RESEARCH ARTICLE 3265
Development 133, 3265-3276 (2006) doi:10.1242/dev.02483
FGF signaling is required for ␤-catenin-mediated induction
of the zebrafish organizer
Shingo Maegawa, Máté Varga and Eric S. Weinberg*
We have used the maternal effect mutant ichabod, which is deficient in maternal ␤-catenin signaling, to test for the epistatic
relationship between ␤-catenin activation, FGF signaling and bozozok, squint and chordin expression. Injection of ␤-catenin RNA
into ichabod embryos can completely rescue normal development. By contrast, when FGF signaling is inhibited, ␤-catenin did not
induce goosecoid and chordin, repress bmp4 expression or induce a dorsal axis. These results demonstrate that FGF signaling is
necessary for ␤-catenin induction of the zebrafish organizer. We show that FGFs function downstream of squint and bozozok to
turn on chordin expression. Full rescue of ichabod by Squint is dependent on FGF signaling, and partial rescue by FGFs is completely
dependent on chordin. By contrast, Bozozok can rescue the complete anteroposterior axis, but not notochord, in embryos blocked
in FGF signaling. Surprisingly, accumulation of bozozok transcript in ␤-catenin RNA-injected ichabod embryos is also dependent on
FGF signaling, indicating a role of FGFs in maintenance of bozozok RNA. These experiments show that FGF-dependent organizer
function operates through both bozozok RNA accumulation and a pathway consisting of ␤-cateninrSquintrFGFrChordin, in which
each component is sufficient for expression of the downstream factors of the pathway, and in which Nodal signaling is required for
FGF gene expression and FGF signaling is required for Squint induction of chordin.
INTRODUCTION
FGF signaling is involved in patterning and tissue formation of early
vertebrate embryos (reviewed by Sivak and Amaya, 2004; Böttcher
and Niehrs, 2005). Expression of XFD, a dominant negative form of
Xenopus FGF receptor 1 (FGFR1), in Xenopus and zebrafish
embryos results in loss of trunk and tail tissues (Amaya et al., 1991;
Amaya et al., 1993; Griffin et al., 1995). FGF signaling has also been
shown to be required for neural induction (Streit et al., 2000; Wilson
et al., 2000; Böttcher and Niehrs, 2005), for organizer-independent
induction of posterior neural tissue (Kudoh et al., 2002; Kudoh et al.,
2004), anterior neurectoderm formation (Hongo et al., 1999) and
posterior mesoderm development (Draper et al., 2003).
Perhaps due to the pleiotropic effects that result from inhibition
of FGF signaling in early embryos, a role of FGF signaling in dorsal
axis establishment – as distinct from mesoderm induction, posterior
development and neural induction – has not been emphasized in
recent reviews (De Robertis and Kuroda, 2004; Niehrs, 2004).
Nevertheless, substantial evidence does indicate that FGF signaling
is involved in establishment of the dorsal axis. In Xenopus, targeted
injections of XFD RNA to blastomeres that give rise to the Spemann
organizer but not to trunk mesoderm itself, result in trunk and tail
deficiencies, but only slightly affect head development (Mitchell and
Sheets, 2001). These results show that FGF signaling is required for
trunk-inducing activities of the Spemann organizer. Ectopic
expression of fgf8 in the zebrafish embryo can induce a partial
secondary axis, but not head (Fürthauer et al., 1997), indicating that
in this organism FGF signaling is sufficient for partial organizer
function. Evidence for the importance of FGF signaling in axis
Department of Biology, University of Pennsylvania, Goddard Labs 316, Philadelphia,
PA 19104-6017, USA.
*Author for correspondence (e-mail: eweinber@sas.upenn.edu)
Accepted 9 June 2006
formation also comes from studies on chordin (chd) and BMP
expression. In both Xenopus (Mitchell and Sheets, 2001) and
zebrafish (Londin et al., 2005), chd expression is dramatically
reduced in embryos blocked in FGF signaling, and dorsalization of
embryos by overexpression of fgf3 does not occur in embryos (dino
mutants) lacking chd function (Koshida et al., 2002).
Also consistent with a role for FGFs in dorsal axis formation is
the relationship between FGF gene expression and potential
upstream signaling pathways. Organizer formation is fully
dependent on ␤-catenin in amphibians, mice and zebrafish
(Heasman et al., 1994; Wylie et al., 1996; Heasman, 2000; Huelsken
et al., 2000). In zebrafish embryos bred from female ichabod
homozygotes, a maternal effect mutation resulting in severe
ventralization and loss of head and trunk, ␤-catenin fails to localize
to dorsal YSL and blastomere nuclei (Kelly et al., 2000). These
deficits are due to impairment of maternal expression of a second ␤catenin gene, ␤-catenin-2, which maps in proximity to the ichabod
mutation and is required for establishment of the early dorsal
signaling center (Bellipanni et al., 2006). ichabod embryos injected
with FGF8 RNA developed trunk and anterior structures such as
forebrain and eyes, although a complete anterior brain and
notochord did not form (Tsang et al., 2004). fgf3 and fgf8, as well as
mkp3, a modulator and itself a target of FGF signaling, are first
normally expressed in the dorsal margin of blastula-stage zebrafish
embryos (Fürthauer et al., 1997; Tsang et al., 2004). ichabod
embryos fail to express these genes in this early dorsal domain,
suggesting that FGF ligands operate downstream of ␤-catenin in
dorsal axis formation (Tsang et al., 2004). ␤-catenin is also required
for expression of XFGF3 in both dorsal and non-dorsal regions of
Xenopus blastulae, and ␤-catenin overexpression causes stimulation
of XFGF3 expression (Schohl and Fagotto, 2003). Activation of the
MAPK pathway is the most common downstream response to FGF
signaling (Böttcher and Niehrs, 2005). MAP kinase phosphatase 3
(MKP3) has been shown to inhibit FGF/MAPK signaling activity
DEVELOPMENT
KEY WORDS: ␤-Catenin, FGF signaling, Dorsal axis formation, Neural induction, Dorsoventral patterning, Anteroposterior patterning,
Organizer, Nodal, Chordin, Zebrafish
by dephosphorylation of MAPK proteins (reviewed by Tsang et al.,
2004) and expression of MKP3 in zebrafish embryos can eliminate
FGF signaling and cause ventralization (Tsang et al., 2004). These
experiments taken together indicate that FGF signaling operates
downstream of ␤-catenin in dorsal axis establishment.
We have utilized the ichabod mutant embryo system to work
out the pathway relationships between these factors, the Nodal
protein gene squint (sqt), and the homeodomain protein gene
bozozok/dharma/nieuwkoid (boz). sqt (Feldman et al., 1998; Erter et
al., 1998; Rebagliati et al., 1998) and boz (Yamanaka et al., 1998;
Koos and Ho, 1998; Fekany et al., 1999) act in parallel downstream
of ␤-catenin (Shimizu et al., 2000; Kelly et al., 2000) to establish the
organizer (Sirotkin et al., 2000; Shimizu et al., 2000). As embryos
bred from homozygous ichabod mothers fail to form a Nieuwkoop
center equivalent region (Kelly et al., 2000), they can be used to
determine which factors and signaling pathway components are
downstream of ␤-catenin, and which aspects of embryonic
patterning are dependent on this pathway (Kelly et al., 2000; Tsang
et al., 2004; Kudoh et al., 2004; Gore et al., 2005). An advantage of
this system is that individual downstream components of the
pathway can be expressed in the absence of expression of other ␤catenin-dependent factors. Using this approach, we show that FGF
signaling is absolutely required for ␤-catenin activation of the
organizer and dorsal axis, that at least one mode of dorsal axis
formation proceeds through the pathway ␤-cateninrSquintrFGF
signalingrChordin, and that FGF signaling is required for dorsal
axis formation at additional steps downstream of ␤-catenin,
including the accumulation of boz transcript and boz induction of
chd.
MATERIALS AND METHODS
Fish husbandry and stocks
To obtain ichabod p1 heterozygous and homozygous fish, we crossed ichabod
homozygous males with ichabod heterozygous females. Embryos derived
from crosses of ichabod homozygous females and brass or AB strain males
are essentially all ventralized and are referred to as ‘ichabod embryos’ in this
study. Only those matings that yielded predominantly the most severely
ventralized phenotypes (Class 1; see Fig. 1B) (Kelly et al., 2000) were used
in this work. Embryos derived from crosses of ichabod heterozygous
females and brass or AB strain have wild-type phenotype and served as
wild-type controls.
In-situ hybridization, RNA synthesis and injection and antisense
oligonucleotide injection
Antisense RNA probes were synthesized and in-situ hybridization
procedures were performed as previously described (Hashimoto et al.,
2004), with BM purple alkaline phosphatase substrate (Roche) used instead
of NBT/BCIP as chromogen in some experiments. Capped mRNAs were
synthesized using the appropriate mMessage mMachine Kit (Ambion),
following the manufacturer’s protocol. Approximately 1 nl of RNA solution
was injected through the intact chorion into the yolk at the base of the
blastomeres of 4-8-cell stage embryos (except for the case of Fig. 8S). The
amounts of mRNA that were injected were as follows: fgf8 (0.1 pg), fgf3 (1
pg), ␤-catenin-1 (50 pg), bozozok (50 pg), squint (10 pg), XFD (200 pg),
d50, (200 pg), and mkp3 (200 pg). The following morpholino antisense
oligonucleotides (MOs) were obtained from Gene Tools (Philomath, OR):
MOsqt (5⬘-ATGTCAAATCAAGGTAATAATCCAC-3⬘), MOcyc (5⬘-GCGACTCCGAGCGTGTGCATGATG-3⬘), MOchd (5⬘-ATCCACAGCAGCCCCTCCATCATCC-3⬘). The pNA-grip antisense oligonucleotide,
pNAboz (N-TTCAAGTGTAGGGGTGCC-C), was used to inhibit bozozok
expression. These specific reagents have all previously been shown to
specifically phenocopy the respective mutant phenotypes (Feldman and
Stemple, 2001; Karlen and Rebagliati, 2001; Nasevicius and Ekker, 2000;
Urtishak et al., 2003), and we confirmed that they produced fully penetrant
phenotypes in wild-type embryos in our experiments and also yielded these
Development 133 (16)
phenotypes when co-injected with ␤-catenin RNA into ichabod embryos
(data not shown). The amounts of these antisense reagents that were injected
were: MOsqt (8 ng), MOcyc (8 ng), MOchd (1 ng) and pNAboz (2.2 ng).
RESULTS
FGF3 and FGF8 can restore anteroposterior
patterning and partially restore dorsoventral
patterning in embryos lacking an organizer
We previously reported (Tsang et al., 2004) that ichabod embryos
do not express fgf3 and fgf8 in the prospective organizer region and
that they can be partially rescued by FGF3 or FGF8 RNA injection
to form eyes and/or some anterior neurectoderm, but not notochord
of fully normal anterior brain (Tsang et al., 2004). As targeted
injection of FGF RNA into one cell at the 4-8-cell stage can elicit
secondary axis formation (Fürthauer et al., 1997), and rescue of
ichabod is more efficient with ␤-catenin RNA if injected in this
fashion, we repeated our experiments with injections into one cell of
ichabod embryos at this stage. By contrast with our earlier
experiments, in which at most only 7% of the injected embryos
formed normal appearing nervous systems, we obtained a high
percentage of embryos with no neural defects (50% for FGF8 and
21% for FGF3), although notochords still did not form (Fig. 1A).
The higher degree of rescue could have resulted from a higher local
concentration of ligand. Injecting a combination of the two FGFs did
not increase the percentage of embryos with completely normal
anterior neurectoderm and did not induce notochord. These
experiments show that FGF signaling was sufficient to induce
normal-appearing neural tissue of both anterior and posterior
identity, a novel finding. Based on these results, all mRNA and
antisense oligonucleotide injections reported in the following
experiments (with the exception of the RNA injection in Fig. 8S)
were carried out by injection into one cell at the 4-8-cell stage.
To better characterize the phenotype of FGF mRNA-injected
ichabod embryos, we examined the expression of genes involved in
dorsoventral (DV) and anteroposterior (AP) patterning (Fig. 1G-Z).
FGF3 and FGF8 could induce otx2 (Fig. 1H-J) as well as hoxb1b
(Fig. 1M-O) during gastrulation in ichabod embryos, indicating that
FGF signaling is sufficient for correctly patterned expression of
these two markers in the absence of dorsal ␤-catenin. Both FGFs
could induce the formation of at least a partial organizer in ichabod
embryos, as shown by the expression of goosecoid (gsc) (Fig. 1RT) and floating head (flh) (data not shown) in the embryonic shield
region at 50% epiboly. These embryos went on to express gsc in the
prechordal plate at tailbud stage (Fig. 1W-Y). FGF8 is more effective
than FGF3 in inducing all of these markers, in agreement with the
greater degree of rescue of ichabod embryos by FGF8 RNA (Fig.
1A). FGF expression did not result in no tail (ntl) or flh expression
in a recognizable notochord in injected ichabod embryos, although
weak expression of flh was observed in the midline mesoderm at
tailbud stage (data not shown). FGFs appear to be sufficient to
induce notochord precursor cells, but not for their maintenance. By
contrast, FGF expression was sufficient to induce and maintain
posterior and anterior neurectoderm, organizer and prechordal plate
in embryos deficient in dorsal ␤-catenin-mediated signaling.
FGF signaling is required for ␤-catenin-mediated
organizer formation
To examine whether FGF signaling is required for ␤-catenin
activation of organizer formation, we tested the effect of FGF
signaling pathway inhibitors on ␤-catenin RNA rescue of ichabod
embryos. To block FGF signaling, we injected RNA for the
dominant negative version of FGFR-1, XFD (Amaya et al., 1991),
DEVELOPMENT
3266 RESEARCH ARTICLE
FGF signaling and organizer formation
RESEARCH ARTICLE 3267
Fig. 1. FGF3 and FGF8 can partially
restore AP and DV patterning in
ichabod embryos. (A) Partial rescue of
ichabod embryos injected with RNAs for
FGF3 (orange) or FGF8 (green), compared
with uninjected ichabod embryos (blue)
or ␤-catenin-injected embryos (red).
(B-F) Phenotypic classes of 24 hpf
ichabod embryos (Kelly et al., 2000;
Tsang et al., 2004) used for the
classification in A and in subsequent
figures. Class 1 embryos lack head and
trunk; class 1a lack all structures anterior
to spinal cord; class 2 lack structures
anterior to hindbrain; class 3 form
deficient forebrain; class 4 lack notochord
(as do the previously mentioned classes)
but have a complete AP axis. Only class 5
embryos (not shown) appear normal at
24 hpf. The arrowhead in E indicates the
position of the midbrain/hindbrain
boundary; the arrow in F points to an
eye. (G-Z) FGFs restore expression of
markers of anterior and posterior
neurectoderm and organizer. FGF3 and
FGF8 induce otx2 (H-J), hoxb1b (M-O)
and gsc (R-T,W-Y). Expression is
compared to wild-type (G,L,Q,V),
uninjected ichabod (H,M,R,W) and
ichabod embryos injected with ␤-catenin
RNA (K,P,U,Z). Embryos are shown at
70% epiboly (G-P, lateral views), 50%
epiboly (Q-U, animal pole views) and 10
hpf tailbud stage (V-Z, dorsal views).
third method of inhibiting FGF signaling, treatment with SU5402,
an inhibitor of FGF receptor activity (Mohammadi et al., 1997). In
ichabod embryos co-injected with ␤-catenin RNA and SU5402,
induction of gsc was severely inhibited (Fig. 2J,M). On the basis of
three independent methods of inhibiting FGF signaling, we conclude
that such signaling is required for ␤-catenin to induce organizer
genes and rescue organizer-deficient embryos.
Squint acts upstream of FGF in organizer
formation and Nodal signaling is required for
␤-catenin induction of FGFs
As ␤-catenin can induce dorsal expression of sqt and boz in the
zebrafish embryo and expression of either sqt or boz can fully rescue
ichabod (Kelly et al., 2000; Shimizu et al., 2000), we next examined
the relationship between FGF signaling and expression of these two
genes during ␤-catenin-induced organizer formation. Dependence
of FGF expression on Nodal signaling has previously been shown
for mesodermal precursors in the zebrafish embryo (Mathieu et al.,
2004). Overexpression of the Nodal proteins Sqt or Cyclops (Cyc)
DEVELOPMENT
along with ␤-catenin RNA into a single blastomere at the 4-8-cell
stage. The ability of ␤-catenin to dorsalize and rescue ichabod
embryos was completely inhibited by XFD, as shown by
morphological examination of 24 hours post fertilization (hpf)
injected embryos (Fig. 2A,E,F). To confirm the specificity of the
XFD effect, we also injected embryos with RNA encoding the nonfunctional FGF receptor, d50 (Amaya et al., 1991), which did not
inhibit ␤-catenin dorsalization and rescue (Fig. 2A,E,G). We also
examined gsc expression at shield stage in embryos injected with
these RNAs. ␤-catenin can induce gsc in ichabod embryos (Kelly et
al., 2000) (Fig. 2J), but XFD completely blocked this induction,
whereas d50 had no effect (Fig. 2K,L). This result suggests that FGF
signaling is required for an early step in formation of the organizer
in response to ␤-catenin. To confirm that FGF signaling is essential
for ␤-catenin dorsalization and rescue, we repeated the experiment
of Fig. 2A, injecting RNA for MKP3, a protein effective in
eliminating FGF signaling in zebrafish embryos (Tsang et al., 2004).
In agreement with the results of XFD expression, MKP3 strikingly
inhibited ␤-catenin rescue of ichabod (Fig. 2B). We also utilized a
3268 RESEARCH ARTICLE
Development 133 (16)
Fig. 2. FGF signaling is required for ␤catenin-induced organizer formation.
(A,B) Distribution of phenotypes of
ichabod embryos injected with RNAs for
␤-catenin alone (red) or co-injected with
RNAs for ␤-catenin and the inhibitor of
FGF signaling (yellow) XFD (A), MKP3 (B)
or the inactive FGF receptor, d50 (light
blue, A). Uninjected ichabod embryos
were severely ventralized (dark blue). ␤catenin can partially or completely rescue
most embryos, but both the dominant
negative FGF receptor, XFD (A) or MKP3
(B) inhibit this rescue. The non-functional
FGF receptor, d50, had no effect on ␤catenin rescue (A). (C-G) Typical
representative embryos are shown for
each condition of treatment. (H-M) FGF
signaling is essential for formation of the
organizer, as assayed by gsc expression.
At 50% epiboly, wild-type embryos
robustly express this gene in the shield
region (H), but ichabod embryos are
devoid of expression (I). ␤-catenin
induction of gsc in ichabod embryos (J) is
abolished by coexpression of XFD (K) or
by injection of SU5402 (M) but not d50
(L). Embryos in H-M are shown in animal
pole views.
FGF signaling is required for AP and DV rescue of
ichabod by Squint but not Bozozok
The experiments described thus far suggest that ␤-catenin rescue of
ichabod embryos proceeds at least in part via the linear pathway, ␤cateninrNodalsrFGFsrorganizer function. As Sqt, but not Boz,
can directly induce FGF genes, we compared the ability of Sqt and
Boz to rescue ichabod under conditions of inhibition of FGF
signaling. When XFD RNA was co-injected with sqt RNA into
ichabod embryos, the ability of Sqt to rescue and dorsalize was
completely inhibited (Fig. 4A-C,I), whereas d50 expression had no
effect (Fig. 4D,I), consistent with the linear pathway mentioned
above. By contrast, Boz could restore a significant degree of
dorsalization as well as anterior neural tube formation (class 3* and
4 embryos; Fig. 4J), but not notochord. Many of the rescued
embryos, however, had abnormal trunk and tail morphologies, even
when they developed two eyes (e.g. Fig. 4G). Thus, FGF signaling
is absolutely necessary for the induction of dorsal and anterior
tissues by Sqt in ichabod embryos, but Boz induction of both dorsal
and anterior structures – although not notochord – is at least partially
independent of FGF signaling.
Chordin induction by FGFs and chordin
dependence of FGF partial rescue of ichabod
We next investigated events downstream of FGF signaling. As
several studies have shown that FGFs can induce the BMP
antagonist gene, chd (Mitchell and Sheets, 2001; Koshida et al.,
2002; Londin et al., 2005), we tested whether FGFs could stimulate
the expression of chd in ichabod embryos. Injection of 1 pg of
FGF3 or 0.1 pg of FGF8 RNA, sufficient for rescue of the complete
DEVELOPMENT
is sufficient to induce fgf3 and fgf8, and early expression of these
FGF genes in presumptive mesoderm does not occur in MZoep
embryos lacking Nodal signaling (Mathieu et al., 2004).
We first examined whether FGF3 or FGF8 could induce sqt and
boz in ichabod embryos and found that neither gene was induced by
either of the FGFs (data not shown). By contrast, Sqt and Boz both
could induce fgf3 and fgf8 in ichabod embryos (Fig. 3A-E,G-K). As
we had previously shown that Boz can induce Sqt in both wild-type
and ichabod embryos (Kelly et al., 2000), we tested whether Boz
might function through Sqt to induce fgf3 and fgf8 expression. Coinjection of boz RNA and an MO targeting sqt (MOsqt) into ichabod
embryos completely blocked the induction of fgf3 and fgf8 obtained
by injection of boz RNA alone (Fig. 3E,F,K,L). Thus, as ␤-catenin
can induce both sqt and boz, and boz can induce sqt under these
conditions, ␤-catenin induction of FGFs proceeds through induction
of Sqt and subsequent Nodal signaling.
To test this further, we examined the effects of inhibiting sqt or
boz on ␤-catenin induction of fgf3 and fgf8 in ichabod embryos
(Fig. 3M-T). In this case, MOs for both sqt and cyc were injected
along with ␤-catenin RNA, in case the two Nodal proteins could
redundantly transduce the ␤-catenin activation effect. Treatment
with the two Nodal MOs completely eliminated ␤-catenindependent FGF gene expression (Fig. 3O,S), whereas co-injection
of boz antisense PNA (pNAboz) (at a concentration that
phenocopied the boz mutation when injected into ichabod embryos
along with ␤-catenin) had only a slight effect (Fig. 3P,T). These
results show that Nodal proteins are required for ␤-catenin
induction of FGFs and that boz is not essential for this part of the
pathway.
FGF signaling and organizer formation
RESEARCH ARTICLE 3269
AP pattern of neurectoderm in ichabod embryos (Fig. 1), induced
expression of chd (Fig. 5D-F,H-L) and repressed bmp2b (Fig. 5PR,T-X) and bmp4 (data not shown) in these embryos. Although we
found that FGF8 and FGF3 both induced chd and repressed bmp2b
during gastrulation, only FGF8 had this effect at 30% epiboly (Fig.
5D,G,J). Expression of FGF3 after injection of 1 pg of RNA neither
induced chd nor repressed bmp2b at this stage (Fig. 5P,S,V). Thus,
under conditions of axis formation, we were unable to dissociate
chd induction from bmp2b repression. We did confirm work of
others (Fürthauer et al., 2004; Londin et al., 2005) that FGF3 could
repress bmp2b at 30% epiboly if 50 pg of RNA was injected (Fig.
5D⬘), and we also found that this repression was independent of chd
expression by co-injecting the RNA and a morpholino
oligonucleotide against chd (MOchd) (Fig. 5F⬘). However, the
physiological significance of effects using this high concentration
of RNA is not clear, as such embryos are hyperdorsalized and do
not form a recognizable axis.
To test to what extent chd was required for FGF3 and FGF8
partial rescue of ichabod embryos, we co-injected MOchd along
with 1 pg of FGF3 RNA or 0.1 pg of FGF8 RNA into these embryos.
Inhibition of chd expression caused a complete block in FGFdependent dorsalization (Fig. 6A,B,E,F,H,I). However, co-injection
of MOchd and ␤-catenin RNA into ichabod embryos resulted in the
much less severe dino mutant phenotype (Fig. 6C,N), indicating ␤catenin is able to induce trunk, anterior neurectoderm and dorsal
mesoderm by chd-independent mechanisms.
FGF signaling is required for induction of chordin
by ␤-catenin, Sqt or Boz
In the section above, we demonstrated that FGFs can induce chd and
that chd is required for FGF partial rescue of ichabod embryos. As
it has been shown that either sqt or boz gene function is sufficient for
some degree of chd expression (Sirotkin et al., 2000; Shimizu et al.,
2000), we tested whether ␤-catenin induction of chd in ichabod
embryos is similarly dependent on Sqt and Boz. ichabod embryos
were co-injected with ␤-catenin RNA and either MOs against sqt
and cyc, pNAboz, or a combination of the three antisense inhibitors.
As expected, chd expression, assayed at 70% epiboly, was
decreased, but not abolished, in embryos blocked in Sqt and Cyc
function and also in embryos blocked in Boz function, and the
combination of inhibition of Nodals and Boz resulted in complete
inhibition of ␤-catenin-induction of chd, demonstrating an absolute
dependence on these factors (data not shown). We also checked to
what extent Boz and Sqt could each independently induce chd in
ichabod embryos. Co-injection of boz RNA and sqt and cyc MOs,
or co-injection of sqt RNA with pNAboz resulted in an intermediate
level of induction of chd (data not shown). These experiments verify
that the previously characterized roles of Boz and Sqt in chd
expression (Sirotkin et al., 2000; Shimizu et al., 2000) are operative
in ichabod embryos.
We next tested to what extent FGF signaling is required for
induction of chd and repression of bmp4 by ␤-catenin, Sqt or Boz
(Fig. 7). ␤-catenin (compare Fig. 7A,B with 7C,F), Sqt (compare
Fig. 7A,B with 7I,L), and Boz (compare Fig. 7A,B with 7O,R) can
induce chd and repress bmp4 in ichabod embryos at 70% epiboly.
The induction of chd and repression of bmp4 by both ␤-catenin (Fig.
7C-H) and Sqt (Fig. 7I-N) was blocked by expression of XFD, but
not d50 control. Boz induction of chd was also blocked by XFD and
not d50 (Fig. 7O-Q), although XFD failed to inhibit the repressive
effect of Boz on bmp4 expression (Fig. 7R-T), consistent with a
direct inhibitory effect of Boz on bmp transcription (shown for
bmp2b) (Leung et al., 2003a), and a result compatible with the roles
of Boz as an antagonist of vox, vent and ved (Melby et al., 2000; Imai
et al., 2001; Shimizu et al., 2002), which are most probably
independent of FGF signaling and chd expression. The lack of chd
expression even when bmp4 is inhibited by Boz expression, is the
one instance of dissociation of chd induction and BMP gene
repression revealed in our studies, indicating that inhibition of BMP
DEVELOPMENT
Fig. 3. FGF3 and FGF8 act
downstream of Squint and
Bozozok. Sqt (D,J), Boz (E,K) and ␤catenin (C,I) can induce expression of
fgf3 (B-E) and fgf8 (H-K). fgf3 and
fgf8 are expressed in the future
dorsal region in wild-type (A,G) but
not in uninjected ichabod (B,H)
embryos. Boz induces fgf3 and fgf8
through Sqt (E,F,K,L). In ichabod
embryos, Boz can induce weak
expression of fgf3 (E) and fgf8 (K),
but this expression is abolished in the
presence of MOsqt (F,L). (M-T) Nodal
proteins, but not Boz, are required
for ␤-catenin induction of fgf3 and
fgf8. The induction of fgf3 and fgf8
observed in ichabod embryos injected
with ␤-catenin RNA (compare M with
N and Q with R) is abolished in the
presence of MOs against sqt and cyc
(O,S) but not by pNAboz (P,T).
Embryos are shown at sphere stage.
3270 RESEARCH ARTICLE
Development 133 (16)
expression is not always sufficient for chd induction. In this case,
FGF signaling appears to be required for Boz induction of chd in
addition to any mechanism that results in BMP gene repression. The
ability of Boz to repress bmp4 expression in the absence of FGF
signaling and chd expression is consistent with the rescue of anterior
and dorsal tissues in ichabod embryos under these conditions (Fig.
4E-H,J).
The effects of XFD on chd induction and bmp4 repression
described above can only partially explain why ␤-catenin and Sqt
cannot dorsalize or rescue ichabod under conditions of inhibition
of FGF signaling. If these were the only effects of XFD, ␤-catenin
and Sqt rescue of ichabod embryos should yield the comparatively
mild dino phenotype, rather than being fully blocked by the
inhibitor (e.g. Fig. 2A,F, Fig. 4C,I). In the section below, we
present an additional effect of inhibition of FGF signaling that may
explain the complete block to ␤-catenin or Sqt rescue of ichabod
by XFD.
FGF signaling is required for ␤-catenin-dependent
bozozok transcript accumulation
Somewhat paradoxically, although FGF signaling is essential for ␤catenin or Sqt to rescue ichabod embryos, and Chd is essential for
FGFs to partially rescue these embryos, ␤-catenin is able to restore
most of the AP and DV pattern in ichabod embryos that are blocked
in chd expression. These findings predict that FGF signaling is
required for dorsal axis formation at steps downstream of ␤-catenin
in addition to activation of chd. As expression of ␤-catenin can
induce boz in ichabod embryos (Kelly et al., 2000) and Boz
expression can restore a significant degree of dorsalization (as well
as anterior neural tube formation) in the absence of FGF signaling
(Fig. 4F,G.J), it was unclear why inhibition of FGF signaling would
result in a complete block in the ability of ␤-catenin or Sqt to induce
an axis in ichabod embryos. The simplest explanation would be that
FGF signaling is required for ␤-catenin-dependent boz expression.
To test this hypothesis, we examined boz expression at sphere stage
in ␤-catenin RNA-injected ichabod embryos in the presence and
absence of XFD (Fig. 8A-D) and found that boz transcripts were
not detected when XFD was expressed, whereas d50 had no
inhibitory effect. In contrast to boz expression, we found no effect
of XFD on sqt expression, indicating that FGF signaling was not
required for all ␤-catenin-activated gene expression. In further
support for a role of FGF signaling in ␤-catenin-dependent boz
expression, we co-injected MKP3 and ␤-catenin RNAs into
ichabod embryos (Fig. 8K,N,Q), or injected MKP3 RNA alone into
wild-type embryos (Fig. 8S). In injected ichabod embryos, MKP3
completely abolished boz expression at 3.5 and 4 hpf sphere stage
(compare Fig. 8M with 8N, and 8P with 8Q), and in the wild-type
embryos, MKP3 greatly reduced the level of boz transcripts at this
stage (compare Fig. 8R,S). However, at 3.0 hpf, the earliest stage
we could detect boz expression in ␤-catenin RNA-injected ichabod
embryos, we found no inhibition of boz expression by MKP3
(compare Fig. 8J,K). Taken together, the results with both XFD and
MKP3 indicate that FGF signaling is essential for accumulation of
boz transcript, but is not required for initial transcription of this
gene. The complete block of ␤-catenin and Sqt rescue of ichabod
embryos when FGF signaling is inhibited can thus be explained by
the finding that FGF signaling is essential for both boz and chd
expression.
DEVELOPMENT
Fig. 4. FGF signaling is essential for rescue of ichabod by
Squint but not by Bozozok. XFD completely blocks rescue
by Sqt (A-D) but not Boz (E-H). Typical examples are shown of
uninjected ichabod embryos (A,E), and ichabod embryos
injected with RNAs for sqt (B), boz (F), sqt and XFD (C), boz
and XFD (G), sqt and d50 (D) or boz and d50 (H). Many
embryos injected with boz and XFD RNAs form eyes (arrow,
G). (I,J) Phenotypic classification of the embryos shown in A-D
(I) or E-H (J). The asterisk indicates embryos similar to those
shown in G and the double asterisk class is highly dorsalized.
Embryos are shown at 24 hpf.
FGF signaling and organizer formation
RESEARCH ARTICLE 3271
DISCUSSION
FGF signaling is required for ␤-catenin induction
of the dorsal axis
We have shown, using expression of the FGF signaling inhibitors
XFD, MKP3 and SU5402 that FGF signaling is required for ␤catenin induction of a dorsal axis in ichabod embryos devoid of a
Nieuwkoop center equivalent. ␤-catenin induction of both gsc and
chd and repression of bmp4 were completely inhibited in ichabod
embryos blocked in FGF signaling by XFD (Fig. 2I-K, Fig. 7C-H).
Moreover, dorsalization and dorsal axis formation in otherwise
highly ventralized ichabod embryos were completely blocked by
both XFD and MKP3 (Fig. 2A-F). XFD or MKP3 expression also
prevented ␤-catenin-dependent accumulation of boz transcript (Fig.
8A-D,L-Q). In addition, treatment with SU5402 inhibited the ␤catenin induction of gsc in these embryos (Fig. 2O). These results
demonstrate that FGF signaling is necessary for ␤-catenin induction
of the zebrafish organizer and in formation of both anterior and
posterior neurectodermal tissues.
In agreement with these results, ␤-catenin can induce early dorsal
expression of fgf3 and fgf8 in ichabod embryos, which lack
expression of these genes in the early prospective dorsal domain
(Tsang et al., 2004). Also indicating a role for FGF signaling, we
found that injection of FGF3 or FGF8 RNA into ichabod embryos
resulted in early expression of gsc (Fig. 1R-T) and flh, properly
patterned expression of otx2 and hoxb1b during gastrulation (Fig.
1H-J,M-O), and subsequent formation of a dorsal axis, with many
embryos developing a fully AP patterned neural tube (Fig. 1A).
These results are consistent with the ability of ectopic FGF8 to
induce a secondary axis in zebrafish embryos (Fürthauer et al.,
1997).
DEVELOPMENT
Fig. 5. Coordinate induction of chd and repression of bmp2b by FGFs. (A-X) ichabod embryos injected with 1 pg FGF3 (G-I,S-U) or 0.1 pg
FGF8 (J-L,V-X) were compared with uninjected ichabod (D-F,P-R) and wild-type embryos (A-C,M-O) for chd (A-L) or bmp2b (M-X) expression. Thirty
percent epiboly and 50% epiboly embryos are shown in animal pole views; 70% epiboly embryos are shown in lateral views, with dorsal to the
right in those embryos with a recognizable dorsal side. In every case of chd induction there was also repression of bmp2b [induction and repression
of these genes in 30% epiboly embryos after injection with FGF8 RNA (J,V) is indicated with arrowheads]. Thirty percent epiboly embryos after
injection with FGF3 RNA, the one condition that did not result in chd induction (compare D,G), also did not show bmp2b repression (compare P,S).
(A⬘⬘-E⬘⬘) ichabod embryos injected with 1 pg (C’,E’) or 50 pg (D’F’) of FGF3 RNA were compared with uninjected ichabod (B’) or wild-type (A’)
embryos for bmp2b expression at 30% epiboly (shown in animal pole views). The lower amount of FGF RNA did not lead to repression of bmp2b
but the higher amount of FGF3 RNA did repress the gene. Co-injection of MOchd (E’,F’) did not affect the level of bmp2b expression at either RNA
input (compare C’,E’ or D’F’).
By contrast to the effects of inhibition of FGF signaling by
interfering with receptor or downstream transduction function,
ventralization has not been reported on loss of function of fgf3 and
fgf8 in the zebrafish (Phillips et al., 2001; Maroon et al., 2002;
Léger and Brand, 2002). This failure might be due to redundancy
with fgf17b and fgf24, which are also expressed in the embryonic
shield (Cao et al., 2004; Draper et al., 2003). However, our attempts
to co-inject MOs specific for any three or all four of these
transcripts always resulted in non-specific early developmental
arrest.
Although previous studies in which FGF signaling was inhibited
by XFD expression throughout whole zebrafish and Xenopus wildtype embryos did not eliminate formation of anterior tissues (Amaya
et al., 1991; Amaya et al., 1993; Griffin et al., 1995; Kroll and
Amaya et al., 1996), there is substantial evidence that FGF signaling
has a crucial role in DV patterning in these embryos (Mitchell and
Sheets, 2001; Fürthauer et al., 1997; Fürthauer et al., 2004; Koshida
et al., 2002; Londin et al., 2005). Here we show, in addition, that
FGF signaling is required for ␤-catenin-mediated dorsal axis
formation, a finding analogous to the demonstration that FGF
signaling is required for Xenopus trunk organizer function (Mitchell
and Sheets, 2001), but we also show that inhibition of FGF signaling
in the zebrafish embryo prevents ␤-catenin induction of anterior
tissue.
Fig. 6. chordin is required for FGF-induced dorsal axis formation
in ichabod embryos. Phenotypic distributions for ichabod embryos
injected with FGF3 (A), FGF8 (B) and ␤-catenin (C) RNAs in the
presence (yellow) or absence (red) of MOchd, compared with
uninjected ichabod embryos (blue) for each. The most common class of
embryo partially rescued by ␤-catenin is dino-like. (D-J) Typical embryos
are shown for each indicated condition.
Development 133 (16)
FGF signaling functions at multiple steps
downstream of ␤-catenin activation
We describe above a requirement for FGF signaling in at least three
distinct steps in the network of response to ␤-catenin that leads to
formation of the dorsal axis (see Fig. 9): (1) chd induction and BMP
gene repression by Nodal proteins; (2) chd induction (but not BMP
repression) by Boz; and (3) maintenance of ␤-catenin-induced boz
transcript.
Fig. 7. FGF signaling is required for induction of chordin by ␤catenin, Squint and Bozozok. chd and bmp4 expression in ichabod
embryos (A,B) is restored to wild-type pattern by injection of ␤-catenin
RNA (C,F), sqt RNA (I,L), or boz RNA (O,R). Co-injection of XFD RNA
inhibits induction of chd by ␤-catenin (D), Sqt (J) and Boz (P), and
dorsal repression of bmp4 by ␤-catenin (G) and Sqt (M). However,
repression of dorsal expression of bmp4 by Boz is observed in XFD RNAinjected embryos (S, compared with R and B). Co-injection with d50
RNA had no effect on ␤-catenin, Sqt, or Boz functions (E,H,K,N,Q,T).
Embryos are at 70% epiboly, shown in lateral view, dorsal to the right.
DEVELOPMENT
3272 RESEARCH ARTICLE
First, FGF signaling has an essential role in the pathway, ␤cateninrSqtrFGFsrChd. We found that ␤-catenin can induce
zygotic expression of sqt (Kelly et al., 2000), that Sqt induces fgf3
and fgf8 (Fig. 3D,J) and that Boz induction of fgf3 and fgf8 (Fig.
3E,K) requires sqt (Fig. 3F,L). ␤-catenin induction of fgf3 and fgf8
is eliminated by sqtMO plus cycMO but not by pNAboz (Fig. 3NP,R-T). We conclude that that Nodal gene expression is necessary
and sufficient for early dorsal fgf3 and fgf8 expression. We found that
␤-catenin, Sqt, FGF3 and FGF8 can each induce chd and repress
bmp4 in ichabod embryos (Fig. 5; Fig. 7A-C,F,I,L), that XFD
inhibits chd induction and bmp4 repression by ␤-catenin and Sqt
(Fig. 7C-N), and that partial rescue of ichabod by FGF3 or FGF8 is
dependent on chd expression (Fig. 6). As FGF3 or FGF8 is capable
of inducing a dorsal axis with full AP pattern in ichabod embryos
(Fig. 1A), we conclude that FGF signaling is necessary and
sufficient for chd expression under conditions in which it is sufficient
to induce a partial dorsal axis. Sqt has previously been shown to
induce chd in zebrafish mesodermal precursors (Mathieu et al.,
2004), and FGFs have been shown to induce chd in early embryos
RESEARCH ARTICLE 3273
(Koshida et al., 2002; Londin et al., 2005). Dorsal axis formation is
thus dependent on the ␤-cateninrSqtrFGFsrChd pathway, with
each component sufficient for expression of the next downstream
factor.
Second, FGF signaling is required for Boz-stimulated chd
expression. It was previously shown that chd expression is
dependent on both Sqt and Boz in zebrafish embryos, and that each
of these factors is sufficient to obtain some chd expression (Sirotkin
et al., 2000; Shimizu et al., 2000). Based on the ability of XFD to
completely eliminate chd expression in Sqt or Boz RNA-injected
ichabod embryos, we conclude that FGF signaling is required for
Sqt and Boz stimulation of chd (Fig. 6I-K,O-Q).
Third, FGF signaling is required for ␤-catenin dependent
accumulation of boz transcript (Fig. 8A-D,L-Q). As ␤-catenin can
induce boz in ichabod embryos, and Boz expression can dorsalize
as well as induce anterior neural tube formation in the absence of
FGF signaling (Fig. 4G,J), it was unclear why inhibition of FGF
signaling would completely block ␤-catenin rescue of ichabod
embryos. One possibility, that FGF signaling was required for ␤Fig. 8. FGF signaling is essential for maintenance but not
initial transcription of ␤-catenin-induced boz RNA.
(A-H) FGF signaling is essential for accumulation of ␤-catenindependent boz, but not sqt, expression at 4 hpf. Neither boz
nor sqt is expressed in ichabod embryos (A,E), but expression of
both genes is restored by injection of ␤-catenin RNA (B,F). sqt
but not boz transcript is expressed in ichabod embryos coinjected with XFD and ␤-catenin RNAs (C,G), whereas both
genes are expressed in embryos co-injected with d50 RNA
(D,H). (I-Q) Inhibition of FGF signaling does not affect the initial
appearance of boz transcript but affects its maintenance.
␤-catenin-induced boz transcript can be detected in ichabod
embryos at 3, 3.5 and 4.0 hpf (J,M,P), but MKP3 inhibition of
FGF signaling results in absence of detectable ␤-catenininduced boz RNA at 3.5 and 4.0 hpf (N,Q), although the
amount at 3.0 hpf is not reduced (K). (R,S) MKP3 also can
inhibit boz RNA accumulation in wild-type embryos. All
embryos are shown in animal pole view.
DEVELOPMENT
FGF signaling and organizer formation
Fig. 9. Roles of FGF signaling in ␤-catenin-dependent dorsal axis
formation. Two versions of the pathways (A,B) are consistent with
experimental data. In both versions, ␤-catenin induces chd by two
separate pathways (solid arrows), one comprising sequential induction
of sqt and genes for FGF ligands, the second dependent on boz
expression and involving FGF signaling in the accumulation of boz
transcript and in Boz induction of chd. In A, FGFs induced by Sqt signal
to repress BMP gene expression, and in B the FGFs act more directly on
chd activation and BMP gene repression is a consequence of Chd
expression. In both models, Boz acts to induce signaling by an FGFdependent mechanism and to repress BMPs in an FGF-independent
manner, and ␤-catenin-induced boz transcript is maintained by FGF
signaling. As Boz acts as a repressor (Leung et al., 2003a; Solnica-Krezel
and Driever, 2001), it is diagrammed to repress unknown factors,
indicated with a generic ‘X’, which in turn repress chd and sqt. Dotted
lines show pathways demonstrated in other studies (reviewed by Schier
and Talbot, 2005). Cross-activation of boz and sqt has previously been
demonstrated in ichabod embryos (Kelly et al., 2000).
catenin-stimulation of boz, was indeed shown to be the case, as
XFD and MKP3 blocked the accumulation of ␤-catenin-induced
boz transcript in early ichabod embryos (Fig. 8A-D,L-Q) and
MKP3 was effective in inhibiting boz expression in wild-type
embryos (Fig. 8R,S). As boz is thought to be a direct target of ␤catenin-activated transcription, based on the presence of Tcf/Lef
consensus binding sites in the boz promoter and analysis of
expression of boz promoter fusion constructs (Ryu et al., 2001;
Leung et al., 2003b), a role for FGFs in boz expression was
unexpected. Indeed, we did not detect an effect of inhibition of
FGF signaling on the initial expression of boz just before high
stage (Fig. 8J,K). FGF signaling thus appears to be required for
stability and maintenance of boz transcript in the early embryo but
not for initial transcriptional activation. The profound combination
of loss of chd and boz function shown here is very similar to that
observed in boz;chd double mutants, which lack both head and
trunk (Gonzalez et al., 2000). These embryos have more
ventralized phenotypes than boz;sqt, boz;cyc, or even boz;sqt;cyc
embryos (Sirotkin et al., 2000; Shimizu et al., 2000), and are
almost as severely affected as ichabod Class 1 mutants (Kelly et
al., 2000).
FGF, Chordin and axis formation
Chd is essential for FGF partial rescue of ichabod embryos, but not
for ␤-catenin-dependent dorsal axis formation. Loss of function of
chd alone (i.e. dino mutants) results in a moderately ventralized
Development 133 (16)
phenotype (Hammerschmidt et al., 1996; Fisher et al., 1997;
Schulte-Merker et al., 1997) and ␤-catenin rescue of ichabod in the
presence of chdMO produces the same phenotype (Fig. 6C,J). The
severity of phenotype of loss of chd function is undoubtedly
mitigated by the expression of other secreted inhibitors of BMP and
Wnt ligands (reviewed by De Robertis and Kuroda, 2004; Schier and
Talbot, 2005), all of which are expressed in the organizer region. As
chdMO can completely prevent FGF rescue in ichabod embryos,
FGFs are probably not sufficient for expression of BMP antagonists
other than Chd.
Fig. 9 summarizes the ␤-catenin inducible, FGF signalingdependent pathways that operate in the zebrafish embryo to induce
chd and repress BMP function. As discussed above, the pathway has
two parallel branches, with one being directly dependent on
induction of sqt and FGF genes, the other on boz. Two versions of
these pathways are illustrated in Fig. 9, one in which FGFs induced
by Squint induce chd as a consequence of inhibition of BMP
signaling (Fig. 9A), the other in which chd is induced more directly
by FGFs (Fig. 9B); our data are consistent with either of these
models.
A potential unifying mechanism that would explain the repressive
effects of FGF signaling on BMP repression and consequent
induction of chd is provided by observations that FGF signaling
promotes MAPK phosphorylation of the linker region of Smad1,
counteracting the effects of BMP receptor-mediated C-terminal
Smad1 phosphorylation (Kretzschmar et al., 1997; Pera et al., 2003;
Kuroda et al., 2005). As BMP gene expression is dependent on a
positive regulatory loop (Kishimoto et al., 1997; Nguyen et al., 1998;
Schmid et al., 2000), and upregulation of chd has been found to be
a consequence of low BMP levels, even in the absence of functional
Chd protein (Schulte-Merker et al., 1997; Yabe et al., 2003), the
FGF-mediated chd induction we observed may be dependent on
interference with BMP signaling by MAPK phosphorylation of
Smad1. Further experiments, however, are required to rule out a
more direct effect of FGF signaling on chd expression in the sqt
branch of the pathway (as in Fig. 9B). FGF-dependent BMP
repression in the absence of chd expression in the early zebrafish
embryo has been reported (Fürthauer et al., 2004; Londin et al.,
2005), but these experiments were carried out using high levels of
injected FGF RNA (25-50 pg), which we found to produce
disorganized, albeit dorsalized, embryos that did not form a
recognizable axis.
Most of our data are consistent with this model, as chd induction
was always accompanied by loss of BMP transcripts (Fig. 5D-X).
However, the finding that chd induction was not observed even
though bmp4 expression was repressed when embryos were injected
with boz RNA under conditions of inhibition of FGF signaling (Fig.
6O-T) suggests that Boz induction of chd cannot solely be mediated
by FGF pathway repression of BMP gene expression. In Fig. 9A,B,
Boz is thus shown to have both FGF-dependent chd-inducing and
FGF-independent BMP-repressing effects.
We thank Joshua Bradner, Jiangyan He, Richard Gill, Jr and Carol Hu for fish
care and general laboratory assistance; Tetsuru Kudoh for reagents; and
Michael Tsang, Mary Mullins and Daniel Kessler for helpful suggestions. This
work was supported by NIH grant HD39272.
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