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First published online July 27, 2006
doi: 10.1242/10.1242/dev.02483
Department of Biology, University of Pennsylvania, Goddard Labs 316, Philadelphia, PA 19104-6017, USA.
* Author for correspondence (e-mail: eweinber{at}sas.upenn.edu)
Accepted 9 June 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SquintFGFChordin, 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.
Key words: ß-Catenin, FGF signaling, Dorsal axis formation, Neural induction, Dorsoventral patterning, Anteroposterior patterning, Organizer, Nodal, Chordin, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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
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 ß-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, ß-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 ß-catenin gene, ß-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 ß-catenin in
dorsal axis formation (Tsang et al.,
2004). ß-catenin is also required for expression of XFGF3 in
both dorsal and non-dorsal regions of Xenopus blastulae, and
ß-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 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 ß-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 ß-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 ß-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 ß-catenin-dependent factors. Using this approach, we show that FGF
signaling is absolutely required for ß-catenin activation of the
organizer and dorsal axis, that at least one mode of dorsal axis formation
proceeds through the pathway ß-cateninSquintFGF
signalingChordin, and that FGF signaling is required for dorsal axis
formation at additional steps downstream of ß-catenin, including the
accumulation of boz transcript and boz induction of
chd.
|
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) 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), ß-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 phenotypes when co-injected with
ß-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 |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) 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 ß-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 ß-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. 1R-T) 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 ß-catenin-mediated signaling.
FGF signaling is required for ß-catenin-mediated organizer formation
To examine whether FGF signaling is required for ß-catenin activation
of organizer formation, we tested the effect of FGF signaling pathway
inhibitors on ß-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), along
with ß-catenin RNA into a single blastomere at the 4-8-cell stage. The
ability of ß-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
non-functional FGF receptor, d50 (Amaya et
al., 1991), which did not inhibit ß-catenin dorsalization and
rescue (Fig. 2A,E,G). We also
examined gsc expression at shield stage in embryos injected with
these RNAs. ß-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 ß-catenin. To confirm that FGF signaling is
essential for ß-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 ß-catenin rescue of ichabod
(Fig. 2B). We also utilized a
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
ß-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 ß-catenin to induce organizer genes
and rescue organizer-deficient embryos.
|
;
Shimizu et al., 2000), we next
examined the relationship between FGF signaling and expression of these two
genes during ß-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) 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. Co-injection 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
ß-catenin can induce both sqt and boz, and boz
can induce sqt under these conditions, ß-catenin induction of
FGFs proceeds through induction of Sqt and subsequent Nodal signaling.
|
FGF signaling is required for AP and DV rescue of ichabod by Squint but not Bozozok
The experiments described thus far suggest that ß-catenin rescue of
ichabod embryos proceeds at least in part via the linear pathway,
ß-cateninNodalsFGFsorganizer 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 AP pattern of neurectoderm in
ichabod embryos (Fig.
1), induced expression of chd
(Fig. 5D-F,H-L) and repressed
bmp2b (Fig. 5P-R,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 FGF-dependent dorsalization (Fig. 6A,B,E,F,H,I). However, co-injection of MOchd and ß-catenin RNA into ichabod embryos resulted in the much less severe dino mutant phenotype (Fig. 6C,N), indicating ß-catenin is able to induce trunk, anterior neurectoderm and dorsal mesoderm by chd-independent mechanisms.
|
;
Shimizu et al., 2000), we
tested whether ß-catenin induction of chd in ichabod
embryos is similarly dependent on Sqt and Boz. ichabod embryos were
co-injected with ß-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 ß-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 ß-catenin, Sqt or Boz
(Fig. 7). ß-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 ß-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 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 ß-catenin and Sqt cannot dorsalize or rescue ichabod under conditions of inhibition of FGF signaling. If these were the only effects of XFD, ß-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 ß-catenin or Sqt rescue of ichabod by XFD.
|
) 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 ß-catenin or Sqt to induce an axis in ichabod
embryos. The simplest explanation would be that FGF signaling is required for
ß-catenin-dependent boz expression. To test this hypothesis, we
examined boz expression at sphere stage in ß-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 ß-catenin-activated gene expression. In further
support for a role of FGF signaling in ß-catenin-dependent boz
expression, we co-injected MKP3 and ß-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
ß-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 ß-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. |
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In agreement with these results, ß-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).
|
; 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 wild-type 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 ß-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 ß-catenin induction of anterior tissue.
|
|
First, FGF signaling has an essential role in the pathway,
ß-cateninSqtFGFsChd. We found that ß-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).
ß-catenin induction of fgf3 and fgf8 is eliminated by
sqtMO plus cycMO but not by pNAboz
(Fig. 3N-P,R-T). We conclude
that that Nodal gene expression is necessary and sufficient for early dorsal
fgf3 and fgf8 expression. We found that ß-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 ß-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
(Koshida et al., 2002;
Londin et al., 2005). Dorsal
axis formation is thus dependent on the
ß-cateninSqtFGFsChd 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).
|
;
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 ß-catenin-dependent dorsal axis formation. Loss of function of
chd alone (i.e. dino mutants) results in a moderately
ventralized phenotype (Hammerschmidt et
al., 1996; Fisher et al.,
1997; Schulte-Merker et al.,
1997) and ß-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.
|
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.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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