RESEARCH ARTICLE 1651
Development 139, 1651-1661 (2012) doi:10.1242/dev.068395
© 2012. Published by The Company of Biologists Ltd
Dynamic in vivo binding of transcription factors to cisregulatory modules of cer and gsc in the stepwise formation
of the Spemann–Mangold organizer
Norihiro Sudou1,2,3, Shinji Yamamoto1, Hajime Ogino2,3 and Masanori Taira1,*
SUMMARY
How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The
Spemann–Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and
VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc)
are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR
analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late
blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and
Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these
data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in
which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1
and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed;
and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple
transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise
integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer.
INTRODUCTION
One of the main questions in the field of developmental biology is
how are multiple developmental cues integrated stepwise with the
transcriptional regulation of cell fate decisions. To date, a large
number of cis-regulatory modules (CRMs) have been identified by
promoter/enhancer analyses in many organisms, and conceptual
gene regulatory networks have been created for various
developmental processes (Davidson and Levine, 2008). However,
little is known about the in vivo status of transcriptional regulation
or the stepwise integration of developmental cues, at least in
vertebrate embryogenesis.
The formation of the Spemann–Mangold organizer in Xenopus
is thought to be established by two major signaling pathways:
Nodal/activin for mesendoderm induction and Wnt/b-catenin for
dorsal determination. The initial state consists of maternal VegT
(mVegT), a T-box transcriptional factor in the vegetal hemisphere
(Lustig et al., 1996; Zhang and King, 1996), and nuclear b-catenin
downstream of canonical Wnt signaling in the dorsal region
(Blythe et al., 2010). At the blastula stage, mVegT and Wnt
signaling coordinately upregulate nodal expression (Xnr5 cluster
genes and Xnr6, called Xnr5 hereafter), which forms a Nodal
gradient in a dorsoventral direction in the presumptive endoderm
(Agius et al., 2000; Takahashi et al., 2000; Takahashi et al., 2006).
1
Department of Biological Sciences, Graduate School of Science, University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2Graduate School of Biological
Sciences, Nara Institute of Science and Technology (NAIST), 8916–5 Takayama,
Ikoma, Nara 630-0192, Japan. 3JST, CREST, Graduate School of Biological Sciences,
Nara Institute of Science and Technology (NAIST), 8916–5 Takayama, Ikoma, Nara
630-0192, Japan.
*Author for correspondence ([email protected])
Accepted 28 February 2012
In the dorsal region, high Nodal signaling and Wnt/b-catenin
signaling are integrated to form the gastrula organizer. During this
process, Nodal signaling induces endoderm- and mesodermspecific transcriptional activators, such as Mix1 (Rosa, 1989);
zygotic VegT (zVegT) and organizer-specific transcription factors,
such as Lim1/Lhx1 (Taira et al., 1992; Taira et al., 1994);
FoxA4/XFKH1 (Dirksen and Jamrich, 1992); and Otx2/Xotx2
(Blitz and Cho, 1995; Pannese et al., 1995; Yamamoto et al., 2003).
Wnt/b-catenin signaling induces expression of the organizerspecific transcriptional activators Siamois (Sia) and its paralog
Twin (Lemaire et al., 1995; Laurent et al., 1997). In addition, the
combination of these signaling proteins induces the production of
a transcriptional repressor, Goosecoid (Gsc) (Watabe et al., 1995;
Wessely et al., 2001). While the organizer is being formed, the
secreted BMP antagonists Noggin, Chordin, Follistatin, Xnr3 and
Cerberus emanate from the organizer region to induce
neuroectoderm in the dorsal animal ectoderm and also dorsalize the
mesoderm to further establish and maintain the organizer. In
addition, Cerberus, which is expressed in the head organizer region,
also inhibits Nodal and Wnt signaling to prevent their
posteriorizing effects (Piccolo et al., 1999). Thus, the outline of
organizer formation and neural induction has been established.
To elucidate the molecular basis of organizer formation in
Xenopus, several groups, including ours, have investigated the
regulation of organizer genes such as (1) gsc by Nodal and Wnt
signaling (Watabe et al., 1995), and (2) lim1 and hhex by Nodal
signaling (Watanabe et al., 2002; Rankin et al., 2011). In addition,
the regulation of target genes for Gsc and Lim1 have been studied,
such as (3) wnt8 and brachyury (bra/Xbra) by Gsc (Latinkic et al.,
1997; Latinkic and Smith, 1999; Yao and Kessler, 2001) and (4)
gsc and cer by Lim1 and Mix1 (Latinkic and Smith, 1999;
Mochizuki et al., 2000; Yamamoto et al., 2003). However, these
DEVELOPMENT
KEY WORDS: ChIP-qPCR, Spemann–Mangold organizer, Xenopus, Gene regulation
1652 RESEARCH ARTICLE
MATERIALS AND METHODS
Embryos and manipulation
Xenopus embryos were obtained by artificial fertilization, dejellied and
incubated in 0.1⫻Steinberg’s solution (Peng, 1991). Embryos were staged
according to the criteria of Nieuwkoop and Faber (Nieuwkoop and Faber,
1967).
Cloning of cer regulatory regions
An about 2 kb DNA fragment of the X. tropicalis cer gene (from –2005 to
+37) was obtained by PCR amplification with genomic DNA and specific
primers (supplementary material Table S1). The amplified fragment was
inserted into the KpnI and XhoI sites of pGL3-EGFP. About 1 kb genomic
DNA fragments of X. borealis and X. mulleri cer genes were isolated by
PCR with degenerated primers designed based on the X. laevis cer
sequence: from –288 to +958 (supplementary material Table S1). Adult X.
borealis and X. mulleri frogs were purchased from the Watanabe breeder
(Japan).
mRNA synthesis and DNA injection reporter assays
Plasmids for mRNA synthesis and reporter constructs are shown in
supplementary material Table S2 and Fig. S9. Synthesis of mRNA and
luciferase assays were carried out as described (Yamamoto et al., 2003).
Reporter DNA was injected at 50 pg/embryo. The statistical significance
(P-value) was calculated using Student’s t-test.
Transgenic and co-transgenic reporter assays using the nuclear
transplantation technique
Transgenesis was performed as described previously (Murray, 1991; Kroll
and Amaya, 1996) with some modifications (Tanaka et al., 2003). Injection
dishes were coated with 2% agarose gel in 0.4⫻Marc’s Modified Ringer
(MMR). Co-transgenesis was performed as described previously (Ogino et
al., 2008). The –62Xt_cer/EGFP construct was used as a basal promoter,
which was digested with PstI at the 5⬘ end of the promoter. PCR fragments
digested with PstI and the digested –62Xt_cer/EGFP construct were mixed
at a 4:1 molar ratio.
Whole mount in situ hybridization
Whole-mount in situ hybridization was carried out as described (Harland,
1991). For hemisections, rehydrated embryos were embedded in 2% low
melting agarose in 0.2⫻PBS containing 0.3 M sucrose and 0.05% Triton
X-100, and bisected through the dorsal-ventral midline with a razor blade
as described (Lee et al., 2001; Hikasa et al., 2002). Bisected embryos were
refixed with MEMFA for 30 minutes before hybridization. Digoxigeninlabeled antisense RNA probes were transcribed from plasmid or PCR
templates (Mamada et al., 2009) (supplementary material Table S2).
Antibodies
Anti-Lim1, Otx2, Sia, Mix1 and VegT rabbit polyclonal antibodies were
generated using the glutathione-S-transferase (GST) gene fusion system,
and specificities of affinity-purified antibodies were verified with western
blotting and immunostaining (supplementary material Fig. S5). Plasmids
for fusion constructs are listed in supplementary material Table S2.
ChIP-qPCR
ChIP experiments were carried out with a chromatin immunoprecipitation
kit (Upstate) as described previously (Kato et al., 2002) with some
modifications. An aliquot of fragmented chromatin preparation (13 g
DNA equivalent to 50-100 embryos) was incubated with 10 g of antiLim1, Otx2, Sia, Mix1 or VegT antibodies or preimmune immunoglobulin
purified by ammonium sulphate precipitation. PCR primer sets for qPCR
are described in Fig. 4 and supplementary material Table S1.
RESULTS
Comparison of the cer genes of Xenopus species
To identify evolutionarily conserved regulatory elements, we
compared nucleotide sequences of the cer gene of X. laevis (Xl_cer)
with that of X. tropicalis (Xt_cer). Both genes have a highly
conserved region at –300/–70 bp upstream of the transcription start
site (supplementary material Fig. S1A). This conserved 5⬘ region
includes the 5⫻TAAT/ABCDE element, but no other conserved
TAAT sites are apparent in 2 kb of the 5⬘ region (supplementary
material Fig. S1B). We further examined evolutionary conservation
of the ABCDE element between four species, X. tropicalis (Xt), X.
laevis (Xl), X. borealis (Xb) and X. mulleri (Xm) (Fig. 1A;
supplementary material Fig. S1C). X. tropicalis is diploid, whereas
the other species are allotetraploid and have two ‘homoeologs’,
called a and b genes, which were supposedly derived from the two
distinct parental species, thereby designated cer-a and cer-b.
DEVELOPMENT
experiments used mainly plasmid DNA injection for luciferase
reporter assays. This approach sometimes does not reflect
endogenous gene regulatory mechanisms, compared with
transgenic reporter assays using nuclear transplantation (Kroll and
Amaya, 1996). Furthermore, the dynamics of in vivo binding of
transcription factors to their regulatory elements have not been
investigated, particularly in terms of the timing and the order of
their binding to target elements for the initiation, maintenance and
cessation of gene expression.
The 5⬘ region of gsc is one of the best-studied transcriptional
regulatory regions. This contains the distal element (DE), the
proximal element (PE) and the upstream element (UE) (Watabe et
al., 1995; Mochizuki et al., 2000). The DE and PE are involved in
the induction of gsc by Nodal and Wnt signaling, respectively
(Watabe et al., 1995). It has been suggested (1) that Sia and Twin
bind to the PE (Laurent et al., 1997); (2) that a TFII-I/Smad2
complex (Ku et al., 2005), a WBSCR11/Smad2/FoxH1 complex
(Ring et al., 2002) or a Mixer/Milk/Smad2 complex (Germain et
al., 2000) transduce Nodal signaling through the DE; and (3) that
Lim1 and Mix1 activate the gsc gene as maintenance factors
through the UE and DE for Lim1 (Mochizuki et al., 2000) or
through the DE and PE for Mix1 (Latinkic and Smith, 1999).
Regarding the regulation of the cer gene, we have shown that the
five consecutive homeodomain core binding sites A, B, C, D and
E (named the 5⫻TAAT element or ABCDE element) in the 5⬘
region are crucial for cer expression, that a complex of Mix1, Sia
and Lim1 binds to the region containing sites A, B and C (named
3⫻TAAT element or ABC element), and that Otx2 binds to site E
(Yamamoto et al., 2003). These studies suggest that the DE, PE and
UE of gsc and the 5⫻TAAT/ABCDE element of cer can integrate
Nodal and Wnt signaling for organizer expression. However, the
question still remains as to how gsc and cer are regulated in vivo
by organizer-expressing transcription factors through these CRMs.
In this paper, we first investigated the regulatory element of the
cer gene by using comparative transgenic reporter and sequence
analyses. We found significant variations in the 5⫻TAAT/ABCDE
element in four Xenopus species and identified conserved T-boxbinding sites that respond to VegT. We also found VegT-response
elements near the DE and PE of the gsc gene. We next performed
chromatin immunoprecipitation coupled with quantitative PCR
(ChIP-qPCR) analysis with anti-Lim1, Mix1, Sia, Otx2 and VegT
antibodies. This analysis revealed dynamic in vivo binding of these
transcription factors to the cer and gsc regulatory modules – named
cer-U1 and gsc-U1, respectively. Finally, we analyzed the
overlapping expression domains of organizer genes, as well as
endodermal and mesodermal genes at the blastula to neurula stages,
showing two distinct origins of organizer gene expression. These
data show, for the first time to our knowledge, the dynamics of in
vivo binding of multiple transcription factors in early vertebrate
development, leading to a new model of stepwise formation of the
Spemann–Mangold organizer.
Development 139 (9)
Stepwise formation of the Spemann organizer
RESEARCH ARTICLE 1653
Because the ABCDE element is important for gene expression
in reporter assays (Yamamoto et al., 2003), these differences
between the homoeologs could cause differences in gene
expression. Therefore, we compared expression levels between
Xl_cer-a and Xl_cer-b using expressed sequence tag (EST) search
with their overlapping coding sequences (supplementary material
Fig. S2A). The data show that 92% (66/72) of ESTs are Xl_cer-a,
suggesting that this gene is transcribed preferentially in the embryo.
Because the ABC element of Xl_cer-a (designated Xla_ABC) is
most deviated from the type of Xt_cer (designated Xt_ABC), we
next examined whether Xla_ABC and Xt_ABC are equally
functional for dorsal expression. To test this, we generated
luciferase reporter constructs fused to the SV40 minimal promoter
(Fig. 1B). The SV40 promoter alone did not show much difference
in expression levels between the dorsal marginal zone (DMZ) and
the ventral marginal zone (VMZ). By contrast, the (Xla_ABC)5 but
not (Xt_ABC)5 construct showed dorsal-specific expression (Fig.
1B). This implies that Xla_ABC evolved from the ancestral type
(supposedly Xt_ABC) to exert such expression by changing, in
part, the nucleotide sequences of both sites B and C. This raised the
question as to which elements other than Xt_ABC might confer
dorsal-specific expression of Xt_cer.
Comparative analysis revealed (1) the X. tropicalis ABCDE element
is likely to be closest to the ancient type among them, because
elements A to E in at least either the a or b gene of the three
allotetraploid species are the same as those of the diploid species X.
tropicalis; (2) that site A is most conserved among them; (3) that sites
B and C in Xl_cer-a are most deviated from the X. tropicalis types;
and (4) that sites D and E seem to be subfunctionalized between cera and cer-b (Fig. 1A; supplementary material Fig. S1B).
VegT response of the cer reporter gene
Because vegt is co-expressed with cer in the endoderm and
mesoderm regions during the blastula to gastrula stages (see Fig.
5), and the T3 sites are in good agreement with a VegT consensus
binding site (Conlon et al., 2001), we examined whether VegT is
involved in regulating the cer gene. We first showed that the
endogenous cer gene was activated by VegT in the animal
ectoderm (supplementary material Fig. S3B). We next tested
DEVELOPMENT
Fig. 1. Comparison of cer promoter sequences and
homeodomain binding sites between Xenopus species.
(A)Evolutionary conservation of ABCDE elements between Xenopus
species. The ABCDE element is schematically aligned for two
homoeologous genes a and b of the X. laevis (Xl), X. borealis (Xb) and
X. muelleri (Xm) cer genes together with the single cer gene of X.
tropicalis (Xt). Phylogenetic relationships were determined by ClustalW
using 5⬘ and exon sequences (supplementary material Fig. S1C)
(DDBJ/Genbank Accession Numbers: Xl_cer-a, AB086394; Xl_cer-b,
AB698643; Xb_cer-a, AB698644; Xb_cer-b, AB698645; Xm_cer-a,
AB698642). Magenta boxes indicate Lim1-binding sites (YTAATNN;
YT or C, NNTA, TG, CA, or GG), green boxes indicate bicoid sites
(TAATCY); white boxes indicate other TAATNN sites. Arrows above and
in the box indicate the direction of TAAT (the double-headed arrow
indicates the TAATTA sequence; boxes with a cross indicate disrupted
TAAT-binding site). (B)Comparison of enhancer activity of the ABC
elements between X. laevis and X. tropicalis cer genes. (Xla_ABC)5/Luc
and (Xt_ABC)5/Luc are five tandemly repeated ABC elements fused to
the SV40 minimal promoter (SV40 prom). Reporter DNA (50
pg/embryo) was injected into the prospective dorsal or ventral marginal
zone (DMZ or VMZ, respectively) and luciferase activity was assayed at
the gastrula stage (stage 10.5). Data are mean±s.d.; *P<0.05; ns, not
significant.
Transgenic reporter assays for the ABCDE element
and T-box sites of the cer gene
To compare the 5⬘ regulatory regions of Xla_cer (designated as
Xl_cer hereafter) and Xt_cer, we used transgenic reporter assays,
because DNA injection reporter analysis in Xenopus does not
always reflect endogenous gene regulatory mechanisms. We first
observed that 2 kb of the 5⬘ regions of both Xl_cer and Xt_cer
recapitulate the endogenous cer expression pattern (Fig. 2A-C).
To narrow down responsive regions, we used co-transgenesis
assays (Ogino et al., 2008) (supplementary material Fig. S3A)
and also examined several deletion constructs (Fig. 2). The data
show that the –452/–62 region of Xt_cer was necessary and that
the –229 region was sufficient (both –229Xl_cer and
–229Xt_cer) for exhibiting cer-like expression. Both the
–229/–166 region and the ABCDE element were required for full
activity (–229-Dabcde, –229-Mabcde, and –166-w of Xl_cer and
Xt_cer), suggesting that the –229/–166 region upstream of the
ABCDE element has additional CRMs for both Xl_cer and
Xt_cer expression.
We searched the –229/–166 region for transcription factor
binding sites, which are conserved between the six orthologs and
homoeologs of the four Xenopus species, and found three putative
T-box recognition sequences (TNNCAC) (Erives and Levine,
2000; Oda-Ishii et al., 2005), named T1, T2 and T3 (supplementary
material Fig. S1C). Therefore, we examined T-box mutant
constructs using transgenic assays. Fig. 2D shows that any one of
the three T-box mutations led to a reduction in dorsal expression of
the reporter gene, suggesting that the three T-box sites work
together in responding to the endogenous T-box transcription
factor(s).
1654 RESEARCH ARTICLE
Development 139 (9)
Fig. 2. Cis-element analysis of X. laevis and X. tropicalis cer
promoter regions using transgenic reporter genes. (A)Deletion
and point mutations of cer promoter-EGFP reporter constructs and their
dorsal expression at the gastrula stage. Constructs of Xl_cer and Xt_cer
promoter regions are schematically shown on the left, and numbers
indicate positions from the transcription start site, whereas numbers in
blue indicate position of Xt_cer when different from those of Xl_cer. A
bent arrow indicates the transcription start site. Fixed embryos were
bisected along the dorsal midline and whole-mount in situ hybridization
was performed for EGFP mRNA. Dorsal expression of reporter genes
was categorized into strong (orange) and partial or weak (pale orange)
expression patterns (supplementary material Fig. S3A), and percentage
incidences of each category are presented by bar graphs. The total
number of embryos examined and those of experiments in the
parentheses are indicated on the right. n.d., not determined.
(B,C)Whole-mount in situ hybridization analysis of reporter expression.
Typical expression patterns of Xl_cer (B) or Xt_cer (C) reporter
constructs are shown. (D)Reporter gene expression of T-box mutant
constructs.
T-box sites and VegT response of the gsc reporter
gene
Because gsc is directly induced by VegT (Messenger et al., 2005),
we examined whether gsc has conserved VegT-binding sites near
the UE, DE and PE. Comparison of the 5⬘ flanking sequences of
the gsc genes of X. laevis and X. tropicalis (designated Xl_gsc and
Xt_gsc, respectively) identified three conserved consensus T-box
sites, T1, T2 and T3, in which T2 (TCTCACCC) most conforms
to the VegT consensus site. Moreover, the DE and PE are almost
perfectly conserved but the UE is not (supplementary material Fig.
S4).
To examine whether the 5⬘ region of gsc might respond to VegT,
gsc reporter constructs were co-injected with vegt mRNA in the
Xenopus embryo. Fig. 3D shows that –1500gsc/Luc and
–492gsc/Luc (Watabe et al., 1995; Mochizuki et al., 2000) strongly
responded to VegT in a dose-dependent manner. Mutations of all
three T-box sites in –492gsc/Luc greatly reduced reporter activation
by VegT (Fig. 3D, the right panel), indicating that VegT activates
the gsc reporter gene through these T-box sites. Based on these
observations, we assume that the region containing the PE, DE and
the three T-box sites forms a CRM, here named gsc-U1
(supplementary material Fig. S4). Thus, gsc is likely to be
regulated by the same set of transcription factors, Sia, Lim1, Otx2,
Mix1 and VegT, as cer is.
DEVELOPMENT
whether luciferase reporter assays using DNA injection could be
applied to analyze the responsiveness of cer reporter constructs to
endogenous and exogenous factors in the embryo. Both
–229Xl_cer/Luc and –229Xt_cer/Luc were specifically activated in
the DMZ (Fig. 3A) and furthermore –229Xt_cer/EGFP was
upregulated by VegT in the animal ectoderm (supplementary
material Fig. S3C), indicating that DNA injection reporter assays
can be used for such analyses.
To examine the synergy between VegT and other transcription
factors, we used a lower dose of VegT, which was not sufficient to
activate the reporter. As shown in Fig. 3B, –229Xt_cer/Luc was
activated synergistically by VegT and a mixture of Mix1, Sia, Lim1
and Otx2 (called 4 mix). To examine the necessity of VegT-binding
sites T1 to T3 in the –229 region (supplementary material Fig.
S1C), one or all of them were mutated (designated MT1, MT2,
MT3 and MT123). Whereas MT1 and MT2 appeared to change the
synergistic activation levels, MT3 and MT123 completely lost
them (Fig. 3B), indicating that T3 is the VegT response site.
Furthermore, among the four, Sia, and to a lesser extent Lim1,
enhanced activation of –229Xl_cer/Luc by VegT (Fig. 3C),
suggesting functional interactions between VegT and Sia/Lim1. It
should be noted that activation of the reporter gene by Mix1 was
not enhanced by VegT, which is in contrast to Sia (Yamamoto et
al., 2003). These data suggest that the T3 site is a main site for
VegT to collaborate with Sia and Lim1 in activating the cer
promoter.
Although we have shown a clear difference in dorsal-specific
expression between (Xla_ABC)5/Luc and (Xt_ABC)5/Luc (Fig.
1B), both –229Xl_cer and –229Xt_cer have sufficient activities to
drive such expression in both transgenic (Fig. 2A) and DNA
injection (Fig. 3A) reporter assays. These data suggest that the
combination of the T-box sites and the ABCDE element forms a
robust functional unit as a whole that tolerates nucleotide changes
in some of its elements. Therefore, we defined a region containing
the T-box sites and the ABCDE element as a CRM, referred to as
cer-U1 (standing for ‘upstream regulatory module 1’;
supplementary material Fig. S1C).
Stepwise formation of the Spemann organizer
RESEARCH ARTICLE 1655
Dynamics of in vivo binding of Lim1, Otx2, Sia, Mix1
and VegT to the cer and gsc regulatory modules
To examine the dynamics of in vivo binding of transcription factors
to cer- U1 and gsc-U1, we performed ChIP-qPCR analysis with X.
laevis embryos using rabbit polyclonal antibodies, which were
raised against recombinant X. laevis Lim1, Mix1, Sia, Otx2 and
VegT proteins and verified for their specificities (supplementary
material Fig. S5). The anti-VegT antibodies recognize both mVegT
and zVegT, and the anti-Lim1 and Mix1 antibodies did not
crossreact with their paralogous proteins Lim5 and Mixer,
respectively (Toyama et al., 1995; Henry and Melton, 1998). As
shown in Fig. 4A, ChIP-qPCR primer sets 1 through 4 were
designed for cer-U1 (primer set 1) and a region within cer intron 1
(primer set 2; a negative control) and for gsc-U1 (primer set 3) and
gsc exon 3 (primer set 4: a negative control). Chromatin was
prepared from late blastula, early gastrula and neurula embryos.
ChIP-qPCR with primer sets 1 and 3, compared with negative
controls with primer sets 2 and 4, revealed that VegT and Sia, but
not Mix1, Lim1 or Otx2, were already bound to the cer-U1 and
gsc-U1 regions at the late blastula stage (Fig. 4B). These results are
consistent with the order of expression of these five transcription
factors (supplementary material Fig. S6A), suggesting that both
VegT and Sia are involved in the initiation of cer and gsc gene
expression at this stage. At the early gastrula stage, all five factors
were bound to the cer-U1 and gsc-U1 regions (Fig. 4B). These in
vivo binding data strongly support the idea that these five
transcription factors are involved in regulating cer and gsc in the
organizer through cer-U1 and gsc-U1, and also suggest
maintenance roles for Mix1, Lim1 and Otx2 in cer and gsc
expression at the gastrula stage.
At the early neurula stage, the binding of all five factors to the
cer-U1 region decreased dramatically to basal levels. In the gsc
gene, binding of Sia, VegT and Mix1 to the gsc-U1 region also
decreased as the expression levels of sia, vegt and mix1
decreased by the early neurula stage. By contrast, the binding of
Lim1 and Otx2 to the gsc-U1 region persisted and if anything
increased (Fig. 4B). At this stage, because lim1 mRNA gradually
disappears in an anteroposterior direction (Taira et al., 1994),
lim1 mRNA is not present in the anterior-most region (Fig. 5E;
supplementary material Fig. S6C, arrow in the lim1 panel at
DEVELOPMENT
Fig. 3. Cooperation of VegT with Mix1, Siamois, Lim1 and Otx2 to activate the cer and gsc enhancers. (A)Dorsal-specific expression of
–229Xl_cer and –229Xt_cer as assayed by DNA injection. Experiments were carried out as described in Fig. 1B. (B)Synergy of VegT with a mixture
of Mix1, Sia, Lim1 and Otx2. Wild-type constructs of –229Xl_cer (white bar) and –229Xt_cer (diagonally striped bar) (left panel) or T-box mutant
constructs of –229Xl_cer (right panel) were co-injected with mRNAs (25 pg mRNA each) for VegT and 4 mix (mix1, sia, lim1 and otx2), as indicated
by a plus mark. Mutated T-box sites in –229Xl_cer-MT1, MT2, MT3 and MT123 are indicated by black diamonds, in which TNNCAC was mutated
to CNNTAC. (C)Synergy of VegT with Sia and Lim1. The –229Xl_cer construct was co-injected with mRNA as indicated with or without mRNA for
VegT. Amounts of injected mRNAs (pg/embryo): vegt, 25; lim1, 25; mix1, 25; sia, 12; otx2, 25. (D)Activation of gsc reporter constructs by VegT.
–1500Xl_gsc or –492Xl_gsc was co-injected with vegt mRNA as indicated. The sequence of –1500Xl_gsc has been deposited in DDBJ/Genbank
(AB698641).
1656 RESEARCH ARTICLE
Development 139 (9)
stage 15). However, the Lim1 protein persisted in the prechordal
plate and notochord until the tailbud stage (supplementary
material Fig. S5N⬘). Thus, our ChIP-qPCR analysis strongly
support the idea that the five transcription factors differentially
and coordinately regulate the cer and gsc genes in the formation
and functioning of the organizer.
Detailed comparison of expression domains of
organizer-expressing genes
Given that the cer and gsc genes are regulated by the five
transcription factors, as suggested above, their expression domains
should overlap each other. The expression domains of these seven
genes have already been reported individually (Taira et al., 1992;
Bouwmeester et al., 1996; Lemaire et al., 1998; Rankin et al.,
2011), but some of their initial or early expression domains have
not been reported, such as gsc, mix1, cer, lim1 and otx2. Therefore,
we performed whole-mount in situ hybridization using bisected
embryos at stages 8-15 for 11 genes (supplementary material Figs
S6, S7), and summarized their expression domains on a reported
blastomere fate map (Bauer et al., 1994). Notably, sia expression
at stages 8 to 8.5, which indicates a blastula chordin- and nogginexpressing (BCNE) region (Kuroda et al., 2004; Ishibashi et al.,
2008), corresponds well to the B1 blastomere-derived region
(Bauer et al., 1994) (Fig. 5A, red cells). During stages 8.5 to 10.5,
sia and gsc expression domains moved vegetally and internally,
together with epiboly and involution of the dorsal animal region,
which fits again to the fate map of B1 blastomere (Fig. 5B-D, red
cells). Concomitantly, sia and gsc expression expanded to the
presumptive dorsal endoderm that corresponds to a part of B1
(red), C1 (blue) and D1 (magenta) blastomere-derived regions (Fig.
5C,D). Thus, it is worth comparing expression patterns and the fate
map.
According to initial expression domains, we divided organizer
genes into two groups: group 1 and group 2. Group 1 genes,
including sia, chd, and gsc, start to be expressed at the mid blastula
stage in a BCNE region, a vegetal part of which will become the
dorsal mesoderm (Kuroda et al., 2004) (Fig. 5A,B; supplementary
material Fig. S6A for sia and gsc). Group 2 genes, including cer,
lim1 and otx2, start to be expressed at the late blastula stage in the
dorsovegetal region (Fig. 5B⬘). Thus, gsc and cer belong to
different groups, but nevertheless, gsc-U1 and cer-U1 responded to
the same set of five transcription factors (Fig. 3), and their in vivo
binding dynamics to these CRMs were very similar to each other
(Fig. 4B). Therefore, it was necessary to examine the timing of
overlapping of expression domains, and the spatial distribution of
their endodermal and mesodermal expression.
Before gsc and cer expressions start, maternal vegt (mvegt)
mRNA is already present and sia starts to be expressed at stage 8
(Fig. 5A; supplementary material Fig. S6A). At stage 8.5, gsc
expression (group 1) starts inside of the sia-expressing BCNE
region (supplementary material Fig. S6A), consistent with the data
that Sia binds to gsc-U1 at the stage 9.5 (Fig. 4B). At stage 8.5,
mvegt mRNA localization did not overlap with gsc, indicating that
VegT is not involved in the initiation of gsc expression in the
BCNE region. At mid-stage 9, zygotic vegt (zvegt) expression
starts in the dorsal marginal zone, and gsc and zvegt mRNA
DEVELOPMENT
Fig. 4. ChIP-qPCR analysis for cer-U1 and gsc-U1.
(A)Schematic diagrams of gene structures of cer and gsc.
Positions of cer-U1 and gsc-U1; primer sets for ChIP-qPCR
are indicated. (B)Dynamics of in vivo binding of
transcription factors to cer-U1 and gsc-U1. Occupancy of
Lim1, Mix1, Sia, Otx2 and VegT proteins at cer-U1 (left
panel) and gsc-U1 (right panel) was examined by ChIPqPCR with primer sets as indicated. qPCR was performed
in triplicate (cerberus) or duplicate (goosecoid) unless
otherwise indicated in parentheses. Magenta or light-blue
lines, specific or preimmune antibodies, respectively, for
ChIP-qPCR; st, stage; data are mean±s.e.m.; *P<0.05;
**P<0.01. Enrichment of cer-U1 and gsc-U1 regions by
ChIP was also examined by PCR/gel electrophoresis
analysis.
Stepwise formation of the Spemann organizer
RESEARCH ARTICLE 1657
Fig. 5. Assignment of expression domains
of organizer-expressing genes.
(A-C)Blastula to gastrula stages. Expression
domains at early blastula (A), mid-blastula (B
for group 1; B⬘ for group 2 genes) and early
gastrula (C) are surrounded by lines, as
indicated on 32-cell blastomere fate maps
reported by Bauer et al. (Bauer et al., 1994);
brown, green, yellow, red, magenta and blue
cells are derived from the B4, A4, A1, B1, C1
and D1 blastomeres, respectively. Group 1
genes, sia, gsc and chd; group 2 genes, lim1,
otx2 and cer. Dotted lines indicate initially
started or expanded expression. (D)Germ
layers and the organizer region. The
mesoderm (green line) is discriminated from
the endoderm (black line) according to the
size of cells (supplementary material Fig. S7).
The superficial mesoderm (Shook et al.,
2004) is indicated by a light magenta. A
combined expression domain of chd, gsc,
lim1 and otx2 is surrounded by a yellow line,
and molecularly defined as the organizer
region in this paper (supplementary material
Fig. S8). (E)Neurula stage. Expression
domains are illustrated on the diagram of
bisected neurula embryos (stage 15) based
on supplementary material Fig. S5N⬘ and Fig.
S6C.
but absent in the posterior mesoderm, whereas lim1 and otx2
expression domains were largely restricted to the dorsal
mesodermal region (supplementary material Fig. S7B). Thus,
expression domains of organizer genes were dynamically changed
from the late blastula to gastrula stages by expansion of expressing
areas from the endoderm to mesoderm (cer, lim1 and otx2) and
vice versa (gsc, sia and chd).
By the neural stage, cer, vegt, sia and mix1 are greatly reduced
in the midline tissues, but the expression of the Lim1 protein and
otx2 and gsc mRNAs in the head organizer region persist to the
neurula stage (Fig. 5E; supplementary material Fig. S5N,N⬘, Fig.
S6C). Therefore, the Lim1, Otx2 and Gsc proteins are likely to coexist in the anterior prechordal region, consistent with the ChIPqPCR data for gsc-U1 at stage 13 (Fig. 4B).
DISCUSSION
Integrated and robust regulation through cer-U1
We have previously shown the importance of the 3⫻TAAT/ABC
element in the –415 region of the X. laevis cer (Xl_cer) gene in
activation by a complex of Lim1, Mix1 and Sia using DNA
injection reporter assays and gel mobility shift assays (Yamamoto
et al., 2003). In the present study, using the transgenic technique
and sequence comparisons between Xl_cer and X. tropicalis cer
(Xt_cer), as well as the X. borealis and X. mulleri cer genes, we
have confirmed that the –415/–472 and –229 regions of Xl_cer and
Xt_cer are sufficient for recapitulating endogenous cer expression
in transgenic embryos (Fig. 2). Although DNA injection reporter
assays showed the difference in responsiveness of the ABC
elements between Xl_cer and Xt_cer (Fig. 1B), transgenic reporter
assays showed similar activity with the –1938/2005 to –229regions
between Xl_cer and Xt_cer (Fig. 2). This implies that cer-U1,
DEVELOPMENT
localizations begin to overlap, which was confirmed by wholemount in situ hybridization with a gsc or vegt probe for each half
embryo (supplementary material Fig. S6A,B).
cer expression (group 2) starts at mid-stage 9 in the dorsovegetal
region where mvegt mRNA exits, and sia expression expands from
the mesodermal region as reported (Crease et al., 1998) (Fig.
5B,B⬘; supplementary material Fig. S6A). Overlapping of cer and
sia expression domains was confirmed by whole-mount in situ
hybridization using a pair of bisected embryos (supplementary
material Fig. S6B). Nodal/activin-inducible mix1, lim1 and otx2
(group 2) start to be expressed in a deep region at mid stage 9
(supplementary material Fig. S6A), but their mRNA localizations
just begin to overlap with gsc and cer in the dorsovegetal region at
mid stage 9 (Fig. 5B⬘), accounting for no binding of Mix1, Lim1
and Otx2 to gsc-U1 and cer-U1 at this stage (Fig. 4B). At stages 10
to 10.5, all mRNA localizations of sia, zvegt, mix1, lim1 and otx2
overlapped with gsc and cer in the dorsal region (Fig. 5C;
supplementary material Fig. S6A, Fig. S7A). Thus, these mRNA
localization patterns appear to be consistent with the ChIP-qPCR
data (Fig. 4B).
To discriminate between endodermal and mesodermal
expressions, DAPI staining was performed for whole-mount in situ
hybridization-stained hemisections, in which nuclear density is
lower in the endoderm than that in the mesoderm, and sox17b
expression was further analyzed to identify the differentiated
endoderm region (Hudson et al., 1997) (supplementary material
Fig. S7). As a result, expression domains of lim1 and otx2 at stages
10 were assigned to both the dorsoanterior endoderm and the dorsal
mesoderm. At this stage, cer expression expanded to the
mesodermal region. At stage 10.5/11, cer expression was seen in
both the anterior endoderm and the anterior-most dorsal mesoderm,
which contains both the VegT response element and the ABCDE
element, is a functionally conserved CRM, which can integrate
various developmental cues and may be robust against nucleotide
changes. Thus, transgenic reporter analyses led to the identification
of T-box sites necessary for dorsal expression of the cer gene. This
finding further let us to find the importance of T-box site for the
gsc gene.
Subdivision of the organizer region
We defined here the organizer region as a domain expressing chd,
gsc, lim1 and otx2 (Fig. 5D; supplementary material Fig. S8). This
organizer region includes the BCNE center-derived mesoderm,
called here the ‘M’ region, and the dorsal endoderm, called here the
‘E’ region (Fig. 5D). These ‘M’ and ‘E’ regions were verified by
whole-mount in situ hybridization for sox17b and DAPI staining
(supplementary material Fig. S7). Tracing back to the blastula
stage, the ‘M’ region is derived from the BCNE region that
expresses sia, chd and gsc, but not mvegt, lim1 or otx2 (Fig. 5A,B).
By contrast, the ‘E’ region is derived from the dorsovegetal region
that expresses mvegt, mix1, cer, lim1 and otx2, but not sia or gsc
(Fig. 5A,B⬘). This means that expression domains of group 1 genes
(sia, chd and gsc) expand from the ‘M’ region to the ‘E’ region
during the late blastula to early gastrula, and vice versa for group
2 genes (cer, lim1 and otx2). The group 1 genes have
characteristics that can be induced in animal caps by Wnt signaling
(sia and chd) (Lemaire et al., 1995; Ishibashi et al., 2008) or by
Wnt and Nodal/activin signaling (gsc) (Crease et al., 1998). The
group 2 genes start to be expressed in the dorsovegetal region at
mid stage 9, and are directly induced by Nodal/activin signaling in
animal caps (Taira et al., 1992; Yamamoto et al., 2003). Thus, the
initial expression domains of group 1 and group 2 genes are well
correlated with the distribution of nuclear b-catenin in the BCNE
region (Schohl and Fagotto, 2002) and Xnr5 expression in the
presumptive dorsal endoderm (Takahashi et al., 2006), respectively.
It should be noted that gsc expression in the BCNE region at stages
8.5-9 (Fig. 5A,B) as well as mesodermal expression of lim1 and
otx2 at mid stage 9 (Fig. 5B⬘) is likely to be induced by Xnr5
secreted from the presumptive dorsal endoderm, the so-called
Nieuwkoop center.
Among organizer genes, cer is exceptional, because this gene is
not directly induced by activin in animal caps (Yamamoto et al.,
2003), in spite of its initial expression in the dorsovegetal region.
It is probable that cer expression is initiated by maternal VegT and
Sia, which is suggested by activation of a cer-U1 reporter by VegT
and Sia (Fig. 3C). Thus, the mechanisms of gene induction of cer
and gsc are different from each other at the blastula stage, but
appear to become similar at the late blastula to gastrula stage,
probably because cer and gsc are regulated by the same set of
transcription factors in the organizer.
Dynamics of in vivo binding of transcription
factors to cer-U1 and gsc-U1
Conventional reporter analysis can show which transcription
factors can bind to regulatory elements and activate or repress
reporter genes, but it does not tell when or how long transcription
factors bind to regulatory elements. In this study, we performed
ChIP-qPCR for cer-U1 and gsc-U1 to analyze the five transcription
factors that were expected to regulate the cer and gsc genes (Fig.
4). Based on the developmental expression patterns of cer and gsc,
and their transcriptional regulator genes vegt, sia, mix1, lim1 and
otx2 (Fig. 5; supplementary material Fig. S6), we interpret the
ChIP-qPCR data as follows. First, both mVegT and Sia bind to cer-
Development 139 (9)
U1 and gsc-U1 at the late blastula stage (mid stage 9) before Lim1,
Otx2 and Mix1 do (Fig. 4B). As we have shown overlapping
mRNA localizations of cer, mvegt and sia, as well as those of gsc,
sia and zvegt, at mid-stage 9 (supplementary material Fig. S6A), it
is possible that both Sia and VegT are directly involved in the
expression of cer and gsc in prospective ‘E’ and ‘M’ regions,
respectively, at this stage (Fig. 6A,D, indicated by asterisks).
However, because vegt and sia mRNAs still stay in the nucleus and
their expression domains just expand from the endodermal to
mesodermal regions (vegt) and vice versa (sia) during the blastula
stage, accumulation of VegT and Sia may not be enough to regulate
gsc and cer, respectively. If this is the case, what is the significance
of the binding of Sia to cer-U1 (cer in Fig. 6D) and of mVegT to
gsc-U1 (gsc in Fig. 6A) at stage 9? It has been proposed that
some transcription factors bind to tissue-specific enhancers before
actual transcription starts and initiate chromatin marking for
transcriptional competence by modifying histones. Such
transcription factors with these kinds of roles are called ‘pioneer
factors’ (Smale, 2010). Therefore, it is possible to speculate that the
T-box protein mVegT binds to gsc-U1 as well as to cer-U1 as a
pioneer factor in the dorsovegetal region to maintain gsc-U1 in a
poised state (Fig. 6A) until gsc expression starts in this region at
the gastrula stage (Fig. 6B). In fact, the T-box sites in the cer-U1
are necessary for dorsal expression in transgenic analysis (Fig.
2A,B), in which reporter DNA is integrated into chromatin,
whereas the TAAT elements in (Xla_ABC)5 are sufficient for dorsal
expression in DNA injection reporter assays (Fig. 1B), in which
reporter DNA is not integrated into chromatin. Thus, it is possible
that mVegT binds to cer-U1 as a maternal factor to maintain it in a
poised state until mid-stage 9 when cer expression begins.
Likewise, Sia may bind to cer-U1, as a pioneer factor, in the
prospective mesodermal region (Fig. 6D), leading to the expression
of cer in the dorsal mesoderm at the gastrula stage (Fig. 6E).
Although mix1 expression starts before gastrulation, the binding
of Mix1 to cer-U1 and gsc-U1 is mainly seen at the gastrula stage.
As Mix1 forms a complex with Lim1 and Sia on the
3⫻TAAT/ABC element (Yamamoto et al., 2003), binding of Mix1
may require both Lim1 and Sia or other co-factors to form a
complex on cer-U1 and gsc-U1 at the gastrula stage (Fig. 6B,E).
Because Mix1 is expressed ubiquitously in the endoderm and
mesoderm, its role may be to determine the level of cer expression,
whereas Lim1 and Sia determine where cer is expressed.
The gsc reporter gene is activated through the distal element
(DE) by Lim1 and its co-factor Ldb1 (Mochizuki et al., 2000),
and more strongly with Ssdp1 (the same as Ssbp3) (Nishioka et
al., 2005), whereas the cer reporter gene is not activated by Lim1
and Ldb1 (Yamamoto et al., 2003). Therefore, the
Lim1/Ldb1/Ssbp3 complex, together with Otx2 bind to DE to
activate gsc but not cer in the prospective prechordal plate (i.e.
the anterior part of the ‘M’ region) from the gastrula to neurula
stages (Fig. 6E,F). In the anterior endoderm at the neural stage,
none of the five transcription factors genes is expressed (Fig. 5;
supplementary material Fig. S6C), thereby showing no
expression of cer and gsc (Fig. 6C). Thus, all data shown in this
and previous papers are consistent and support our model of
gene regulations in the organizer.
Stepwise formation of the Spemann–Mangold
organizer by three developmental cues
We now discuss what the developmental cues are that induce the
organizer in the dorsal blastopore lip. In our model for organizer
formation, the first cue is vegetally localized maternal factors,
DEVELOPMENT
1658 RESEARCH ARTICLE
Stepwise formation of the Spemann organizer
RESEARCH ARTICLE 1659
including mVegT, in the presumptive endoderm; the second cue is
Sia and Twin, which are directly activated by canonical Wnt
signaling in the BCNE center; the third cue involves transcription
factors, including Lim1, Otx2, Mix1 and FoxA4, which are directly
induced by Nodal/activin signaling. Xnr5 is upregulated in the
interior dorsovegetal region by maternal VegT and Wnt signaling
(Takahashi et al., 2000). Subsequently, Xnr1 and Xnr2 are induced
by Xnr5 in the dorsally tilted vegetal surface (Takahashi et al.,
2006), in which the endodermal marker sox17b is induced at early
stage 9 (supplementary material Fig. S6A). Xnr5 also induces gsc
and maintain chd at stage 8.5 in the BCNE-derived region in
combination with Wnt signaling (Fig. 6D), and induces mix1, lim1
and otx2 in the dorsovegetal region at mid stage 9. At late stage 9
to stage 10, Nodal signaling induces zvegt, mix1, lim1 and otx2 in
the ‘M’ region, and gsc and chd in the ‘E’ region (Fig. 5C,D, Fig.
6B,E). In the ‘M’ region, a combination of zVegT, Mix1, Lim1 and
Otx2 together with Sia induces the cer gene (Fig. 6E). Thus, the
order of developmental cues for organizer formation is (1) VegT
(vegetal cue), (2) Wnt/Sia (dorsal cue) and (3) Nodal (endoderm
and mesoderm cue), which are combined in a stepwise fashion to
induce the organizer-expressing transcription factors in the dorsal
mesodermal and endodermal regions, and finally mediate
organizer-specific transcriptional regulation (Fig. 6). Thus, our
detailed expression patterns and in vivo binding data of
transcription factors have uncovered the stepwise formation of the
Spemann–Mangold organizer.
Acknowledgements
We thank for Toshiaki Tanaka, Mari Itoh and Yumi Izutsu for the transgenic
technique; Shoji Tajima for protein syntheses and purification of polyclonal
antibodies; Yoichi Kato for a protocol for ChIP; K. Misawa for pCS2+2FT-4HA-
DEVELOPMENT
Fig. 6. A model of gene regulations for cer and gsc during organizer formation. (A-C)An endoderm-derived region of the organizer (the ‘E’
region) at the blastula to neurula stages. mVegT upregulates Nodal genes in the vegetal region (an arrow with a broken line in A). (D-F)A BCNE
center-derived region of the organizer (the ‘M’ region). Note that at the blastula stage gsc expression is not solely initiated by Wnt signaling via
Sia/Twin, but in cooperation with Nodal signaling via various Smad2 complexes, as indicated by a ribbon arrow (Laurent et al., 1997; Germain et al.,
2000; Ring et al., 2002; Ku et al., 2005) in the BCNE center-derived organizer region. Bent magenta arrows indicate active transcription; bent black
lines with a vertical line indicate no transcription; asterisks indicate possible binding of Sia or zVegT.
NLS-globin; Hiroki Kuroda and Shuji Takahashi for discussion; and Aaron Zorn
for the sia plasmid and a whole-mount in situ hybridization protocol. We also
thank the National Bioresource Project for Xenopus tropicalis.
Funding
This work was supported in part by Grants-in-Aid for Scientific Research from
the MEXT (Ministry of Education, Culture, Sports, Science and Technology of
Japan) [M.T.]; by Global COE Program (Integrative Life Science Based on the
Study of Biosignaling Mechanisms), MEXT [N.S.]; by Grant-in-Aid for Scientific
Research on Innovative Areas from the MEXT [#21200064 to H.O.]; and by
CREST [H.O.].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.068395/-/DC1
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Stepwise formation of the Spemann organizer