal Cap Is Specified to Form CNS
In embryology, the test of whether cells are speciﬁed toform a particular tissue is to culture them in isolation fromthe rest of the embryo. Dorsal animal cap explants fromembryos injected with CerS mRNA expressed multiple neuralmolecular markers at stage 26, whereas animal or ventral
explants did not (Figure 3A and 3B). CerS was required toinhibit mesoderm formation; when identical explants wereprepared without CerS mRNA injection, mesoderm contamination from the marginal zone was detected (data notshown). Neural differentiation could also be obtained in theabsence of CerS mRNA when additional care was taken to
avoid mesodermal contamination. As shown in Figure 3C,
small explants from the top half of the BCNE region were
excised, sandwiched together, and cultured in saline solution
for 3 d. The sandwich procedure allows such small explants tosurvive in culture for long periods of time. Dorsal BCNE
explants differentiated into histotypic CNS, including grayand white matter (Figure 3D), whereas similar explants fromventral ectoderm differentiated into epidermis (Figure 3E).
These results demonstrate that dorsal animal cap cells are
already speciﬁed to form CNS at the blastula stage.

BCNE Tissue Is Required for Brain Formation
To test whether the BCNE center is required for brain
formation, we ﬁrst deleted ventral or dorsal regions of theanimal cap. Deletion of the dorsal region, but not of theventral animal cap, resulted in headless embryos (Figure 4A
and 4B). Since Xenopus is one of the best-studied vertebrate
embryos, it was surprising that this requirement of a region of
the blastula for CNS formation had not been reported
previously. To investigate this further, we replaced thedeleted fragment with various ectodermal grafts. The brain
defects could be rescued by transplantation of dorsal, but notventral, animal cap grafts (Figure 4C and 4D). Ectoderm fromthe animal pole was unable to rescue the ablated dorsal
animal cap (Figure 4F). However, similar animal poles fromlithium chloride (LiCl)–treated embryos, in which b-Catenin
is stabilized and transcription of BCNE genes activated, wereable to rescue head formation (Figure 4E).
Despite this requirement for brain development, blastula
dorsal animal caps grafted into the ventral side of a hostblastula were only able to form weak secondary axes (Figure
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Neural Induction in Xenopus

Figure 2. The BCNE Center Contributes to Forebrain and Midline Structures(A) Method used for lineage tracing of the BCNE center with biotin-dextran amine (BDA) labeled grafts.
(B) Sagittal section of a recently grafted BCNE at stage 9.
(C) Chd mRNA expression at stage 9.
(D) BCNE descendants at stage 10.5.
(E) Chd mRNA expression at stage 10.5.
(F) BCNE center descendants at stage 11.
(G) Dorsal view of BCNE descendants at neural plate stage 14.
(H) Double staining of transplanted BCNE region with nuclear lacZ mRNA and epidermal ectoderm of the host with epidermal cytokeratin (epi)probe in light red at stage 14.
(I) Transverse section at the level of the trunk at stage 16. Abbreviations: fp, ﬂoor plate; no, notochord.
(J–L) Transverse sections at stage 40. Abbreviations: fp, ﬂoor plate; hb, hindbrain; he, heart; le, lens; mb, midbrain; no, notochord; ov, otic vesicle;
re, retina.
(M) Dorsal view of 6-d embryo transplanted with a BCNE graft from CMV-GFP transgenic embryos. Abbreviations: br, brain; fp, ﬂoor plate; on,
optic nerve; op, olfactory placode.
(N) Side view at 4 d showing labeled retina and brain. Abbreviation: br, brain.
DOI: 10.1371/journal.pbio.0020092.g002

4G). Chd-MO, which blocks the activity of Spemann grafts(Oelgeschläger et al. 2003), also inactivated BCNE grafts (Figure
4H). However, an important difference with the mature
Spemann organizer was that BCNE cell transplants self-differentiated into spinal cord and muscle in these weak axes andwere unable to induce CNS in neighboring cells as the Spemannorganizer does (data not shown). We conclude that the dorsal
animal cap BCNE center is required for brain formation.
However, when transplanted into ectopic sites, BCNE tissue has
only weak effects and does not induce neural tissue.

Anterior CNS Formation in the Absence of Mesoderm
Requires Chd and Nog
We next investigated whether BCNE center signals are
required for the anterior CNS that forms in embryos lacking
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mesoderm and Spemann organizer. CerS mRNA was injected
at the 4-cell stage and the BCNE region marked with BDA atthe 64-cell stage (Figure 5A; see also Figure S1). These
mesoderm-less embryos developed forebrain tissue andprominent cyclopic eyes, which were derived from thelineage-labeled BCNE cells (Figure 5B and 5C). To test
whether there was a requirement for Chd in these embryos,
we injected Chd-MO at the 2-cell stage. When Chd was
knocked down, BCNE cells developed into epidermis insteadof CNS (Figure 5D and 5E). Brain and eye formation could be
rescued by overexpression of Chd mRNA lacking the region
targeted by Chd-MO (Figure 5F and 5G).
Molecular analyses conﬁrmed that mesoderm-less embryos
injected with Chd-MO did not express anterior neural tissue
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Neural Induction in Xenopus

show that the brain tissue formed in embryos lacking
mesoderm and Spemann organizer derive from BCNE cells.
The formation of anterior CNS in mesoderm-less embryos
requires the expression of Chd and Nog in prospective
neuroectoderm.

Neural Induction by b-Catenin Requires Chd

Figure 3. The Blastula Dorsal Animal Cap Is Specified to Form CNS(A) Experimental diagram showing embryos injected with CerS mRNA
from which three regions of the animal cap were dissected at blastula,cultured until stage 26, and processed for RT-PCR. The size of the
explants was 0.3 mm by 0.3 mm in these samples. Abbreviations: A,
animal pole; D, dorsal region; V, ventral animal cap.
(B) RT-PCR analysis of animal cap fragments; note that anterior brain
markers were expressed in the dorsal fragments in the absence of
mesoderm (a-actin) and endoderm (endodermin, Edd) differentiation.
Abbreviations: A, animal pole; D, dorsal region; V, ventral animal cap.
(C) Experimental diagram of the small animal cap sandwich experiments; these embryos were not injected with CerS. In this case, the sizeof the explants was 0.15 mm by 0.15 mm leaving a 0.15-mm gap fromthe ﬂoor of the blastocoel to avoid contamination from mesodermforming cells. Fragments from two explants were sandwiched
together (explants are too small to heal by curling up) and cultured
in 13 Steinberg’s solution until stage 40. Abbreviations: VSW, ventral
sandwich; DSW, dorsal sandwich.
(D) Histological section of dorsal animal cap explant (dorsal
sandwich). These sandwiches differentiated into histotypic forebrain
tissue including white and gray matter (4/17). Abbrevations: DSW,
dorsal sandwich; gm, gray matter; wm, white matter.
(E) Histological section of a ventral animal cap sandwich. All
sandwiches differentiated into atypical epidermis (n = 20). Abbreviations: ae, atypical epidermis; VSW, ventral sandwich.
DOI: 10.1371/journal.pbio.0020092.g003

markers such as Otx2, Rx2a, En2, and Krox20 (Figure 5H,
compare lanes 3 and 4). However, spinal cord (HoxB9) or panneural markers (N-tubulin, neural cell adhesion molecule[NCAM]) were still expressed, indicating that only anterior
neural differentiation was eliminated by Chd-MO and thatposterior neural induction continues to take place. We also
generated a Noggin antisense morpholino oligo (Nog-MO)
reagent, which, like Chd-MO, inhibited brain markers (Figure
5H, lane 5). Nog-MO was slightly weaker than Chd-MO, buteven a combination of both morpholinos did not eliminate
posterior neural markers (Figure 5H, lane 6). These results
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It has recently been discovered that microinjection of bcatenin mRNA is able to induce neural tissue in Xenopus animal
caps (Baker et al. 1999). Stabilization of b-Catenin has a dual
effect, inhibiting the transcription of BMPs (Baker et al. 1999;
Leung et al. 2003) and increasing expression of the BMP
antagonists Chd and Nog in the blastula animal cap (Wessely et
al. 2001). We next tested the effect of b-Catenin knockdown
on CNS differentiation. As shown in Figure 5I and 5J, b-catMO oligos (Heasman et al. 2000) blocked formation ofhistological anterior brain and eye structures in CerS
mesoderm-less embryos. Importantly, anterior CNS formation could be restored by overexpression of either b-catenin or
Chd mRNA in these embryos (Figure 5K–5N). We conclude
that brain formation in the absence of mesoderm requires
the early b-Catenin signal.
To investigate whether neural induction by b-Catenin inanimal cap explants required Chd, the b-Catenin pathway wasactivated by b-catenin mRNA, dominant negative glycogensynthase kinase-3 (dnGSK3) mRNA, or LiCl. These treatments
induced multiple neural markers in animal caps (Figure 5O,
lanes 3–5). Microinjection of Chd-MO inhibited the expression of anterior neural markers (Six3, Otx2, Rx2a, En2), but notof posterior or pan-neural ones (HoxB9, NCAM) (Figure 5O,
lanes 6–8). The results indicate that neural induction by theb-Catenin signal requires expression of its downstream target
gene Chordin.

Anterior Neural Induction in Keller Explants Requires Chd
Is the expression of Chd in prospective neuroectoderm atblastula responsible for the ‘‘planar’’ neural induction signals(Figure 6A) described by earlier workers? To investigate this,we used Keller sandwiches (Keller and Danilchick 1988;
Doniach et al. 1992; Ruiz i Altaba 1992), in which neural tissue
develops without contacting underlying mesoderm (Figure
6B). Marking of the BCNE region with lineage tracer
indicated that Keller sandwiches contain cells that expressed
Chd in prospective neuroectoderm at blastula (Figure 6C).
Keller sandwiches expressed anterior CNS gene markers(Figure 6D and 6F). However, explants prepared fromembryos injected with Chd-MO failed to express Otx2 orKrox20 in anterior neuroectoderm, while retaining Otx2
expression in endoderm (compare Figure 6D and 6E). Ntubulin expression, which marks differentiated neurons, wasinhibited by Chd-MO in the anterior CNS, but persisted inprospective spinal cord (Figure 6F and 6G; results summarized in Figure 6H and 6I). These results show that theanterior CNS formation observed in Keller explants lacking
underlying mesoderm requires Chd.
Molecular analyses of Keller explants conﬁrmed that brain
markers were inhibited by Chd-MO, while pan-neural andspinal cord markers were less affected (Figure 6J, compare
lanes 2 and 3). As before, posterior neural induction did not
require Chd. The origin of this posterior neural differentiation is due to ﬁbroblast growth factor (FGF) signaling(Hongo et al. 1999; Pera et al. 2003), since it could be blocked
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Neural Induction in Xenopus
Figure 4. The Dorsal Animal Cap Is
Required for Brain Formation(A) Ventral animal cap deletion (DV)produces a normal embryo.
(B–F) Dorsal animal cap deletion (DD)results in loss of anterior brain structure.
The headless phenotype of dorsal animal
cap deletions was rescued by dorsal
animal cap grafts (C) and animal pole
grafts obtained from LiCl-treated embryos (E), but not by ventral animal cap
transplants (D) or animal pole transplants (F). The average dorso-anterior
indices (DAI) were 4.89 ([A] n = 28), 3.52([B] n = 25), 4.90 ([C] n = 10), 3.63 ([D] n= 19), 4.90 ([E] n = 12), and 3.50 ([F] n =10).
(G) Transplantation of the dorsal animal
cap into the ventral animal cap region ofa host embryo induced weak secondary
axes (65.4%, n = 26). The embryo shown
here was one of the strongest axes
obtained.
(H) Activity of BCNE transplanted ventrally was blocked by Chd-MO (n = 15).
DOI: 10.1371/journal.pbio.0020092.g004

in explants injected with dominant negative FGF receptor 4a(dnFGFR4a) mRNA (Figure 6J, lanes 4 and 5). Importantly,
anterior CNS markers were still expressed in Keller sandwiches when mesoderm induction was blocked by CerS mRNA
(Figure 6J, lane 6) and could be blocked by Chd-MO (Figure 6J,
lane 7). Since mesoderm-less CerS Keller explants lack an
endomesodermal Spemann organizer, their sole source of
Chd is the BCNE center. Taken together, these experiments
indicate that the anterior neural induction observed in Keller
explants, known as ‘‘planar’’ induction, results from theactivity of Chd-expressing cells located in the presumptive
neuroectoderm at the blastula stage.

Chordin and Cerberus Cooperate in Brain Induction
Do vertical signals from endomesoderm cooperate with the
BCNE center in brain differentiation? The endomesoderm
secretes growth-factor antagonists with head-patterning
activity, such as Cer, Frzb-1, Crescent, Dickkopf-1, Chd, andNog (Harland 2000; De Robertis et al. 2000). Several of these
secreted antagonists are expressed in the anterior endoderm,which is homologous to the mouse anterior visceral endoderm (Beddington and Robertson 2000). We chose to study
one of these antagonists, the head-inducer Cer, because it isexpressed in the anterior endoderm of the Spemannorganizer (Bouwmeester et al. 1996) and in the Nieuwkoop
center, but not in the BCNE center (see Figure 1E).
Two recent studies have described morpholino antisense
oligos targeting Cer. In both, Cer did not appear to berequired for head development on its own, but cooperated
when coinjected with other factors (Hino et al. 2003; Silva et
al. 2003). Xenopus laevis genes frequently have pseudoalleles
thought to have originated from hybridization between two
different Xenopus species in the course of evolution (Kobel
and Du Pasquier 1986). Examination of the EST database
showed that a second Cer allele existed, and that the publishedmorpholinos had three and four mismatches with it,
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respectively (Figure 7A) (Silva et al. 2003; Hino et al. 2003).
We therefore designed a new morpholino oligo, Cer-MO,
targeting both X. laevis pseudoalleles (Figure 7A). Cer-MO
inhibited head formation in Xenopus embryos, which could be
rescued by Cer mRNA lacking the targeted 59-leader sequence(data not shown).
To test whether Cer and Chd cooperated, we targeted CerMO to dorsal endomesoderm and Chd-MO to dorsal neuroectoderm at the 8-cell stage (Figure 7B). At the doses used,microinjection of Chd-MO or Cer-MO alone resulted in partial
reductions of the anterior CNS (Figure 7C–7H). However,when neuroectodermal and endomesodermal progenitor
cells were injected with Chd-MO and Cer-MO, respectively,
brain differentiation did not occur (Figure 7I and 7J). In
histological sections, these embryos lacked brain structures
but still developed spinal cord, somites, and notochord (datanot shown). The results suggest that formation of the brain
requires partly overlapping distinct factors in different cell
layers. Chd is required in prospective neuroectoderm topredispose cells to form anterior CNS and cooperates withvertical signals from the underlying endomesoderm that
include Cer (Figure 8).

Discussion
The results presented here are consistent with thefollowing sequence of events during CNS development in
Xenopus. A dorsal b-Catenin signal triggered by the earlycortical rotation of the egg (Gerhart et al. 1991; De Robertis
et al. 2000) induces the expression of anti-BMP molecules
such as Chd and Nog in a group of cells located in the dorsal
animal region at the blastula stage (see Figure 8). The dorsal
prospective neuroectoderm is already speciﬁed to form CNS
at blastula (see Figure 3). Remarkably, transplantation studies
showed that this BCNE center was required for brain
formation in vivo (see Figure 4). The normal fate of theseMay 2004 | Volume 2 | Issue 5 | Page 0628

Neural Induction in Xenopus
Figure 5. The CNS of Mesodermless
Embryos Derives from BCNE Cells andRequires Chd, Nog, and b-Catenin(A) Experimental design. Embryos inwhich mesoderm induction was inhibited (by injection of 600 pg of CerSmRNA into the vegetal pole) weresectioned at stage 38 and stained withhematoxylin-eosin or for microinjected
BDA lineage tracer marking the BCNE
region.
(B and C) Embryos injected with CerSmRNA alone (n = 40). Abbreviation: br,brain.
(D and E) Embryos injected with 17 ng of
Chd-MO in addition to CerS (n = 21).
Abbreviation: epi, epidermis.
(F and G) Coinjection of 17 ng of ChdMO and CerS, followed by 100 pg of Chd
mRNA together with the lineage tracer(n ¼ 19). Abbreviation: br, brain.
(H) Expression of anterior CNS markers
in mesodermless embryos requires Chd
and Nog. RT-PCR analysis of CerS
mRNA–injected embryos at tailbud stage
26. Markers of anterior brain (Otx2), eye(Rx2a), midhindbrain boundary (En2),
hindbrain (Krox20), and cement gland(XAG) were inhibited by injection of ChdMO, Nog-MO, or both. A pan-neuralmarker (NCAM) and a neuronal marker(N-tubulin) were partially inhibited, andthe posterior neural marker HoxB9 was
not affected. a-actin serves as a mesoderm marker to show that CerS blocked
mesoderm in these embryos and ODC asmRNA loading control. The effects of
the Nog-MO described here can be
rescued by full-length Nog mRNA lacking
the 59 leader sequence targeted by theantisense morpholino (data not shown).
(I and J) b-cat-MO (13.6 ng) together with
CerS mRNA (n = 15). Abbreviation: epi,
epidermis.
(K and L) Rescue of b-cat-MO by 800 pg
of b-catenin mRNA. Abbreviation: br,brain.
(M and N) Rescue of the b-cat-MO
phenotype by 100 pg of Chd mRNA (n¼ 8).
(O) Chd is required for the anterior
neural induction caused by b-Catenin.
Neural and cement gland markers were
induced in animal cap explants byactivation of b-Catenin signal by the
injection of 600 pg b-catenin mRNA,
dnGSK3 mRNA, or LiCl treatment (lanes
3–5). Markers of anterior brain (Six3,
Otx2), eye (Rx2a), midhindbrain boundary (En2), hindbrain (Krox20), and cement gland (XAG) were inhibited by ChdMO (lanes 6–8). Although inhibition was
not detected for the posterior neuralmarker HoxB9 and the pan-neural marker NCAM, the neuronal marker N-tubulin was inhibited. a-actin and a-globin are dorsal and ventral
mesoderm markers, respectively, used to show the absence of mesoderm formation, and ODC serves as loading control.
DOI: 10.1371/journal.pbio.0020092.g005

dorsal animal cap cells during development is to give rise toanterior CNS, ﬂoor plate, and notochord (see Figure 2). We
note that our previous term ‘‘preorganizer’’ (Wessely et al.
2001) was somewhat inadequate. Since BCNE grafts areunable to induce CNS in neighboring cells after transplantation, they lack organizer activity.
The Nieuwkoop center arises at the same stage as the BCNE
center, but in more vegetal cells (see Figure 8). The
PLoS Biology | http://biology.plosjournals.org

Nieuwkoop center expresses secreted factors such as mesoderm-inducing Xnrs and Cer and in later development gives
rise to the endoderm that underlies the anterior CNS (seeFigure 8). By inhibiting Chd in presumptive neuroectoderm
and Cer in the endomesodermal substratum (see Figure 7), wewere able to provide evidence that partly overlapping
functions of distinct growth-factor antagonists secreted bydifferent germ layers cooperate in CNS formation.
May 2004 | Volume 2 | Issue 5 | Page 0629

Neural Induction in Xenopus

Figure 6. Anterior Neural Induction in Keller Explants Requires Chd(A) Proposed vertical and planar signals in neural induction (following Ruiz i Altaba 1993).
(B) Diagram of Keller explant preparation and subsequent elongation of the endomesoderm by convergent extension (Keller 1991).
(C) The neural and mesodermal regions of Keller explants contain descendants of BCNE cells (in blue) marked by blastomere injection at the 64cell stage.
(D) Expression of Otx2 and Krox20 in Keller explants (n = 7).
(E) Injection of 17 ng Chd-MO completely blocked Otx2 and Krox20 expression in neural regions, while expression of Otx2 in anterior endoderm
was not affected (n = 10).
(F) The differentiated neuron marker N-tubulin is expressed in Keller explants (n = 8).
(G) Partial inhibition of N-tubulin by injection of Chd-MO (n = 7).
(H and I) Summary of the effects of Chd-MO in Keller explants. Abbreviations: SC, spinal cord; CG, cement gland; Epi, epidermis.
(J) RT-PCR analyses of the effect of Chd-MO in Keller explants; samples injected with (plus) or without (minus) Chd-MO are indicated. Lane 1,
whole embryos; lanes 2–7, Keller sandwiches. Note that expression of the neural markers NCAM and N-tubulin in Keller sandwiches was abolishedby co-injection with 200 pg of dnFGF receptor 4a (dnFGF4a) mRNA and 17 ng of Chd-MO (lane 5). Injection with 600 pg of CerS mRNA, which
eliminates mesoderm but not BCNE formation, does not affect neural induction in this assay (lane 6).
DOI: 10.1371/journal.pbio.0020092.g006

Neural Induction Starts at Blastula
At the blastula stage, gene expression in the BCNE region
causes a neural predisposition in the prospective brain tissue
itself. When prospective neuroectoderm is explanted atblastula and cultured in the absence of mesoderm, it candevelop into histotypic neural tissue (see Figure 3D). Once
the Nieuwkoop center induces a Spemann organizer in dorsal
mesoderm, a cocktail of growth factor antagonists is secretedby the endomesoderm. These Spemann organizer molecules
require Nodal-related signals in order to be produced at
gastrula (Agius et al. 2000; Wessely et al. 2001). These
endomesodermal factors include Cer (an inhibitor of Nodal,
PLoS Biology | http://biology.plosjournals.org

Wnt, and BMP signals); anti-Wnts such as Frzb-1, Crescent,and Dickkopf, and anti-BMPs such as Follistatin, Chd, andNog (De Robertis et al. 2000). Some molecules, like Chd andNog, are expressed both in the dorsal animal cap at blastulaand in the Spemann organizer at gastrula, whereas others,
like Cer, are expressed only in the Nieuwkoop center and its
endomesodermal descendants. In amphibians, the ability of agastrula dorsal lip to induce a complete CNS when transplanted into a host gastrula had been taken as an indicationthat neural induction occurs at gastrula. We now show thatbrief expression of Chd and Nog during blastula stages,
triggered by the maternal b-Catenin signal, is required for
May 2004 | Volume 2 | Issue 5 | Page 0630

Neural Induction in Xenopus

Figure 8. Double-Assurance Model for Brain Formation by the BCNE andNieuwkoop Centers
Blastula Chd- and Nog-expressing cells are located in the dorsal animal
region, while the Nieuwkoop center is found in the dorsal-vegetal
region. At gastrula, the anterior endoderm derived from the
Nieuwkoop center is found in close apposition to the prospective
anterior CNS. See text for discussion.
DOI: 10.1371/journal.pbio.0020092.g008

Redundant Signals in Neural Induction

Figure 7. A Double-Assurance Mechanism in Xenopus Neural Induction
That Requires Chordin and Cerberus(A) A new Cer-MO is complementary to both Cer pseudoalleles, while
two MOs reported by other authors (Hino et al. 2003; Silva et al. 2003)
match only one allele, having three or four mismatches, respectively,
with the other allele. The Cer-MO used in the present study inhibits
head formation in intact embryos (data not shown), while the othertwo do not (Hino et al. 2003; Silva et al. 2003).
(B) Experimental procedure and cell lineages at 32-cell and earlygastrula (stage 10.5) for dorsal-animal (FDA, green) and dorsal-vegetal(TRDA, red) blastomeres microinjected at the 8-cell stage.
(C and D) Uninjected embryos.
(E and F) Dorsal-animal injection with 8.5 ng of Chd-MO alone
partially inhibited head formation; green ﬂuorescence was seen in
anterior CNS.
(G and H) Dorsal-vegetal injection with 17 ng of Cer-MO also
inhibited brain formation partially; red ﬂuorescence may be seen in
anterior endomesoderm.
(I and J) Injection with 8.5 ng Chd-MO dorsal-animally and 17 ng CerMO dorsal-vegetally blocked brain formation, but not spinal cord andsomites (histological sections not shown).
DOI: 10.1371/journal.pbio.0020092.g007

brain formation. This requirement becomes apparent when
additional signals from underlying endomesoderm areblocked by inhibition of Nodal signaling or by preventing
involution in Keller dorsal explants. We conclude that
Xenopus neural induction starts in presumptive neuroectoderm shortly after midblastula. This is in agreement withother recent work in Xenopus (Baker et al. 1999; Gamse andSive 2001; Wessely et al. 2001).
PLoS Biology | http://biology.plosjournals.org

Multiple secreted growth factors antagonists participate in
CNS induction (Harland 2000; De Robertis et al. 2000), andtheir activities can be redundant. In the mouse, Chd and Nog
mutants have normal neural plates, but in Chd-/-;Nog-/embryos, development of the forebrain fails (Bachiller et al.
2000). In Xenopus and zebraﬁsh, loss of Chd in the whole
embryo results in animals that still are able to form anterior
CNS, although its size is reduced (Schulte-Merker et al. 1997;
Oelgeschläger et al. 2003). This contrasts with the strong
requirement for Chd revealed here when neural induction isdriven by a single signaling center in Xenopus. In mesodermless embryos, (CerS-injected) blastula dorsal animal cells are
the sole source of Chd, and anterior CNS differentiation canbe completely inhibited by Chd-MO (see Figure 5). Chd is also
required for the neural inducing activity of Spemannorganizer grafts (Oelgeschläger et al. 2003). Multiple neuralinducing molecules, such as the above-mentioned growth
factor antagonists, have been identiﬁed and may compensate
for the loss of Chd in intact embryos. In addition other signals
located outside of dorsal signaling centers, such as FGFs andinsulin-like growth factors (IGFs) may participate in neuralinduction (see below).
Could redundant signals from prospective neuroectoderm
and endomesoderm also function in neural induction inother vertebrates? This seems possible in the case of the chickembryo. Chick Chd is initially expressed in the unincubated
egg in epiblast central cells just anterior to Koller’s sickle(Streit et al. 1998), a region that may correspond to the
Xenopus blastula Chd-expressing region. The progeny of thisregion of the chick epiblast contributes to the prospective
forebrain and moves anteriorly during development. The
descendants of the chick early Chd-expressing brain progenitors migrate at all times in front of the organizer, which islocated at the tip of the primitive streak and has a caudalizing
inﬂuence (Foley et al. 2000). In zebraﬁsh, mutant embryos
lacking Nodal signaling still form brain tissue in the absenceof a mesodermal organizer and express Chd (Gritsman et al.
1999). In the mouse embryo, transplantation experiments at
the gastrula stage support a role for different germ layers inbrain induction (Tam and Steiner 1999). However, expression
May 2004 | Volume 2 | Issue 5 | Page 0631

Neural Induction in Xenopus

of mouse Chd and Nog has only been analyzed from earlyprimitive streak stage on (Bachiller et al. 2000). Studies on theexpression of these BMP antagonists in prestreak or periimplantation mouse embryos, or on the earliest nuclear
localization of b-Catenin protein, will be required todetermine whether a region homologous to that of the
Xenopus blastula Chd-expressing region exists in mammalianembryos.

Neural-Inducing Signals in Chordates
In amphibians the default model of neural induction
proposes that BMPs expressed in ectoderm cause epidermal
induction. When animal cap cells are dissociated, they
become neuralized (reviewed by Weinstein and HemmatiBrivanlou 1999). When exogenous BMP is added to dissociated animal cap cells, epidermal differentiation is restored.
The present work with morpholinos that inhibit Chd, Nog,and Cer highlights the importance of BMP signaling
regulation in Xenopus. The BCNE center appears shortly after
midblastula and is required for anterior CNS formation whenendomesodermal signals are inhibited. Induction of posterior
neural tissue can still take in the absence of Chd and Nog (e.g.,
Figure 5H). Formation of this posterior neural tissue can beblocked by dominant-negative FGF receptor 4a (see Figure6J). In Xenopus, FGF and IGF signaling are able to induce
neural differentiation in animal caps (Hardcastle et al. 2000;
Pera et al. 2001; Richard-Parpaillon et al. 2002), and latecanonical Wnt signals are known to inhibit anterior brain
formation (Kiecker and Niehrs 2001).
In chick and ascidian embryos, current models of neuralinduction highlight the role of FGF and Wnt in neuralinduction and de-emphasize a role for BMP regulation(Wilson and Edlund 2001; Stern 2002; Bertrand et al. 2003).
We are unable to discuss in depth here the relative
importance of the different signaling pathways in variousorganisms (reviewed in Wilson and Edlund 2001). It is clear,
however, that multiple pathways cooperate in neural development. For example, in the chick embryo, Wnt or BMP
antagonists applied to cells at the border region between
epidermis and CNS expand the neural plate, and FGF
signaling represses BMP4 expression in the neuroectoderm.
In addition, the anti-neural effects of intermediate levels of
an FGF antagonist can be reversed by the addition of chick
Chd (Wilson and Edlund 2001). One of the difﬁculties incomparing neural induction between Xenopus and otherchordates concerned the different timing of events. We now
ﬁnd a requirement for critical signals triggered by b-Cateninin the prospective neuroectoderm just after midblastula.
Thus, the neural induction process seems to start at blastulain all chordates (Wessely et al. 2001; Wilson and Edlund 2001;
Stern 2002; Bertrand et al. 2003). In addition, new molecularmechanisms are being discovered that help explain how
disparate signaling pathways—such as those of FGF, IGF, andanti-BMPs—can be integrated during development. Tyrosine
kinase receptors such as those for FGF and IGF have recently
been found to inhibit the BMP pathway effector protein
Smad1 by phosphorylation via mitogen-activated protein
kinase (MAPK) (Pera et al. 2003; Sater et al. 2003). Neuralinduction by the BMP antagonist Chd requires the extra
boost in Smad1 inhibition provided by FGF and IGF signaling(Pera et al. 2003). This molecular mechanism exempliﬁes one
way in which signaling pathways hitherto considered entirely
PLoS Biology | http://biology.plosjournals.org

independent might be integrated in embryonic cells (Massagué 2003). Primary neural induction in the chordate
embryo has been an area of active investigation for many
years and we can expect this to continue for the foreseeable
future.

A Role for Ectoderm in Amphibian Neural Induction
The role of the ectoderm in amphibian neural induction
has been the subject of much debate (Spemann 1938;
Holtfreter and Hamburger 1955; Nieuwkoop and Koster1995). Gene marker studies in Xenopus had noticed a
predisposition of dorsal ectoderm for neural induction by
mesoderm (Sharpe et al. 1987; London et al. 1988), but arequirement for any speciﬁc genes had not been addressed.
In addition, it was known that the dorsal animal cap responds
much better to the mesoderm-inducer Activin (Sokol andMelton 1991). These earlier ﬁndings can now be reinterpreted as reﬂecting the effects of the early b-Catenin signalthat induces expression of genes such as Chd, Nog, Xnr3, andSiamois. Chd is not only expressed in the organizer region
during gastrulation, but also in the dorsal animal cap region
during blastula, and this is required for neural speciﬁcation.
In this study we have provided evidence that presumptive
neural plate material can differentiate into CNS in theabsence of a mesodermal substratum. The BCNE center isrequired for brain formation in the embryo, but requires thecooperation of endomesodermal signals such as Cerberus. A
requirement of gastrula prospective neuroectoderm forneural plate formation had been proposed by earlier workers
on the basis of defect experiments (Goerttler 1925; Lehmann1928). However, these results were disputed (Holtfreter 1933;
Spemann 1938; Holtfreter and Hamburger 1955; Hamburger
1988; Nieuwkoop and Koster 1995), and vertical induction by
the endomesodermal Spemann organizer was attributed thepreeminent role in amphibian neural induction. A possible
explanation for why the role of the prospective neuroectoderm remained unrecognized for so many years of
research on the experimental embryology of neural inductionis that, unlike Spemann’s organizer, the BCNE center lacks
inducing activity when transplanted to ectopic sites. The
availability of new tools to investigate the function ofindividual genes—such as b-catenin, Chd, and Cer—has now
provided evidence that both ectodermal and endomesodermal signals are required for primary embryonic induction in
Xenopus.

Materials and Methods
Embryo manipulations. Xenopus embryos obtained by in vitro
fertilization were cultured in 0.13 modiﬁed Barth’s medium (Sive etal. 2000). For BCNE transplantation and deletion experiments,
dissections were performed in 13 Steinberg’s solution (Sive et al.
2000). BCNE grafts were 0.3 mm squares isolated from the dorsal
animal cap just above the ﬂoor of the blastocoel. They were excised atearly stage 9 (6.75–7.25 h after fertilization at room temperature),
before extensive epiboly movements begin, just one division after thelarge-cell blastula stage (stage 8), and could be monitored by the
thickness of the animal cap and cell size. Embryos were cultured in 13
Steinberg’s solution until healing (0.5–1 h) and then changed into
0.13 Barth’s solution. Embryo stages were according to Nieuwkoop
and Faber (1994).
Keller sandwiches were prepared at early stage 10. The dorsal
sector of the gastrula was excised at an angle of 308 from the dorsal
midline, from the dorsal lip up to the animal pole, using stainless steel
forceps. Two explants were sandwiched and cultured in 1 x Steinberg
solution for 12 h for in situ hybridization, 1 d for RT-PCR analysis,
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Neural Induction in Xenopus
and 2 d for morphological analysis. For RT-PCR analyses, RNA waspooled from ﬁve Keller explants, ﬁve animal caps, or single embryos.
The RT-PCR conditions and primers, as well the protocol for wholemount in situ hybridization, are described in http://www.hhmi.ucla.
edu/derobertis/index.html.
Lineage tracing. To fate map BCNE descendants, an improved
lineage tracing method was developed. Embryos were injected with 1–
4 nl of 1% BDA (Molecular Probes, Eugene, Oregon, United States) in
H2O and cultured explants or embryos were ﬁxed for at least 1 h in
MEMFA (Sive et al. 2000). Subsequently, embryos were placed for 24 h
in 70% ethanol, 1 h in 100% ethanol, 1 h in 100% isopropanol, 12–16
h in 100% xylene, and 1 h in parafﬁn at 658C before embedding. We
found that overnight incubation in xylene improved sectioning of
early embryos, which are rich in yolk. Sections were cut at 8–10 lm
and dewaxed in 100% xylene, 100% ethanol, and 70% ethanol for 2
min each. Next, sections were washed twice in binding buffer (100mM Tris–HCl, 150 mM NaCl [pH 7.5]) for 5 min and incubated inbinding buffer containing streptavidin-coupled alkaline phospatase(Roche, Basel, Switzerland) at a dilution of 1:5,000 overnight at room
temperature. Afterwards, slides were washed twice with binding
buffer, once with reaction buffer (100 mM Tris–HCl, 100 mM NaCl,
50 mM MgCl2, pH 9.5) for 5 min and incubated overnight withreaction buffer containing 10% BM purple solution (Roche) in a
Coplin jar in the dark at 48C. Staining was stopped by incubation in
Stop solution (100 mM Tris-HCl, 1 mM EDTA, pH 7.4), and sections
dehydrated in 100% methanol and completely air-dried before
mounting in Vectashield medium (Vector Laboratories Inc., Burlingame, California, United States). In some experiments nuclear lacZmRNA (kind gift of R. Harland), ﬂuorescein dextran amine or Texas
red dextran amine were used as lineage tracers.
RNA injections. To generate synthetic mRNAs, the plasmids pCS2CerS, pCS2-Chd, pCS2-b-catenin, and pCS2-dnGSK3 were linearizedwith NotI and transcribed with SP6 RNA polymerase as described
previously (Piccolo et al. 1999). The following amounts of mRNA wereused for microinjections: 600 pg (150 pg four times into the vegetal
region at 4-cell stage) for CerS, 100 pg (50 pg twice into dorsal-animal
region at 4-cell stage) for Chd, 800 pg (400 pg twice into the dorsalanimal region at 8-cell stage) for b-catenin, and 600 pg (300 pg twice
into the dorsal-animal region at 8-cell stage) for dnGSK3 mRNA.
Morpholino oligos. Morpholino oligos were as follows: Chd-MO1(59-ACG TTC TGT CTC GTA TAG TGA GCG T-39) and Chd-MO2 (59ACA GCA TTT TTG TGG TTG TCC CGA A-39) (Oelgeschläger et al.
2003); Nog-MO (59-TCA CAA GGC ACT GGG AAT GAT CCA T-39)(this work); b-cat-MO (59-TTT CAA CCG TTT CCA AAG AAC CAG G39) (Heasman et al. 2000); Cer-MO (59-ACT TGC TGT TCC TGC ACTGTG C-39) (this work); and a control-MO (59-CCT CTT ACC TCAGTT ACA ATT TAT A-39) (Oelgeschläger et al. 2003). The
morpholino oligos were resuspended to prepare a 1 mM stock
solution (SS) that was then further diluted in sterile water to give aworking solution: Chd-MO solution (Chd-MO1-SS:Chd-MO2-SS:H2O =1:1:6), b-cat-MO solution (b-cat-MO-SS:H2O = 1:4), Nog-MO solution(Nog-MO-SS:H2O = 1:1), Cer-MO solution (Cer-MO-SS:H2O =1:1),

control-MO solution (control-MO-SS:H2O = 1:1), and Chd-MO/NogMO solution (Chd-MO1-SS:Chd-MO2-SS:Nog-MO-SS:H2O = 1:1:4:10).
A total of 8 nl (two times 4 nl or four times 2 nl) morpholino solutions
were injected at the 2-cell stage or 4 nl (two times 2 nl) of morpholino
solution injected at the 8-cell stage.

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PLoS Biology | http://biology.plosjournals.org

Supporting Information
Figure S1. The BCNE Region Can Be Reliably Marked at the 64-Cell
Stage Using an Improved BDA Lineage-Tracing Method(A–H) Microinjection of individual 32-cell blastomeres does notfaithfully recapitulate the lineage of BCNE grafts at gastrula(compare with Figure 2B and 2D). Note that the lineage of D1
includes part of the Nieuwkoop center and contributes to anterior
endoderm at gastrula. This 32-cell map is in general agreement withpreviously published fate maps (Dale and Slack 1987; Bauer et al.
1994); the minor differences observed are explained by our choice ofbatches of regularly cleaving embryos (Klein 1987) with tightly
adhering small animal blastomeres. Abbreviation: AE, anterior
endoderm.
(I–L) Diagram indicating the injection of the lower daughter of B1
and the upper daughter of C1 at the 64-cell stage (I), which reliably
identify BCNE descendants at stage 9 (J), stage 11 (K), and stage 36 (L).
Arrowheads indicate the blastopore. Abbreviations: fp, ﬂoor plate;no, notochord.
Found at DOI: 10.1371/journal.pbio.0020092.sg001
Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/
Genbank/index.html) accession numbers discussed in this paper arefor b-catenin (M77013), Cer (U64831), Cer pseudoallele (BG160114), Chd(L35764), Goosecoid (M81481), and Nog (M98807).

Acknowledgments
We thank Dr. D. D. Brown for transgenic X. laevis; Dr. H. Okamoto,Dr. D. Wilkinson, Dr. R. Harland, and Dr. D. Kimelman for DNA
constructs; U. Tran and A. Cuellar for technical assistance; and E.
Neufeld, L. Zipursky, S. Millard, E. Pera, and C. Cofﬁnier forcomments on the manuscript. This work was supported by the
National Institutes of Health (HD21502–18) and the Howard Hughes
Medical Institute, of which EMDR is an investigator.
Conﬂicts of interest. The authors have declared that no conﬂicts ofinterest exist.
Author contributions. HK and EMDR conceived and designed theexperiments. HK and EMDR performed the experiments: HK, OW,
and EMDR analyzed the data. HK, OW, and EMDR contributed
reagents/materials/analysis tools. HK and EMDR wrote the paper. All&
the authors collaborated on the work.

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Neural Induction in Xenopus
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May 2004 | Volume 2 | Issue 5 | Page 0634