Mechanisms of Development 124 (2007) 775–791
www.elsevier.com/locate/modo
Chordin expression, mediated by Nodal and FGF signaling,
is restricted by redundant function of two b-catenins
in the zebrafish embryo
Máté Varga 1, Shingo Maegawa, Gianfranco Bellipanni, Eric S. Weinberg
*
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
Received 20 April 2007; received in revised form 30 May 2007; accepted 31 May 2007
Available online 12 June 2007
Abstract
Using embryos transgenic for the TOP-GFP reporter, we show that the two zebrafish b-catenins have different roles in the organizer
and germ-ring regions of the embryo. b-Catenin-activated transcription in the prospective organizer region specifically requires b-catenin-2, whereas the ventrolateral domain of activated transcription is abolished only when both b-catenins are inhibited. chordin expression during zebrafish gastrulation has been previously shown in both axial and paraxial domains, but is excluded from ventrolateral
domains. We show that this gene is expressed in paraxial territories adjacent to the domain of ventrolateral b-catenin-activated transcription, with only slight overlap, consistent with the now well-known inhibitory effects of Wnt8 on dorsal gene expression. Eliminating both
Wnt8/b-catenin signaling and organizer activity by inhibition of expression of the two b-catenins results in massive ectopic circumferential expression of chordin and later, by formation of a distinctive embryonic phenotype (‘ciuffo’) that expresses trunk and anterior neural markers with correct relative anteroposterior patterning. We show that chordin expression is required for this neural gene expression.
The Nodal gene squint has been shown to be necessary for optimal expression of chordin and is sufficient in some contexts for its expression. However, chordin is not normally expressed in the ventrolateral germ-ring despite robust expression of squint in this domain. We
show the ectopic circumferential expression of chordin and other dorsal genes to be completely dependent on Nodal and FGF signaling,
and to be independent of a functional organizer. We propose that whereas the axial domain of chordin expression is formed by cells that
are derived from the organizer, the paraxial domain is the result of axial-derived anti-Wnt signals, which relieve the repression that otherwise is set by the Wnt8/b-catenin/vox,vent pathway on latent germ-ring Nodal/FGF-activated expression.
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: b-Catenin; Wnt8; Vox; Vent; Nodal signaling; FGF signaling; Dorsoventral patterning; ichabod (ich)
1. Introduction
Dorsoventral patterning of the zebrafish embryo, as in
other vertebrates, is a consequence of signaling by canonical Wnt, BMP, Nodal, and FGF pathways (reviewed in
Harland and Gerhart, 1997; Schier and Talbot, 1998,
2005; De Robertis and Kuroda, 2004). Both mesodermal
and ectodermal dorsoventral tissue identities are set at least
*
Corresponding author. Tel.: +1 215 898 4198; fax: +1 215 898 8780.
E-mail address: eweinber@sas.upenn.edu (E.S. Weinberg).
1
Present address: Department of Anatomy and Developmental Biology,
University College London, London WC1E 6BT, UK.
in part by the level of BMP signaling (Wilson et al., 1997;
Dosch et al., 1997; Nguyen et al., 1998; Eimon and
Harland, 1999; Barth et al., 1999; Ramel et al., 2005),
which itself is under the control of the secretion of
anti-BMP factors produced in the dorsal organizer region
(reviewed in Harland and Gerhart, 1997; Yamamoto and
Oelgeschlager, 2004). These BMP antagonists include
Chordin (Sasai et al., 1994, 1995; Piccolo et al., 1996),
Noggin (Smith and Harland, 1992; Zimmerman et al.,
1996; Groppe et al., 2002) and Follistatin (HemmatiBrivanlou et al., 1994; Fainsod et al., 1997).
b-Catenin-mediated signaling is essential in the development of the vertebrate dorsal embryonic signaling center.
0925-4773/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mod.2007.05.005
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M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
A functional b-catenin gene is required for formation of the
Spemann organizer in amphibians (Heasman et al., 1994,
2000; Wylie et al., 1996) and for normal axis formation
in the mouse (Haegel et al., 1995; Huelsken et al., 2000).
In the zebrafish, severe ventralization of embryos bred
from females homozygous for the maternal effect ichabod
(ich) mutation is accompanied by a failure of nuclear localization of b-catenin in dorsal cells and the dorsal YSL
(Kelly et al., 2000). These effects are due to an impairment
of maternal expression of a second b-catenin gene, b-catenin-2, which maps in proximity to the ich mutation and is
required for organizer and axis formation (Bellipanni
et al., 2006).
In both amphibians and teleosts, chordin (chd) expression in the organizer region is clearly dependent on b-catenin. In Xenopus, dorsal expression of chd prior to the onset
of gastrulation requires an active b-catenin pathway, and
ectopic b-catenin expression is sufficient to induce expression of chd and other BMP antagonists (Wessely et al.,
2001). In the zebrafish, chd is first expressed in the midblastula on the future dorsal side of the embryo and later
in the embryonic shield in both axial and paraxial domains
(Miller-Bertoglio et al., 1997; Schulte-Merker et al., 1997;
Shimizu et al., 2000). chd expression is absent or greatly
reduced in ich embryos at all stages, but can be induced
by b-catenin produced from injected RNA (Kelly et al.,
2000; Bellipanni et al., 2006; Maegawa et al., 2006). Inhibition of b-catenin function by expression of a dominant negative form of Tcf3 (dnTcf3) (Pelegri and Maischein, 1998)
or by expression of axin (Shimizu et al., 2000) results in
elimination of chd expression at blastula stages (but not
at shield stage, see Section 3).
Wnt signaling pathways are not only important in establishment of the organizer, but also have a modifying or
opposing role during gastrulation of teleost and amphibian
embryos. wnt8 is expressed in the ventrolateral germ-ring
of zebrafish embryos (Kelly et al., 1995) as bicistronic transcripts (Lekven et al., 2001) and is required for formation
of ventrolateral and posterior mesoderm, spinal cord and
posterior brain (Lekven et al., 2001; Erter et al., 2001;
Momoi et al., 2003; Ramel and Lekven, 2004). Ventrolateral Wnt signaling during gastrulation, as well as the earlier
dorsal b-catenin-mediated gene activation, can be visualized in zebrafish embryos carrying a transgene in which
GFP is expressed from a b-catenin-responsive promoter
(Dorsky et al., 2002). In Xenopus, wnt-8 is expressed in a
wide vegetal region including the ventrolateral marginal
zone (Christian et al., 1991; Smith and Harland, 1991;
Christian and Moon, 1993) and has a similar ventralizing
and posteriorizing role (Christian and Moon, 1993; Hoppler et al., 1996; Fredieu et al., 1997). Wnt8 represses organizer function in the zebrafish embryo by inducing the
expression of the homeobox genes vox/vega1 and vent/
vega2 and this induction requires b-catenin (Ramel and
Lekven, 2004). The zygotic expression of a third member
of this homeobox family, ved (Shimizu et al., 2002; Gilardelli et al., 2004), is also partially under wnt8 control (Ramel
et al., 2005). These homeobox proteins act as redundant
transcriptional repressors to limit the expression of genes
such as goosecoid (gsc), bozozok (boz) and chordin (chd)
to the organizer region (Kawahara et al., 2000a,b; Melby
et al., 2000; Imai et al., 2001; Ramel and Lekven, 2004;
Ramel et al., 2005; Shimizu et al., 2005). Similar ventral
homeobox genes (Xvents) also function in Xenopus to
repress organizer gene expression (including gsc and chd)
(Onichtchouk et al., 1996; Hoppler and Moon, 1998;
Melby et al., 1999; Trindale et al., 1999). Loss of function
of zebrafish wnt8 or of both vox/vega1 and vent/vega2
results in expansion of axial mesoderm with similar expansion of chd and other dorsal markers to the lateral germring (Lekven et al., 2001; Erter et al., 2001; Imai et al.,
2001; Ramel and Lekven, 2004; Ramel et al., 2005). These
results are consistent with the finding that in Xenopus, Vox
can directly repress chd transcription (Melby et al., 1999).
Transcription of vox and vent is also under control of
BMP signaling, but mainly starting at 70–75% epiboly
(Kawahara et al., 2000a; Melby et al., 2000; Imai et al.,
2001; Ramel and Lekven, 2004). Thus, in contrast to the
dependence of chd expression on b-catenin in the organizer
and dorsal regions of the embryo during blastula stages,
chd expression during early and mid-gastrula is under negative control of Wnt signaling and b-catenin (through Vox
and Vent) in the ventrolateral germ-ring of the zebrafish
embryo.
b-Catenin induction of chd in the zebrafish is dependent on the homeodomain protein Bozozok and the
Nodal-related factor Squint. squint (sqt) (Feldman
et al., 1998; Erter et al., 1998; Rebagliati et al., 1998)
and bozozok/dharma/nieuwkoid (boz) (Yamanaka et al.,
1998; Koos and Ho, 1998; Fekany et al., 1999) act in
parallel to establish the organizer (Sirotkin et al., 2000;
Shimizu et al., 2000; reviewed in Schier and Talbot,
2005). These two genes are induced by b-catenin (Kelly
et al., 2000; Shimizu et al., 2000; Maegawa et al.,
2006) and embryos lacking both genes fail to express
chd, although single mutant embryos still can express
chd transcript (Sirotkin et al., 2000; Shimizu et al.,
2000; Maegawa et al., 2006). As in amphibians, zebrafish
embryos lacking mesendoderm, due to loss of function of
Nodal genes (in this case, sqt and cyclops) or absence of
One-eyed pinhead, can still express chd and form neurectoderm (Feldman et al., 1998; Gritsman et al., 1999;
Shimizu et al., 2000; Maegawa et al., 2006). Expression
of chd has been shown to be under the control of
FGF signaling in both Xenopus (Mitchell and Sheets,
2001) and zebrafish (Londin et al., 2005; Maegawa
et al., 2006). Moreover, dorsalization of zebrafish
embryos by FGF3 does not occur in embryos (chordino
mutants) lacking chd function (Koshida et al., 2002),
and we have recently shown that FGF signaling is
required for induction of chordin by both Boz and Sqt
(Maegawa et al., 2006). There is thus a firm basis for
a pathway of chd induction by b-catenin mediated by
both Nodal pathway and FGF pathway signaling.
M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
Insight into the multiple roles of b-catenin in the patterning of zebrafish embryos has come from work with
the maternal effect mutant ich. Embryos bred from homozygous ich females are highly ventralized and fail to form a
functional organizer (Kelly et al., 2000). The zebrafish genome contains two b-catenin genes and formation of the
dorsal organizing center was found to be dependent on
maternal expression of b-catenin-2 but not b-catenin-1 (Bellipanni et al., 2006). The deficiency in b-catenin-2 function
is due to a genome rearrangement in close proximity to bcatenin-2 (S. Maegawa, M. Nimmakayalu, B. Emanuel,
E.S. Weinberg, unpublished results). Wild-type embryos
treated with antisense morpholino oligonucleotides
(MOs) directed against b-catenin-2 transcript (bcat2MO),
but not against b-catenin-1 transcript (bcat1MO), show
ventralized phenotypes similar to that of the ich mutant,
and bcat2MO treatment of ich embryos results in a shift
towards the most ventralized phenotypes (Bellipanni
et al., 2006). When ich embryos were treated with bcat1MO
(or with both bcat1MO and bcat2MO), or when wild-type
embryos were treated with both MOs, a new phenotype
was observed (termed ‘ciuffo’), which is characterized by
a protrusion of tissue extending from the vegetal end of
the yolk, robust expression of neurectodermal markers in
apparently correct anteroposterior order, and formation
of neurons (Bellipanni et al., 2006). In ich embryos, which
lack a functional organizer and have low amounts of
maternal b-catenin-2 transcript, b-catenin-1 expression
alone is able to repress the expression of all neural markers
tested. However, in wild-type embryos, inhibition of bcatenin-1 does not lead to an expansion of neural tissue
or patterning defects. Thus, only when expression of both
b-catenin genes is inhibited do we find a de-repression of
neural markers (Bellipanni et al., 2006). That chd was
expressed ectopically around the germ-ring of such
embryos starting at 50% epiboly (Bellipanni et al., 2006)
is consistent with the formation of neurectoderm in these
treated embryos, but the network of control of ventrolateral chd expression was not clear.
We show here that in contrast to the specific requirement for b-catenin-2 in organizer formation, the two zebrafish b-catenins act redundantly in response to Wnt8 to
induce expression of vox and vent and to repress and limit
chd expression to the dorsal and paraxial regions of the
embryo. We demonstrate that ventrolateral b-catenin-activated transcription of a reporter gene expressed from a
promoter with multiple Tcf3 sites (TOP-GFP) is abolished
only when both b-catenins are inhibited, whereas inhibition
of only b-catenin-2 results in failure to activate transcription in the dorsal organizing center domain. We show that
chd expression is normally excluded from the ventrolateral
domain of Wnt/b-catenin signaling activity. Elimination of
this signaling results in a massive circumferential ectopic
expression of chd and development of embryos with the
‘ciuffo’ phenotype. Consistent with previous work in which
wnt8 or vox and vent expression has been inhibited directly
(Erter et al., 2001; Imai et al., 2001; Lekven et al., 2001;
777
Ramel and Lekven, 2004; Ramel et al., 2005) or indirectly
(Reim and Brand, 2006), we showed that in ich embryos
lacking maternal b-catenin-2, inhibition of b-catenin-1
expression leads to ventrolateral germ-ring expression of
chd (Bellipanni et al., 2006). In addition, we now show that
at mid-gastrula, inhibition of both b-catenins, wnt8, or vox
and vent results in a broad circumferential chd expression
domain that encompasses approximately half of the
embryo. The normal pathway of repression of chd in the
ventrolateral germ-ring is thus dependent on Wnt8 (Lekven
et al., 2001; Erter et al., 2001) induction of Vox and Vent
(Imai et al., 2001; Ramel and Lekven, 2004), shown here
to be mediated by both b-catenins. We also present
evidence that the ectopic germ-ring chd expression is completely dependent on germ-ring-derived Nodal and FGF
signaling. Thus, without a functional Wnt8 signaling
pathway, the same factors that normally induce chd on
the dorsal side of the embryo are able to function ventrolaterally to induce the gene.
2. Results
2.1. Dorsal TOP-GFP expression is dependent only on
b-catenin-2 whereas b-catenin-1 or b-catenin-2 is sufficient
for later germ-ring TOP-GFP expression
To test our hypothesis that germ-ring Wnt8 signaling
can be mediated by either of the two b-catenins, we made
use of the Tg(TOP:GFP)w25 transgenic line of zebrafish
in which b-catenin-dependent transcriptional activity can
be monitored by expression of a GFP reporter driven from
a promoter containing multiple Tcf/Lef elements (Dorsky
et al., 2002). As already shown (Dorsky et al., 2002), bcatenin-mediated gene activation can be observed in both
the incipient embryonic shield and in the ventrolateral
germ-ring at 50% epiboly (Fig. 1A). At 70% epiboly,
germ-ring gene activation increases and the dorsal domain
disappears (Fig. 1E). When bcat1MO is injected into these
embryos, there is some decrease in the ventrolateral gene
response at 50% epiboly, but no decrease in dorsal activity
(Fig. 1B). Injection of bcat2MO, in contrast, completely
abolishes dorsal activity and also decreases the ventrolateral activity at this developmental time (Fig. 1C). At 70%
epiboly, when only the ventrolateral germ-ring activity
can be detected in untreated embryos (Fig. 1E), both
bcat1MO and bcat2MO decrease the signaling response
in dorsolateral regions (Fig. 1F and G), but when both
MOs are injected, the b-catenin-mediated signaling activity
completely disappears (Fig. 1H). We conclude that b-catenin-mediated transcription in the organizer is completely
dependent on expression of b-catenin-2, consistent with
our previous finding that maternal b-catenin-2 expression
is required for formation of the organizer (Bellipanni
et al., 2006). We further conclude that the ventrolateral
germ-ring signaling is dependent on both b-catenins, confirming our previous finding that repression of chd in this
domain, coincident with expression of wnt8 (Kelly et al.,
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M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
Fig. 1. The ventrolateral domain of Wnt/b-catenin signaling is dependent on both b-catenin-1 and b-catenin-2, whereas organizer b-catenin-mediated gene
activation is dependent only on b-catenin-2. (A–H) TOP-GFP embryos were injected with bcat1MO (MO1) (B and F), MO2 (C and G), or both bcat1MO
and bcat2MO (MO2) (D and H) and compared to uninjected embryos (A and B) after in situ hybridization with probe for GFP at 50% epiboly (A–D) or
70% epiboly (E–H). Embryos are shown in animal view (A–D) or lateral view (E–H). (I and J) chd expression is excluded from the ventrolateral Wnt/bcatenin signaling domain. TOP-GFP embryos at 70% epiboly were assayed by two color in situ hybridization for both GFP (blue) and chd (red)
expression. A single embryo is presented in both animal pole (I) and vegetal pole (J) views to show the complementarity of chd and Wnt signaling domains
at this stage. The arrows in (I and J) point to a slight region of overlap of the chordin expression and Wnt signaling domains. All embryos are shown with
dorsal to the right; (E–H) animal pole is to the top.
1995; Erter et al., 2001; Lekven et al., 2001), was relieved
only when expression of both b-catenins is inhibited
(Bellipanni et al., 2006). In contrast to the apparently equal
involvement of both b-catenins in ventrolateral Wnt
signaling at mid-gastrula, the germ-ring expression at
50% epiboly appears to be more dependent on b-catenin2 than b-catenin-1.
2.2. Exclusion of chordin expression from the mid-gastrula
wnt signaling domain
To examine the relationship of chd expression to the
ventrolateral domain of Wnt8 signaling, we performed
two-color double in situ hybridizations to assay both chordin and GFP transcripts in TOP-GFP embryos. At 70%
epiboly, chd is expressed in a butterfly like pattern with
an axial domain and winged paraxial domains, and is not
expressed in the ventrolateral germ-ring (Miller-Bertoglio
et al., 1997). In TOP-GFP embryos assayed at this stage,
chd expression is clearly present in dorsal and paraxial
regions which do not exhibit b-catenin-activated transcription (Fig. 1I and J). Marginal ventrolateral Wnt8/b-catenin
signaling decreases towards the dorsal side of the embryo
and fades out in the region where paraxial chd expression
domain can be detected. At 70% epiboly, the most lateral
extent of paraxial chordin expression only slightly overlaps
the boundary of marginal b-catenin-activated transcription
(arrows in Fig. 1I and J). These patterns are consistent with
an inhibitory threshold of Wnt8 signaling on dorsal gene
expression.
2.3. Inhibition of Wnt-b-catenin signaling causes ectopic
expression of dorsal markers in the germ-ring
We previously reported circumferential ectopic germring expression of gsc and chd at 50% epiboly in ich
embryos treated with bcat1MO (Bellipanni et al., 2006).
These embryos, deficient in expression of both b-catenin1 and b-catenin-2, exhibit the ‘ciuffo’ neurectodermal-containing protrusions at 24 hpf. As ectopic chd expression is
M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
first noticeable in the germ-ring of these embryos only at
50% epiboly (1.3 h later than when expression of the gene
is first detected in wild-type embryos, Miller-Bertoglio
et al., 1997), it was of interest to look somewhat later
in gastrulation to see if this expression is sustained.
Although expression is not observed in untreated or
bcat2MO-injected ich embryos at 70% epiboly (Fig. 2B
and D), a massive amount of ectopic chd transcript is
found all around the germ-ring at 70% epiboly in
bcat1MO-treated ich embryos (Fig. 2C). A similar circumferential expression of chd was observed in wild-type
embryos treated with bcat1MO and bcat2MO (data not
shown). We conclude that either one of the two b-catenins
is sufficient to prevent the ectopic expression of chd, but
when expression of both b-catenins is inhibited, this
repression is relieved. The pattern of expression is quite
different from the winged-type pattern (dorsal and flanking paraxial domains) observed in wild-type embryos at
this stage (Fig. 2A; Miller-Bertoglio et al., 1997). We
often observed a somewhat wider band of expression in
ich embryos treated with both MOs (Fig. 2E), suggesting
that even the low amounts of endogenous maternal bcatenin-2 in ich embryos (Bellipanni et al., 2006) may
act to repress chd expression to some extent. We also
examined the expression of noggin1 (nog1) in embryos
lacking one or both b-catenins. Like chd, nog1 expression
is first observed at 4 hpf in the presumptive organizer area
in wild-type embryos and is then strongly expressed in
axial mesendoderm (Fürthauer et al., 1999; Fig. 2F). A
more diffuse transverse stripe of nog1 expression extending into paraxial mesoderm can be detected at the end
of gastrulation (Fürthauer et al., 1999; not shown in
779
Fig. 2F, which is at an earlier stage). The gene is not
expressed in untreated or bcat2MO-injected ich embryos
(shown at 70% epiboly in Fig. 2G and I), but is first
detected in bcat1MO-injected ich embryos as a faint ring
of ectopic expression in the germ-ring at 50% epiboly
(5.3 hpf) (not shown), and like chd, is massively ectopically expressed at 70% epiboly all around the germ-ring
(Fig. 2H). These data extend our findings that the two
b-catenins mediate the repression of dorsal-specific genes
in the germ-ring domain during early gastrula stages,
(Bellipanni et al., 2006), but in addition, we demonstrate
that relief of this repression results in dorsal gene expression in a extremely wide marginal band coincident with
the zone of ventrolateral Wnt signaling at 70% epiboly
(Fig. 1E). This band of expression is much wider and
more intense than in other studies in which lateral ectopic
expression of chd has been observed (Pelegri and Maischein, 1998; Shimizu et al., 2000, 2002; Imai et al., 2001;
Ramel and Lekven, 2004; Ramel et al., 2005; Bellipanni
et al., 2006), and the strong expression extends completely
around the embryo. In these prior studies, the massive
mid-gastrula expression was not reported, as the latest
embryonic stage examined for chd expression was the
shield stage. We also examined expression of bmp4 in
ich embryos treated with both b-catenin MOs and found
that although inhibition of b-catenins resulted in a
decrease in bmp4 expression, there was still considerable
bmp4 transcript throughout the embryo (compare
Fig. 6M,S with 6P,V). The remaining bmp4 expression
in the marginal zone represents an unusual case of uncoupling of the feedback control that regulates the reciprocal
expression of chd and BMP genes.
Fig. 2. Elimination of b-catenin signaling induces the ectopic expression of dorsal genes around the germ-ring. At 75% epiboly, wild-type embryos have
characteristic patterns of chd (A) and nog1 (F) expression, whereas ich embryos do not express these genes (B and G). Injection of bcat1MO (MO1) into ich
embryos induces expression of chd (H) and nog1 (H) around the germ-ring, but bcat2MO (MO2) has no effect (D and I). Co-injection of the two MOs into
ich embryos causes somewhat increased expression of chd (E) and nog1 (J) around the germ-ring. Concentrations of MOs injected were 3 mM for the single
MO1 or MO2 injections and 3 mM each for the double MO injection. Wild-type embryos are shown in dorsal view (A and F) and all other embryos are
shown in lateral views. Expression (or lack of) was identical for all embryos for each treatment (>16 in each case) except for (I) in which 1/19 embryos
showed very weak expression.
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M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
2.4. Chordin is required for the ‘ciuffo’ phenotype and for
expression of neural markers when both b-catenins are
inhibited
To test whether the ectopic expression of chd is essential for establishment of the neurectoderm in ‘ciuffo’
embryos, we inhibited expression of both chd and the
two b-catenins in ich embryos. Injection of an MO
against chd (chdMO) into ich embryos resulted in a flatter tail protrusion (compare Fig. 3A and B) and, as in
untreated mutant embryos (Fig. 3E, I, and M), no
expression of the neural markers emx1 (Fig. 3E) and
krox20 (Fig. 3I). (That this MO yielded a flatter tail
region may indicate a low level of tail chd expression
in ich embryos that has not been detected by other
means.) When ich embryos were injected with bcat1MO
and bcat2MO to eliminate expression of both b-catenins,
the embryos developed massive ‘ciuffo’ protrusions at
24 hpf (Fig. 3C and Bellipanni et al., 2006), in which patterned expression of emx1 (Fig. 1G) and krox 20
(Fig. 1K) could be observed. emx1 expression was found
close to the yolk (arrow, Fig. 3G), indicating the position
of tissue with telencephalon identity (Morita et al., 1995),
and krox20 expression was found further toward the tip
of the protrusion, often in two stripes or rings (arrowheads, Fig. 3K), corresponding to tissue of rhombomere
3 and 5 identity (Oxtoby and Jowett, 1993). These results
show that the embryo can express neural markers with
correct relative antero-posterior pattern, with posterior
oriented toward the tip of the ‘ciuffo’ protrusion, in
embryos inhibited in expression of both b-catenins.
The role of Chordin in expression of these markers was
tested by co-injection of chdMO along with bcat1MO and
bcat2MO. This treatment resulted in suppression of the
‘ciuffo’ phenotype, with all embryos developing a broad
tail-like appendage (much larger than that formed by the
uninjected ich embryos) and failure to form the necrotic
animal pole protrusion (found in both untreated and
chdMO-injected ich embyos) (Fig. 3D). The expression of
neural markers in these embryos was suppressed (emx1,
Fig. 3H; krox20, Fig. 3L), with virtually all embryos examined showing no detectable expression (27/27 embryos for
emx1, 26/28 embryos for krox20).
We also examined expression of myoD in these four classes of embryos (Fig. 3M–P) and found that this gene,
which is normally expressed in the tail regions of ich
embryos (Fig. 3M) is not inhibited by chdMO, bcat1MO
and bcat2MO, or a combination of all three MOs
(Fig. 3N–P). The strong effect of the triple MO injection
on neural marker expression without concomitant inhibition of myoD expression indicates that the loss of neural
marker expression is a specific response to the chdMO,
and is not merely due to toxicity or other non-specific
effects of the administration of multiple MOs. We
conclude, therefore, that chd expression is required for formation of hindbrain and anterior neurectoderm in embryos
lacking expression of the two b-catenins.
To determine whether the effects of inhibition of chd
resulted in ectopic expression of neural markers in even
earlier ‘ciuffo’ embryos, we assayed expression of otx2
(Fig. 4A–C) in 10 hpf embryos, comparing uninjected ich
embryos, bcat1MO and bcat2MO ‘ciuffo’ embryos, and
embryos co-injected with chdMO along with bcat1MO
and bcat2MO. As expected, otx2 was not expressed in ich
embryos (Fig. 4A). Injection of the two bcat MOs into
ich embryos clearly resulted in induction of otx2 in an
equatorial band extending completely around the embryo
(Fig. 4B), and the additional injection of chd MO completely suppressed this induction (Fig. 4C). We also
assayed for expression of the epidermal marker foxi1 at this
stage in the three types of embryos (Fig. 4D–F). foxi1 is
expressed ubiquitously in ich embryos, with the most
intense expression found toward the vegetal pole
(Fig. 4D). Injection of the two bcat MOs into ich embryos
resulted in a marked suppression of expression of foxi1
(Fig. 4E), except in the animal pole region which most
probably gives rise to the epidermal tissue covering the
yolk cell in 24 hpf ‘ciuffo’ embryos. Intense foxi1 expression was restored in ich embryos injected with all three
MOs (Fig. 4F). These experiments suggest that the lack
of neural gene expression in 24 hpf ‘ciuffo’ embryos treated
with chdMO (Fig. 3H and L) is not due to the loss of neurectodermal cells that might have been present in these
embryos at earlier developmental times. Analysis of
10 hpf embryos clearly shows ‘ciuffo’ embryos can robustly
express an early neural marker, and that expression of this
marker is completely abolished on inhibition of chd. The
lack of chd expression appears to respecify ectoderm from
neurectodermal to epidermal identity.
2.5. Wnt8 signaling, mediated by vox/vent and b-catenin-1/2, prevents expression of chordin in the germ-ring
If the Wnt8/Vox/Vent pathway acts to prevent germring chd expression, we would predict that loss of function
of wnt8 or of vox and vent in ich embryos would have the
same effect on chd expression as the loss of expression of
the two b-catenins. Co-injection into ich embryos of MOs
that target the two wnt8 transcripts (Fig. 5B) or co-injection of MOs targeting vox and vent transcripts (Fig. 5D)
resulted in the same intense broad circumferential band of
chd expression at 70% epiboly as observed in ich embryos
injected with bcat1MO or bcat1MO + bcat2MO (Fig. 2C
and E). chd expression was not observed in embryos injected
with just one or the other Wnt8 MO or with voxMO or
ventMO alone (data not shown), indicating that either
wnt8 ORF product or either Vox or Vent alone is sufficient
to repress the ectopic chd expression in ich embros, as has
been shown in wild-type embryos (Lekven et al., 2001; Imai
et al., 2001; Ramel and Lekven, 2004). To test whether
b-catenin depletion had an effect on vox and/or vent transcript levels, we examined the expression of these two genes
in ich embryos injected with bcat1MO + bcat2MO. Each
of these genes is expressed ubiquitously in untreated ich
M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
781
Fig. 3. chordin expression is required for expression of neural markers when both b-catenins are inhibited. 24 hpf untreated ich embryos (A, E, I, and M),
and ich embryos injected with chdMO alone (B, F, J, and N), bcat1MO and bcat2MO ([MO1 + MO2] C, G, K, and O), or all three MOs (D, H, L, and P)
were photographed alive (A–D) or after in situ hybridization with probe for emx1 (E–H) krox20 (I–L), or myoD (M–P). ich embryos, which are highly
ventralized if untreated (A), do not express emx1 (E), or krox20 (I), but do express myoD in the tail region (M). When depleted of b-catenin-1 and bcatenin-2 expression by MO treatment, embryos develop the ‘ciuffo’ phenotype (C, bracket indicates the protrusion that has very different morphology
from the tail region of ich embryos, indicated by arrow in A) and express emx1 (G, arrow points to single band of expression), and krox20 (K, arrowheads
point to two bands of expression), in addition to myoD (O). ich embryos injected with chdMO alone remain highly ventralized, with a broader but less
protrusive tail (B, arrow) than in untreated ich embryos, and show no induction of emx1 (F) or krox20 (J). When ich embryos are injected with chdMO as
well as the two b-catenin MOs, the posterior protrusion is no longer ‘ciuffo-like’, but rather is tail-like, and the anterior necrotic protrusion (at the end of
the embryo opposite the arrow) seen in ich embryos (A) and embryos treated with chdMO alone (B) fails to form (D). The embryos treated with the three
MOs are inhibited in both emx1 (H) and krox20 (L) expression but do express myoD in the tail region (P), indicating that the failure of emx1 and krox20
expression in the triple MO-injected embryos was not due to non-specific toxic effects of the MOs. Over 24 embryos were examined for each marker and
each condition, with results identical to embryo shown in the panel (except for panel N in which 1 of 24 embryos did not express myoD and panel K in
which 2 of 28 embryos did not express krox20). Concentrations of MOs injected were 3 mM for each of the b-catenin MOs and 0.6 mM chdMO.
embryos (Fig. 5E and G), as would be expected in embryos
lacking boz expression (Kawahara et al., 2000a, 2000b;
Imai et al., 2001). In ich embryos injected with the two bcatenin MOs, both vox and vent were found to be downregulated at 70% epiboly compared to their expression in
uninjected ich control embryos (compare Fig. 5E to 5F
and 5G to 5H). bcat1MO or bcat2MO treatment of
wild-type embryos did not reduce vox and vent transcript
levels (data not shown), indicating that either of the two
b-catenins is sufficient to mediate wnt8 activation of vox
and vent. Taken together, these results show that even
in the absence of the organizer, wnt8 represses the expres-
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M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
Fig. 4. chordin expression is required for otx2 expression and suppresses foxi1 expression in 10 hpf embryos inhibited in expression of both b-catenins.
Untreated ich embryos (A and D) and ich embryos injected with bcat1MO and bcat2MO ([MO1 + MO2] (B and E), or additionally with chdMO (C and
F) were assayed for expression of otx2 (A–C) or foxi1 (D–F). Inhibition of expression of the two b-catenins results in induction of the anterior neural
marker otx2 at the expense of expression of the epidermal marker foxi1. The inhibition of Chd in such embryos prevents otx2 expression and restores the
foxi1 expression. Concentrations of MOs injected were as in Fig. 3 and embryos are shown in lateral views.
Fig. 5. Germ-ring expression of chordin is repressed by Wnt8 through the transcriptional repressors Vox and Vent. ich embryos co-injected with MOs
targeted against both ORFs of wnt8 (B) or with MOs against both vox and vent (D) show ectopic expression of chd around the germ-ring. Uninjected ich
embryos do not express chd (A and C). Suppression of b-catenin signaling in the germ-ring of ich embryos by injection of bcat1MO + bcat2MO
(MO1 + MO2) causes a decrease of vox and vent expression (compare E with F, and G with H). Embryos are shown in lateral (A–D) or animal (E–H)
views at 70% epiboly. Concentrations of MOs injected were 0.6 mM of each of the two Wnt8MOs, 0.7 mM of each of voxMO and ventMO, and 3 mM of
each of the two b-catenin MOs.
sion of chd by activation of vox and vent expression and
that either of the two b-catenins can transduce the
Wnt8 signal. These conclusions are consistent with the
finding that only the combined inhibition of both b-catenin transcripts can inhibit endogenous Wnt signaling in
wild-type embryos (Fig. 1H).
Both the combination of vox and vent MOs or of the
two Wnt8 MOs, when injected into ich embryos, also gave
rise to many embryos with a ‘ciuffo’-like phenotype (data
not shown). These embryos all expressed krox20, whereas
none of the uninjected control ich embryos expressed this
marker (data not shown). These results indicate that
M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
wnt8, the two b-catenin genes, and vox and vent all act in a
pathway of repression of germ-ring ectopic chd, which
when expressed result in neurectoderm formation in the
absence of the organizer.
2.6. Nodal and FGF signaling is required for ectopic germring chordin and goosecoid expression
We next investigated the factors that act to induce chd
expression in the absence of Wnt8 signaling. The ventrolateral germ-ring normally expresses several genes (sqt, fgf3,
fgf8) that are expressed somewhat earlier in the presumptive organizer. chd expression in the organizer is partially
dependent on a pathway in which FGFs are induced by
sqt, and in turn FGFs then signal cells to activate chd
(Maegawa et al., 2006; see Section 3). Both sqt and FGF
genes are also expressed in the germ-ring of ich embryos,
indicating that this domain of expression is independent
of the organizer. We therefore considered the possibility
that the very same Nodal and FGF pathway that activates
chd in the organizer could potentially function in the germring to yield ectopic chd transcripts. In wild-type embryos,
we hypothesize that this latent activity is prevented by signaling by Wnt8, acting through the two b-catenins.
To test this hypothesis, we blocked either Nodal or FGF
signaling in bcat1MO + bcat2MO treated ich embryos and
examined chd expression at 70% epiboly (Fig. 6A–F).
sqtMO or cycMO, alone, each reduced the level of ectopic
germ-ring chd expression produced when the two b-catenins are inhibited (data not shown), and the combination
of sqtMO and cycMO completely eliminated this expression (compare Fig. 6D with 6E). This result demonstrates
that ectopic germ-ring chd expression that occurs when gastrula wnt8 signaling is eliminated is totally dependent on
Nodal signaling. To test for the role of FGF signaling,
we injected mkp3 RNA, which is effective in blocking the
transduction of FGF signals in zebrafish embryos (Tsang
c
Fig. 6. Germ-ring expression of chordin and goosecoid in embryos with
impaired Wnt8/b-catenin signaling is dependent on Nodal and FGF
signaling pathways. Circumferential germ-line expression of chd (D) and
gsc (J) in ich embryos depleted of b-catenins by co-injection of bcat1MO
and bcat2MO (MO1 + MO2) was eliminated in embryos co-injected with
additional MOs against sqt and cyc (E and K) or with mkp3 RNA to
inhibit FGF signaling (F and L). Uninjected control ich embryos and ich
embryos injected solely with sqtMO + cycMO or with mkp3 RNA did not
express chd (A–C) or gsc (G–I). The widespread ubiquitous expression of
bmp4 in ich embryos (M and S) is somewhat reduced when the embryos
are co-injected with bcat1MO and bcat2MO (P and V). Injection of ich
embryos with sqtMO + cycMO (N and T) or with mkp3 RNA (O and U)
does not reduce bmp4 expression. Injection of the four MOs, sqtMO +
cycMO + bcat1MO + bcat2MO (Q and W), or mkp3 RNA + the two bcat
MOs (R and X), also did not reduce bmp4 expression, except in the latter
case, the expression was less uniform with greater expression towards the
margin. Results for eve1 expression were essentially identical to that of
bmp4 (data not shown). Although only one embryo is shown per panel,
results were completely uniform for all embryos in each panel (n > 25 in
each case). Embryos are all at 70% epiboly and are shown in animal views
(A–R) and for the embryos of (M–R), also shown in lateral view (S–X).
783
et al., 2004). MAP kinase phosphatase 3 (MKP3) has been
shown to inhibit FGF/MAPK signaling activity by dephosphorylation of MAPK proteins (reviewed in Tsang
et al., 2004). Expression of mkp3 completely eliminated
the ectopic germ-ring chd expression produced in ich
embryos injected with the two b-catenin MOs (compare
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M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
Fig. 6D with 6F). Similar results were obtained for goosecoid (gsc) expression (Fig. 6G–L). To test whether the
administration of multiple MOs causes non-specific toxic
effects that would lead to failure of chd or gsc expression,
we also examined the effects of these treatments on bmp4
(Fig. 6M–X) and eve1 (data not shown) expression. Both
of these markers were robustly expressed under exactly
the same conditions that resulted in failure to express chd
and gsc. Thus, both Nodal and FGF signaling are required
for the activation of chd and gsc expression that occurs on
inhibition of Wnt8-b-catenin signaling. As we have already
demonstrated in the previous section that wnt8 and vox and
vent can repress germ-ring chd expression in ich embryos
(that still express b-catenin-1), it is likely that Wnt8 signaling counteracts latent Sqt/FGF activation of chd.
3. Discussion
3.1. b-Catenin-1 and b-catenin-2 are each sufficient to
transduce wnt signaling and prevent chordin expression in the
germ-ring
Two distinct areas of b-catenin-mediated gene activation, a dorsal domain in the prospective organizer and a
ventrolateral germ-ring domain correlated with Wnt8 signaling, can be visualized in TOP-GFP transgenic zebrafish
embryos (Dorsky et al., 2002; Fig. 1A). These two domains
have opposite consequences for dorsoventral patterning of
the zebrafish embryo: the organizer promotes dorsal fates,
and the ventrolateral Wnt8 signaling ventralizes mesoderm
and posteriorizes neurectoderm. Using MOs specific for
each b-catenin, we show that ventrolateral germ-ring
TOP-GFP activity at 70% epiboly is decreased by either
bcat1MO or bcat2MO, but is eliminated only when expression of both b-catenins is inhibited (Fig. 1F–H) (at 50%
epiboly, it is more dependent on b-catenin-2; Fig. 1A–C).
In contrast, the early dorsal domain of TOP-GFP activity
is completely dependent on b-catenin-2 (Fig. 1A–C), as
expected from our results showing that maternal b-catenin-2 expression is required for formation of the organizer
(Bellipanni et al., 2006). This difference in regulation cannot be due to lack of expression of b-catenin-1 in the dorsal
region, as transcripts of both genes are detected in the
dorsal and ventrolateral germ-ring embryonic domains
(Bellipanni et al., 2006). This finding suggests that there
may be functional differences between the two b-catenins,
differences in stability or sub-cellular localization of the
two proteins in the dorsal region, or a block to translation
of b-catenin-1 in this region. In wild-type embryos, chd
expression and TOP-GFP expression only overlap slightly
(Fig. 1I and J). Class 1–3 ich embryos, deficient in maternal
b-catenin-2 expression, do not express chd at all (Kelly
et al., 2000; Bellipanni et al., 2006; Fig. 2B). However,
when these embryos are also depleted of b-catenin-1 activity by bcat1MO injection, massive chordin expression is
induced circumferentially in a wide band extending from
the germ-ring (Fig. 2C). nog1 and gsc are also induced in
this region in bcat1MO-injected ich embryos (Fig. 2H; Bellipanni et al., 2006). Injection of bcat1MO or bcat2MO
alone do not cause induction of these genes in wild-type
embryos, but the presence of the two MOs together also
induces circumferential expression of chd in wild-type
embryos (data not shown). Thus, expression of either of
the two b-catenins is sufficient to repress chd expression
in the germ-ring. Only when expression of both b-catenins
is prevented (either by MO treatment and, for b-catenin-2,
as a result of the ich mutation), is chd expressed in this
region.
3.2. In the absence of organizer, de-repression of chordin is
required for patterned expression of neural markers
When expression of both b-catenins is inhibited,
embryos form a distinctive phenotype (‘ciuffo’), in which
epiboly movements propel tissue away from the vegetal
pole in a protrusion that expresses trunk and anterior neural markers. These neural genes are expressed in correct
anteroposterior pattern, with posterior markers expressed
towards the tip of the protrusion and the most anterior
markers expressed close to the yolk (Fig. 3G and K; Bellipanni et al., 2006). The ‘ciuffo’ phenotype and expression of
emx1 and krox20 neural markers was completely suppressed in 24 hpf embryos, and expression of otx2 was suppressed in 10 hpf embryos, when chd MO was co-injected
into ich embryos along with MOs for both b-catenins (compare Fig. 3G,K with Fig. 3H,L; and Fig. 4B with 4C).
Moreover, induction of otx2 in ‘ciuffo’ embryos and its
repression in ‘ciuffo’ embryos inhibited in chd expression
was accompanied by the reciprocal repression and induction of the epidermal marker foxi1. chd expression is thus
required in ‘ciuffo’ embryos for neural specification of ectoderm, which is reversed to epidermal identify in the absence
of Chd. Inhibition of chd expression in the embryos
injected with MOs or both b-catenins, however, does not
fully restore the ich phenotype (compare Fig. 3A and D),
indicating that not all the effects of loss of both b-catenins
are mediated by chd.
The massive expression of chd in bcat1MO + bcat2MO-treated ich embryos would be expected to cause
a profound inhibition of BMP signaling due to direct
binding of Chd to ligand (Piccolo et al., 1996). The neural
induction default model, based on experiments with
amphibian embryos, posits that neural induction occurs
in ectoderm in the absence of BMP (Wilson and Hemmati-Brivanlou, 1995, 1997; reviewed in Harland, 2000). Our
results suggest that inhibition of BMP signaling is sufficient for neuralization in zebrafish as well, whereas in
chick, Chordin and Noggin cannot induce ectopic neural
markers (reviewed in Streit and Stern, 1999a,b). We find
that neuralization can also be induced in ich embryos
by injection of a MO for bmp2b, circumventing the need
for chd induction and that such neurectoderm also exhibits correct relative anteroposterior marker expression (M.
Varga, S. Maegawa, E.S. Weinberg, unpublished observa-
M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
tions). These results are similar to those of Reversade
et al. (2005), who showed that large amounts of neurectoderm with a degree of normal AP pattern could be
formed in Xenopus embryos lacking organizer, if expression of BMP genes was inhibited.
bcat1MO + bcat2MO-treated ich embryos (‘ciuffo’) do
not resemble swirl/bmp2b mutant embryos, which neuralize
the entire ectoderm in its normal position, not in a protrusion at the vegetal pole as in ‘ciuffo’ embryos (Mullins
et al., 1996). swirl/bmp2b embryos have expanded organizer activity, and although dorsalized, still retain an axis
and dorsoventral pattern, whereas ‘ciuffo’ embryos are
completely radialized and have a distinctive vegetal protrusion. There are at least two possible reasons for this difference. First, as BMP signaling is involved in convergence
movements (von der Hardt et al., 2007), the circumferential
expression of chd in gastrulating ‘ciuffo’ embryos would
reorient the BMP activity gradient from vegetal to animal
(instead of ventral to dorsal), thus increasing the migration
of cells towards the vegetal pole and subsequently past the
pole into a protrusion, which effectively would replace the
dorsal axis as the destination of converging cells. These
‘ciuffo’ embryos exhibit a significant level of BMP transcript throughout the embryo (shown for bmp4 expression
at 70% epiboly in Fig. 6V). Such BMP expression would be
absent in swirl embryos. Second, there may be functions of
one or the other b-catenins required for convergence movements, thus eliminating restraints on epiboly in ‘ciuffo’
embryos.
3.3. Repression of chordin by Wnt8 signaling
How Vox and Vent act to repress chd is not yet known,
but this repression is clearly initiated by Wnt8/b-catenin-1
and -2 induction of vox and vent. vent/vox/ved and wnt8
expression is low or absent in the dorsal marginal area that
expresses chd at 40% epiboly and shield stages (Kawahara
et al., 2000a,b; Ramel and Lekven, 2004; Shimizu et al.,
2005). Here, consistent with the studies of Ramel and Lekven (2004) done at shield stage, we show that in embryos
lacking organizer and expression of both b-catenins (ich
embryos injected with bcat1MO or wild-type embryos
injected with bcat1MO + bcat2MO), wnt8 (ich embryos
injected with Wnt8-ORF1&2MOs), vox and vent (ich
embryos injected with vox and vent MOs), the same broad
intense ectopic circumferential expression of chd is
observed bordering the mid-gastrula germ-ring (Figs.
2C,E, 4B,D, 7E,E 0 ). Ectopic chd expression in ich embryos
treated with bcat1MO (or both bcat1MO and bcat2MO) is
much more intense than previously reported in wild-type
embryos inhibited in wnt8 or vox and vent expression or
in mutants for these genes (Imai et al., 2001; Ramel and
Lekven, 2004; Ramel et al., 2005), but these studies used
earlier embryonic stages than the 70% epiboly embryos
that we investigated.
Relief of inhibition of germ-ring chd and gsc during
gastrulation is not only observed when both b-catenins
785
are inhibited (Bellipanni et al., 2006; this work), but also
when embryos express a dominant negative (dn) Tcf3 (Pelegri and Maischein, 1998; Lewis et al., 2004) or overexpress
axin (Shimizu et al., 2000), each of which would be
expected to inhibit the function of both b-catenins. These
studies are also consistent in showing that inhibition of
b-catenin (b-catenin-2 in our work) early in development
abolishes organizer-dependent expression of chd. The
expression of dnTcf3 (Pelegri and Maischein, 1998) and
axin (Shimizu et al., 2000) led to different effects depending
on the time of development. At sphere stage, expression of
these proteins resulted in the elimination of dorsal markers,
including chd, whereas at shield stage, expression of dorsal
markers was expanded. The work we present here clarifies
the role of the b-catenins at these two developmental times.
At sphere stage, activation of dorsal genes is completely
dependent on b-catenin-2, whereas at shield stage, the
repression of dorsal genes can be mediated by either of
two b-catenins in the ventrolateral germ-ring. A decrease
in b-catenin function or levels by a mechanism that does
not discriminate between the two b-catenins, thus has
opposite outcomes at the two developmental stages.
Maintenance of vox and vent expression during late
blastula in both non-marginal and marginal zones is also
dependent on maternal pou2 (spg) function and MZspg
embryos show ectopic ventrolateral expansion of chd,
nog1, and gsc (Reim and Brand, 2006). This pathway acts
in parallel to Wnt8 during late blastula stages but would
most likely not have been perturbed in the embryos we
treated with b-catenin MOs. vox and vent expression
becomes dependent on BMP signaling, predominantly
starting at 70–75% epiboly (Kawahara et al., 2000a; Melby et al., 2000; Imai et al., 2001), but decreases in expression of these genes is more severe in wnt8;swr(bmp2b)
mutants than in wnt8 mutants alone (Ramel and Lekven,
2004), indicating that even at shield stage, vox and vent
expression depend on at least three pathways controlled,
respectively, by wnt8, bmp2b, and pou2. The effects on
dorsal gene expression that we observe at 70% epiboly
as a consequence of b-catenin MO treatment of ich
embryos are most likely entirely due to elimination of
Wnt8 signaling.
3.4. Germ-ring potential for chordin expression
During zebrafish organizer formation both FGF signaling (Maegawa et al., 2006) and either Nodals or Bozozok are required for the dorsal induction of chd (Sirotkin
et al., 2000; Shimizu et al., 2000; Maegawa et al., 2006).
Dependence of FGF expression on Nodal signaling has
previously been shown for mesodermal precursors in the
zebrafish embryo (Mathieu et al., 2004; Bennett et al.,
2007) and we have demonstrated that this is also the case
for prospective organizer tissue (Maegawa et al., 2006).
Several studies have shown that FGFs can induce chd
(Mitchell and Sheets, 2001; Koshida et al., 2002; Londin
et al., 2005), and we have found a requirement for FGF
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M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
Fig. 7. Two-step model of chordin gene regulation in the zebrafish gastrula. (A) During early- and mid-blastula stages, b-catenin-2 enters the nuclei of the
dorsal YSL and dorsal blastomeres triggering the transcriptional activation of boz and sqt, which in turn induce axial expression of chd (expressed in the
region shown in dark blue) in a process dependent on FGF signaling. boz is shown activating chd by inhibition of a repressor, X, which could be one or
more of the Vox/Vent/Ved class of homeobox repressor proteins. Approximately 30 min after the first dorsal appearance of sqt and FGF transcripts,
Nodal signals from the YSL induce sqt expression in the lateral and ventral germ-ring, and germ-ring Sqt induces expression of FGF genes in this region.
(B) By mid-gastrula stage, axial mesoderm (shown in dark blue) continues to express chd and is also the source of anti-Wnt secreted proteins such as Fzb1.
These anti-Wnt and anti-BMP proteins will diffuse laterally and form a region (shown in beige) in which Wnt and BMP signaling is inhibited In cells
around the germ-ring, wnt8 is induced by Nodal signaling. A broad band of potential germ-ring derived chd expression (light blue) is established by sqt/cyc
acting through FGFs. In the paraxial regions where Wnt8 is inhibited by anti-Wnt proteins (light blue/grey stipes), chd is expressed, whereas in the more
lateral and ventral regions, Wnt8, acting through Vox and Vent, inhibits the expression of chd. Indicated in grey typeface and arrows are those parts of the
pathway that normally do not operate in that particular region due to the normal inhibitory network.
signaling in b-catenin and Sqt induction of chd (Maegawa
et al., 2006). Since both sqt and FGF genes are expressed
in the germ-ring of wild-type and ich embryos, we tested
whether the same pathway that activates chd in the organizer could also induce chd transcripts in the germ-ring.
Indeed, blocking either Nodal or FGF signaling in bcat1MO + bcat2MO treated ich embryos (Fig. 6A–F) pre-
vented the germ-ring ectopic chd expression that
normally results from inhibition of both b-catenins. A
similar requirement for Nodal and FGF signaling was
also demonstrated for the induction of germ-ring gsc
expression in bcat1MO + bcat2MO injected ich embryos
(Fig. 6G–L). In wild-type embryos, this latent activity
can be prevented by signaling by Wnt8, acting through
M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
vox and vent and the two b-catenins. Our evidence suggests that Wnt8 signaling in the germ-ring is required
to inhibit the induction of chordin by a pathway dependent on germ-ring expression of both Nodal and FGF
ligands.
3.5. Control of axial and paraxial domains of chordin
expression
We propose a model to explain the restriction of chd
expression to axial and paraxial domains, and the control
of expression within each of these domains. During early
stages of development (Fig. 7A), dorsal canonical Wnt/bcatenin signaling (driven by b-catenin-2) induces the
expression of early dorsal marker genes (e.g., boz, sqt)
(Fekany et al., 1999; Shimizu et al., 2000). chd is induced
dorsally by Sqt and Boz (Sirotkin et al., 2000; Shimizu
et al., 2000) and this activation is dependent on FGF signaling (Maegawa et al., 2006). Slightly later than the time
of first dorsal expression of sqt, signals emanating from
the YSL induce the expression of Nodal genes in germ-ring
blastomeres (Chen and Kimelman, 2000) (Fig. 7B). The
germ-ring Nodals will in turn induce the expression of
FGF genes (Gritsman et al., 1999; Mathieu et al., 2004)
and wnt8 (Erter et al., 2001), which activates expression
of the transcription of vox and vent (Ramel and Lekven,
2004) (Fig. 7B). As both Vox and Vent are known repressors of chd (Kawahara et al., 2000a,b), it would be expected
that they have this effect in the whole germ-ring, except for
the most dorsal/axial region, where wnt8 is not expressed
(Lekven et al., 2001). However, chd is expressed in extensive paraxial domains at this stage (e.g, Figs. 2A and
7A). We propose that this later paraxial expression
(Fig. 7B, striped region) is dependent on germ-ring sqt
and FGF expression, and not directly on organizer-derived
sqt and FGFs. We posit that in these paraxial regions, the
potential inhibition of chd expression by Wnt8 and Vox/
Vent is inhibited by secreted anti-Wnt proteins derived
from axial tissues (e.g., fzb1, Fig. 7B). This model is substantiated in wild-type embryos by: (1) increases in the
chd paraxial domain obtained by increasing the amount
of the Wnt antagonist Fzb1 by injecting fzb1 RNA (frzb
– Zebrafish Information Network) RNA (Shinya et al.,
2000) (data not shown) and reducing the transduction of
Wnt8 signaling by injection of MOs targeted against vox
and vent or by using vox;vent mutant embryos (data not
shown; Imai et al., 2001; Ramel and Lekven, 2004, and
Ramel et al., 2005); (2) elimination of all paraxial and all
but a small amount of axial chd expression when Nodal
activity was eliminated by injection of antivin RNA (data
not shown); and (3) by decrease in axial chd expression
by injection of boz-pNA or in boz mutant embryos (data
not shown; Shimizu et al., 2000). These experiments are
all consistent with the two-step model of chd expression,
which defines a non-expressing ventrolateral zone, a
germ-ring Nodal-dependent/axial anti-Wnt factor-dependent paraxial zone, and an axial zone dependent on orga-
787
nizer genes. chd is thus expressed in an initial step in the
organizer and axial regions dependent on the b-catenin2 fi Sqt [and Boz] fi FGF pathway (Fig. 7A), and a later
paraxial step in which is also dependent on germ-ring Sqt
and FGFs (Fig. 7B). Chd expression from both of these
domains is available to diffuse and bind to BMP ligands,
creating a dorsoventral gradient of BMP activity thought
to be a major determinant in dorsoventral patterning of
the embryo (reviewed in Schier and Talbot, 2005).
3.6. Comparative aspects of chordin expression
The relevance of the model of control of chd expression
presented above to control of this gene in other model
animals is still not clear. Unfortunately, expression pattern data from other teleost species (Medaka, Fugu, and
Tetraodon) is not yet available for the genes involved.
However, a number of findings in Xenopus are useful to
consider. Xenopus chd is expressed strongly in the dorsal
lip during gastrulation (Stages 10–11). At Stage 11.5 chd
expression, though still dorsal, becomes a bit more diffuse,
and continues to be expressed in axial mesoderm (but not
paraxial mesoderm) even during neurulation (Sasai et al.,
1994). Repressive effects of ventral Xvent1 and Xvent2
factors on chd and gsc expression have been demonstrated
(Gawantka et al., 1995; Onichtchouk et al., 1998; Melby
et al., 1999) and some of these ventral factors are upregulated by Xwnt8 (Hoppler and Moon, 1998; Friedle and
Knöchel, 2002). Despite these similarities, however, inhibition of b-catenin by MOs in Xenopus does not result
in a circumferential upregulation of chd (Reversade
et al., 2005). The most plausible explanation for this
may be that there are a high number of functionally
redundant ventralizing homeobox factors in Xenopus.
The Xvent1 and Xvent2 subfamilies have several members: Xvent1 (Gawantka et al., 1995), PV.1 (Ault et al.,
1996), Xvent1B (Rastegar et al., 1999), Xvent2 (Onichtchouk et al., 1996), Xbr-1b (Papalopulu and Kintner,
1996), Xom (Ladher et al., 1996), Vox (Melby et al.,
1999), Xvent2B (Rastegar et al., 1999). These factors
appear to be regulated by different mechanisms. While
all can be activated by BMP signaling, this regulation
can be direct (Xvent2B) or indirect, through other intermediate molecules such as GATA-2 (Xvent1B) (Friedle
and Knöchel, 2002). Thus, far, only three members of
the Xvent subfamilies have been shown to be under
Xwnt8 control [xvent1, vox (Hoppler and Moon, 1998)
and Xvent1B (Friedle and Knöchel, 2002)]. It is possible
that some of the other ventralizing homeodomain factors
are not regulated by Wnt signaling. Therefore, even in a
b-catenin-MO treated Xenopus embryo, one or more of
these factors would still be able to repress the expression
of dorsal genes in ventrolateral regions.
In the chick, chd is first found in the pre-primitive streak
embryo in the posterior epiblast just anterior to Koller’s
sickle and in a few middle layer cells, both of which contribute to the organizer, and is then expressed in the ante-
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M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
rior tip of the primitive streak, in Hensen’ node, and subsequently in the head process and notochord (Streit et al.,
1998). Chick chd can be induced by a combination of
Vg1, Wnt1, and the secreted protein Tsukushi (Ohta
et al., 2004), but the network of control of chd expression
has not yet been elucidated. As in the zebrafish, both Nodal
and FGF signaling are implicated as essential for induction
of chd in the chick embryo (Betochhini and Stern, 2002;
Bertocchini et al., 2004). Wnt-3a, Wnt5a, and Wnt-8c are
all expressed in the primitive streak and node region
(Hume and Dodd, 1993; Hollyday et al., 1995; Baranski
et al., 2000; Rodrı́guez-Esteban et al., 2001). Loss-of-function experiments have not been reported that would
explain which of the Wnts are involved in induction of dorsal genes, including chd, and which if any might be involved
in repression of these genes in posterior (i.e., ventrolateral)
regions of the streak. The roles of Chd in the chick are
somewhat different than observed in frogs and fish.
Although Chd can induce an ectopic primitive streak in
the chick, it is not sufficient for generation of somites and
intermediate mesoderm or for neural induction (Streit
et al., 1998; Streit and Stern, 1999a,b). In the mouse
embryo, chd is first expressed in the anterior primitive
streak and then in the node and in axial mesoderm, but
not in the anterior visceral endoderm (Bachiller et al.,
2000). In mouse embryos, Wnt3 is expressed in the primitive streak and around the node, (Liu et al., 1999) and
Wnt3-/- embryos lack these tissues and do not express a
variety of dorsal markers (Liu et al., 1999). Wnt3a is also
expressed in the primitive streak and dorsal posterior node
(Nakaya et al., 2005), but null mice do form a primitive
streak and node but have an ectopic neural tube instead
of posterior somites, suggesting that this gene is required
for the cell fate decision between paraxial mesoderm and
neurectoderm (Takada et al., 1994; Yoshikawa et al.,
1997). Expression of chd has not been reported in these
mutants. Phenotypes of loss-of-function have not been
reported for other Wnt genes expressed in the primitive
streak during gastrulation, which include Wnt8 (Bouillet
et al., 1996) and Wnt2b (Kemp et al., 2005). In mouse
embryos, as in the chick, the relationship of expression of
each Wnt species to the expression and repression of chd
is still unkown.
Taken together, these data suggest that particular
aspects of the mechanism of chd regulation (e.g. expression in the paraxial mesoderm) appear to be rather specific to zebrafish or perhaps teleosts. Other vertebrates
appear to use different mechanisms to fine-tune the
expression of chd to modulate BMP signaling during gastrulation. In the absence of experimental data from other
fish species, it is not possible to know whether the paraxial mesodermal expression of chd expression is a general
characteristic of teleosts. The comparison of chd regulation between different fish species, as well as between fish
and frog, could provide important insights into the evolution of BMP regulation in different phylogenetic
lineages.
4. Experimental procedures
4.1. Zebrafish strains and husbandry
Wild-type embryos were derived from AB parents. ichabodp1 (ich)
embryos were obtained by breeding homozygous ich females with brass
males. Only those ich females that reproducibly yielded severely ventralized (Class 1 and 1a; see Kelly et al., 2000; Maegawa et al., 2006) embryos
were used in this work. Adult zebrafish were maintained under standard
conditions (Westerfield, 1993). To examine endogenous b-catenin activity,
we monitored GFP expression in Tg(TOP:GFP)w25 embryos.
4.2. DNA clones, RNA preparation, hybridization, and RNA
injection
The following clones were used to prepare antisense probes for hybridization and sense RNAs for embryo injection: goosecoid in pBS-SK (Stachel et al., 1993), chd in pCS2+ (Miller-Bertoglio et al., 1997), noggin1 in
pCS2+ (Fürthauer et al., 1999), vox (vega1) and vent (vega2) in pBS
(Kawahara et al., 2000a,b), mkp3 in pCS2+ (Tsang et al., 2004), krox20
(Oxtoby and Jowett, 1993), emx1 (Morita et al., 1995), foxi1 in pCS2+
(Solomon et al., 2003), otx2 in pCS2+ (Li et al., 1994), and gfp in
pCS2+ (Dorsky et al., 2002). Antisense RNA probes were synthesized
and single probe in situ hybridization was carried out as previously
described (Bellipanni et al., 2006). For two color in situ hybridization,
we combined a modification of the protocol of Teraoka et al. (2004) with
our standard method as follows: After staining with Purple AP substrate
(Roche) was completed, samples were washed three times in PBST and
treated with 0.1 M Glycine–HCl (pH 2.2), 0.1% Tween-20 for 10 min at
room temperature to incativate the phosphatase. After several PBST
washes, embryos were incubated in 1:1000 dilution of alkaline-phosphatase-conjugated anti-fluorescein Fab fragments (Roche) overnight at 4C.
Subsequently, samples were washed six times with PBST (for 15 min each)
and three times with 0.1 M Tris–HCl (pH 8.2)/0.1% Tween-20 (5 min
each). Finally, embryos were stained with Fast-Red (Roche) following
the manufacturer’s protocol.
Sense RNAs to be injected into embryos were synthesized and capped
using an mMessage mMachine Kit (Ambion), following the manufacturer’s protocol. These RNAs were stored in dH2O at 80 C and injection solutions were prepared by diluting a 2· stock of RNA in dH2O
with an equal volume of Dulbecco’s modified phosphate-buffered saline
containing 5% phenol red (Sigma). Approximately 1 nl of RNA solution
was injected through the intact chorion into the yolk at the base of blastomeres of 1–4 cell embryos, using an agar mold to hold the embryos.
The concentration of mkp3 mRNAs injected was 0.4 lg/ll.
4.3. Morpholino antisense oligonucleotide injections
Morpholino antisense oligonucleotides (MOs) were obtained from
Gene Tools (Philomath, OR). b-catenin-1-MO1 (bcat1MO) (Bellipanni
et al., 2006), b-catenin-2-MO1 (bcat2MO) (Bellipanni et al., 2006),
bmp2bMO (Imai and Talbot, 2001), chdMO (Nasevicius and Ekker,
2000), wnt8-ORF1MO and wnt8-ORF2MO (Lekven et al., 2001), sqtMO
(Feldman and Stemple, 2001), and cycMO (Karlen and Rebagliati, 2001)
were manufactured by Gene Tools (Philomath, OR) and the sequences of
each are as published. The amounts of these antisense reagents that were
injected per embryo were as follows: bcat1MO and bcat2MO (3 mM each,
when injected alone or together), bmp2bMO (0.4 mM), sqtMO and cycMO (0.6 mM each, when injected alone or together), chdMO (0.6 mM),
wnt8MOs (0.6 mM each, injected together), ventMO and voxMO
(0.7 mM each, when injected alone or together).
Acknowledgements
This work was supported by National Institute of
Health Grant RO1-HD39272 to E.S.W. We are grateful
M. Varga et al. / Mechanisms of Development 124 (2007) 775–791
to Richard Dorsky for arranging for us to obtain the
Tg(TOP:GFP)w25 line of fish and to Tetsurohito Kudoh
for the foxi1 plasmid. We thank Mary Mullins and Daniel
Kessler for helpful discussions and Joshua Bradner, Jianyan He, Richard Gill Jr., and Carol Hu for fish care
and general laboratory assistance.
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