1 Division of Developmental Biology, Cincinnati Children's Research Foundation,
3333 Burnet Avenue, Cincinnati, OH 45229, USA
2 Biomolecular Medicine Group, Department of Biological Sciences, University of
Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
3 Department of Cell Biology, Harvard Medical School, Boston, MA 02115,
USA
* Author for correspondence (e-mail: heabq9{at}chmcc.org)
Accepted 5 August 2004
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SUMMARY |
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Key words: Foxh1, Fast1, Antisense, Nodal, Xnr5, Xnr6, Xnr3, Xenopus
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Introduction |
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In Xenopus, experiments with activator and repressor constructs,
and blocking antibodies, suggested that FoxH1 activates mesendodermal gene
expression and controls gastrulation movements
(Watanabe and Whitman, 1999).
Loss of function experiments using a morpholino approach had a less extreme
effect on development, but also indicated a potential role for FoxH1 in
gastrulation movements, since the convergence extension movements of
activin-induced animal caps were blocked by FoxH1 depletion
(Howell et al., 2002
). A
second early zygotic member of the Fox family, Fast3, was also found to be
expressed specifically during the gastrula stage. Loss of function experiments
on Fast3 showed similar phenotypes to those caused by morpholino-induced
depletion of FoxH1 (Howell et al.,
2002
). FoxH1 has been shown to bind to DNA in the absence of
activated Smads and interacts relatively weakly with activated Smads compared
to Fast3 (Howell et al.,
2002
), raising the possibility that maternal FoxH1 may have other,
Smad-independent functions.
Since controversy remains on the relative requirement for FoxH1 in pattern
formation and nodal signal transduction in vertebrates, we have specifically
analysed the contribution of maternal Foxh1 in Xenopus early
development using an antisense oligo-mediated approach. This approach has been
useful in demonstrating that the maternal T-box transcription factor VegT is
necessary and sufficient for the establishment for both mesodermal and
endodermal germ layers (Zhang et al.,
1998), and that the cytoplasmic protein ß catenin establishes
the dorsal axis by relieving the repressive effects of the HMG box
transcription factor XTcf3, on target genes such as goosecoid
(Houston et al., 2002
).
Here we depleted Foxh1 mRNA from stage 6 oocytes using an antisense oligonucleotide and assayed the effect on development. We show that maternal FoxH1-depleted embryos are headless and lack axial structures. FoxH1 depletion results in a severe inhibition of the activation of a FoxH1 reporter ARE-luciferase. Even so, nodal responsiveness is not lost in animal caps, and mes-endodermal gene expression continues in FoxH1-depleted embryos. We find that the expression of the organizer gene Xnr3, which is a direct target of the maternal Wnt signaling pathway, is most sensitive to FoxH1 depletion. Using Foxh1/XTcf3 double depletions, we show that FoxH1 is required, together with XTcf3 de-repression by ß-catenin, to activate Xnr3 expression in a Smad2-independent fashion. In contrast, we find that maternal FoxH1 inhibits the ectopic expression of Xnr5 and 6 in the ventral vegetal area of the late blastula. We conclude that FoxH1 is required to regulate the spatio-temporal patterns of Xnr3, 5 and 6 expression.
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Materials and methods |
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Fixation and histology
For histology and X-gal staining, embryos were fixed in MEMFA for 2 hours,
rinsed in PBS and stained using X-gal. For histology, embryos were dehydrated,
embedded in low-melt wax, serially sectioned at 20 µm and stained with
Haematoxylin and Eosin.
In situ hybridization
Embryos for in situ hybridizations for Xnr5 and Xnr6 were
prepared by fixing whole blastulae for 1 hour in MEMFA, bisecting the embryos
along the dorsal-ventral axis with a scalpel blade, fixing for one additional
hour in MEMFA, washing and storing in 100% ethanol. The in situ hybridizations
were performed as described (Harland,
1991) using BM Purple as substrate (Roche) with two exceptions.
The RNase A/T1 digestion was omitted from the protocol and the
anti-digoxigenin antibody was diluted in MAB-blocking buffer then pre-absorbed
with embryonic acetone powder before embryo incubation.
Oligos and mRNAs
The antisense FoxH1 oligo used was an 18-mer
5'-C*A*G*CTTCATCGCATC*C*A*G-3'
where * indicates a phosphorothioate bond, and other linkages were
phosphorodiester bonds. The oligo was resuspended in sterile, filtered water
and was injected in doses of 2.5-5 ng per oocyte. The oligos for depletion of
VegT
(5'-C*A*G*CAGCATGTACTT*G*G*C-3')
and XTcf3
(5'-C*G*A*G*GGATCCCAGTC*T*T*G*G-3')
were used as described previously (Houston
et al., 2002; Zhang et al.,
1998
). The oocytes were cultured immediately at 18°C.
Foxh1 mRNA was synthesized by linearizing the plasmid vector
pCS2+FoxH1 with NotI, and transcribing the linear template with SP6
polymerase in the presence of cap analog and GTP using the Megascript kit
(Ambion). RNA was ethanol precipitated and resuspended in sterile, distilled
water for injection.
Western blot analysis
Western blot analysis with anti-phospho-Smad2 antibody was used after
affinity purification from crude antisera (Peter ten Dijke), and using
secondary goat anti-rabbit IgG-HRP antibody (Boehringer Mannheim). Western
analysis was carried out as described by Lee et al.
(Lee et al., 2001).
Luciferase assay
The firefly luciferase reporter construct pGL3-ARE-luciferase, consisting
of three repeats of the activin response element (ARE) containing the FoxH1
binding sites from the regulatory sequence of the Mix.2 gene was used
as described previously (Huang et al.,
1995). It was injected into specific cells of the early embryo as
described in the text, in doses of 50 pg, together with 10 pg of control HSTK
Renilla luciferase plasmid. Pools of four or five embryos or five
animal caps were collected in triplicate for each injection mixture at stage
10. Luciferase assays were performed using the Dual Luciferase Reporter Assay
system (Promega). Five embryos were homogenized in 100 µl of lysis buffer,
and cleared by microcentrifugation. The supernatant (20 µl) was assayed in
50 µl of assay mixture, and luciferase activity was measured for 10 seconds
with an analytical luminescence laboratory monolight 2010. Firefly luciferase
activity was normalized to Renilla activity. Each experiment was
repeated at least twice, and single representative experiments are shown.
Analysis of gene expression using real-time RT-PCR
Total RNA was prepared from oocytes, embryos and explants using proteinase
K and then treated with RNase-free DNase as described
(Zhang et al., 1998).
Approximately 1/6 embryo equivalent of RNA was used for cDNA synthesis with
oligo (dT) primers followed by real-time RT-PCR and quantitation using the
LightCycler System (Roche) as described in Kofron et al.
(Kofron et al., 2001
). The
primers and cycling conditions used are listed in
Table 1. Relative expression
values were calculated by comparison to a standard curve generated by serial
dilution of uninjected control cDNA. Samples were normalized to levels of
ornithine decarboxylase (ODC), which was used as a loading control. Samples of
water alone, or controls lacking reverse transcriptase in the cDNA synthesis
reaction, failed to give specific products in all cases. Experiments were
repeated at least twice on different oocyte and embryo batches to ensure that
the pattern of gene expression described was reproducible from one experiment
to the next.
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Results |
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To test the effect of FoxH1 depletion on the activation of the ARE-luciferase by endogenous TGFß signaling in the early embryo, we injected ARE-luciferase into the vegetal area, or equatorial region of control or FoxH1-depleted embryos at the four-cell stage, and analyzed the level of induction of luciferase activity at the early gastrula stage. Fig. 1D shows that the ARE-luciferase is activated to similar levels when injected equatorially or vegetally into control embryos, and this activation is significantly reduced in depleted embryos.
These results show that the loss of FoxH1 is sufficient to severely reduce the activity of the FoxH1-reporter construct in response to both exogenous and endogenous ARE-inducing signals.
Maternal FoxH1 is required for head formation
To examine the effects on morphogenesis of FoxH1-depletion, we injected the
antisense oligo into defolliculated oocytes, cultured them for 48 hours, to
allow the target mRNA and the injected oligo to degrade the mRNA, and then
matured and fertilized the eggs by the host-transfer technique
(Zuck et al., 1998).
FoxH1-depleted embryos developed normally through gastrulation but axial
defects became apparent during neurulation and were obvious at the tailbud and
tadpole stages (Fig. 1E,F). In
seven experiments, 108 of 114 control uninjected embryos developed normally,
while only four, FoxH1-depleted embryos were normal and 109 were headless or
had more extreme axial deficiencies, including a dose-dependent shortening of
the axis as shown in Fig. 1E.
In histological sections taken at the tailbud stage of headless embryos, three
germ layers were visible, but axial structures were abnormal. In particular,
the notochord was absent or reduced (5/5 cases examined) and somites fused
across the midline (arrow in Fig.
1G). While initial gut formation appeared normal, gut looping was
disrupted. Heart tissue developed, but was also abnormal
(Fig. 1F). Using real-time
RT-PCR analysis of late neurula stage embryos, we confirmed that mesodermal
and endodermal tissues were specified. MyoD (somite marker) and Xsox17
(endoderm marker) were relatively normally expressed, whereas anterior
endodermal (Pdx1) and heart marker (Nkx2.5) expression was reduced (data not
shown). Although the formation of anterior structures was most impaired, the
anterior-posterior axis was not altered, since the
ß-galactosidase-labeled progeny of ventral cells injected at the
four-cell stage were found predominantly in the posterior and trunk of both
control and FoxH1-depleted embryos (Fig.
1H and data not shown).
Maternal FoxH1 regulates Xnr3
Since this phenotype strongly resembled that caused by partially blocking
the maternal Wnt signaling pathway by depleting maternal ß catenin
(Heasman et al., 1994), we
first confirmed that maternal ß catenin mRNA levels were
unaffected in FoxH1-depleted embryos (data not shown). Next we examined the
expression in FoxH1-depleted embryos of the known targets of the maternal
XTcf3/ß catenin signaling pathway, including Xnr3, siamois,
goosecoid and chordin. FoxH1 depletion resulted in a loss of the
expression of the organizer gene, Xnr3 (reduced to less than 10% in
6/6 experiments). Expression of other organizer genes, particularly
chordin, was also reduced but was not as dramatically or consistently
affected as Xnr3. We confirmed that these changes were not simply due
to a delayed onset of expression, by comparing the expression patterns at
2-hour intervals over an 8-hour period, during which Xnr3 expression
peaks and falls in control, uninjected embryos
(Fig. 2A). Xnr3
expression did not reach wild-type levels in FoxH1-depleted embryos at any
stage during gastrulation. This shows that FoxH1 is required for the
activation of ß catenin/XTcf3 target genes, and that
Xnr3 is most sensitive to its depletion.
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For rescue experiments, oligo 10-injected and control oocytes were incubated for 48 hours (to allow oligo and mRNA degradation) and then 15 and 30 pg Foxh1 mRNA was injected in the vegetal area. Oocytes were matured and fertilized and allowed to develop to the tailbud stage. Siblings were frozen at the gastrula stage for the analysis of molecular markers of axis formation. Foxh1 mRNA significantly rescued the expression of Xnr3, and other organizer genes, in a dose-responsive fashion (Fig. 2C); 30 pg also significantly rescued head formation in 80% of embryos (8/10 cases) compared to sibling FoxH1-depleted embryos which had 100%-reduced heads or headless phenotype (15/15 cases; Fig. 2D). The experiment was repeated with a similar result. These results indicate that the embryo is extremely sensitive to the level of expression of Foxh1 mRNA, and confirms that FoxH1 regulates the expression of Xnr3, and head formation.
In previous studies we have shown that the expression of Xnr3 is
regulated by maternal ß catenin, which blocks the repression of
Xnr3 expression by the maternal HMG box protein XTcf3
(Houston et al., 2002). We
next tested whether there was genetic interaction between XTcf3 and FoxH1, by
partially depleting maternal stores of XTcf3 and Foxh1
mRNAs, both singly and together. Fig.
3A shows that while partial FoxH1 depletion alone (2.5 ng oligo)
caused a reduction of Xnr3 expression, double-depleted embryos lose
Xnr3 expression completely. This experiment was repeated three times
with the same result. The organizer gene, goosecoid, has been shown
to be a target of FoxH1 in zebrafish
(Sirotkin et al., 2000
). In
comparison to Xnr3, goosecoid was reduced but not eliminated in
XTcf3/FoxH1 embryos (Fig.
3A). Double-depleted XTcf3/FoxH1 embryos had
more severe axial defects, than those caused by the depletion of XTcf3 or
FoxH1 alone (Fig. 3B).These
results show that the wild-type level of Xnr3 expression requires the
combinatorial activity of maternal FoxH1 transcriptional activation together
with XTcf3 de-repression by ß catenin.
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The nodal signaling pathway has been shown in mouse, zebrafish and
Xenopus embryos to be important for head and trunk mesoderm
formation, as well as the establishment of the endoderm germ layer (reviewed
in Whitman, 2001;
Schier, 2003
). FoxH1 is
considered to be a major effector of the nodal signaling pathway (reviewed by
Osada et al., 2000
;
Schier, 2003
;
Whitman, 2001
), although
several studies have also reported FoxH1-independent nodal signaling routes
(Germain et al., 2000
;
Ohkawara et al., 2004
;
Sirotkin et al., 2000
).
Fig.1C,D showed that FoxH1
depletion severely reduced FoxH1-reporter activity in response to activin or
to endogenous inducing signals. To examine the extent to which loss of
maternal FoxH1 affected the embryo's response to TGFß signals, we
dissected wild-type or FoxH1-depleted animal caps at the mid-blastula stage
and treated them with activin (10 µg/ml) for 4 hours before assaying for
gene expression at the mid-gastrula stage. Alternatively, we injected
Xnr1 mRNA (2 pg), into wild-type or FoxH1-depleted embryos at the
two-cell stage, dissected caps at the mid-blastula stage and analysed them at
the mid-gastrula stage for the expression of genes normally upregulated by
TGFß signaling (Mix.2, Xnr1, Fgf8, Xbra, goosecoid and
chordin; Fig. 4A). Since depletion of maternal FoxH1 substantially reduced the FoxH1 binding
construct, ARE-luciferase, from responding to activin, we expected that FoxH1
depletion would cause a reduction in the induction of nodal response genes in
this assay. Surprisingly, Mix.2, Xnr1, Fgf8, Xbra, goosecoid and
chordin were induced normally in maternal FoxH1-depleted animal caps
treated with activin protein or Xnr1 mRNA
(Fig. 4A), and sibling caps
cultured to the tail-bud stage elongated to similar extents as controls (data
not shown). To confirm this result, we tested, within one experiment, the
degree of reduction of ARE-luciferase activity, and the level of induction of
nodal-response genes in sibling animal caps and correlated these with the
phenotype of sibling embryos at the tailbud stage.
Fig. 4B-D confirmed that FoxH1
depletion severely limited the ability of activin to activate the ARE,
returning luciferase levels to the control, non-induced state, and causing a
headless phenotype. However it did not prevent Xnr1 inducing Mix.2,
chordin, Fgf8, Xbra and goosecoid in animal caps. This
demonstrates that the induction of nodal response genes in animal caps by Xnr1
and activin does not depend on maternal FoxH1 or the formation of ARF. These
zygotic genes are not only `nodal response genes' but are activated by other
pathways as well.
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FoxH1 inhibits the expression of Xnr5 and 6 mRNA in the ventral vegetal region of the blastula
Since the upregulation of Xnr5 and 6 mRNAs by FoxH1
depletion was unexpected, we first showed that this effect could be rescued by
the injection of 15 or 30pg of Foxh1 mRNA into FoxH1 depleted oocytes
(Fig. 5A). Next we examined
when and where the over-expression of Xnr5 mRNA caused by FoxH1
depletion occurred. Fig. 5B
shows that the expression of Xnr5 mRNA is detected at the
mid-blastula stage in both control and FoxH1-depleted embryos and is enhanced
in FoxH1-depleted embryos two hours later, at the late blastula stage. To
confirm that FoxH1 depletion caused increased expression of Xnr5 and 6, we
carried out in situ hybridization on hemisected embryos at the late blastula
stage. Xnr5 and 6 are not abundant mRNAs and are difficult to detect by in
situ (Takahashi et al., 2000).
Their expression is vegetally localized and nuclear at the late blastula stage
and is enhanced by FoxH1 depletion (Fig.
5C). Next we examined the expression of Xnr5 and
6 in animal, equatorial and vegetal explants
(Fig. 5D), and also in dorsal
and ventral half embryos at the late blastula and early gastrula stages
(Fig. 5E). We found that
maternal FoxH1 depletion caused an increased expression of Xnr5 and
6 in the vegetal mass, specifically on the ventral side (arrows in
E). The experiment was repeated twice with the same result. This shows that
maternal FoxH1 normally prevents the ectopic expression of Xnr5 and
6 mRNA in ventral vegetal cells.
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Maternal FoxH1, XTcf3 and VegT regulate Xnr5 and 6expression
Previous studies have shown that Xnr5 and 6 expression is
repressed in the early embryo by maternal XTcf3, and that their activation
requires both ß catenin and VegT
(Hilton et al., 2003;
Xanthos et al., 2002
). We
therefore asked whether removing both maternal XTcf3 and FoxH1 would act in an
additive fashion to enhance Xnr5 expression. This was not the case.
In two experiments, depletion of either FoxH1 or XTcf3 enhanced the expression
of Xnr5, but the double depletion did not increase its expression
further (Fig. 6A).
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The mis-regulation of Xnr3 and Xnr5 mRNA in FoxH1 depleted gastrulae contributes to their abnormal development at the tailbud stage
Since Xnrs are known to be potent signaling molecules, we reasoned that the
downregulation of Xnr3 and upregulation of Xnr5 and
6 mRNA may be responsible for the later abnormal development of
FoxH1-depleted embryos.
In previous studies, we and others have shown that the depletion and
over-expression of Xnr3 mRNA results in embryos that have reduced
heads and abnormalities in convergence extension movements
(Smith et al., 1995;
Yokota et al., 2003
). Here we
examined the effect of injecting Xnr3 mRNA into one dorsal cell of
four-cell-stage FoxH1-depleted embryos.
Fig. 7A shows that
Xnr3 mRNA expression partially rescues FoxH1-depleted embryos in
causing head formation, but does not rescue correct elongation of the body
axis (lower row), while the same dose in control embryos causes convergence
extension defects (upper row).
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Discussion |
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In these experiments, the degree of depletion of Foxh1 mRNA by the antisense oligo was to 10-20% of control levels. Since the available antibody is not sensitive enough to detect endogenous FoxH1 protein (data not shown), the extent to which the protein was reduced could not be measured directly. Instead, protein activity was measured by the reduction in the ARE-luciferase activity in FoxH1-depleted embryos compared to controls. Considering that some residual mRNA remained, the extent of reduction of ARE-luciferase activity was surprising. One explanation of the reduction of ARE-luciferase activity could be that maternal protein is only translated in the oocyte, and that the available protein is broken down during the 48-72-hour culture period before fertilization. This explanation is supported by the fact that a shorter incubation time causes a less severe phenotype (data not shown).
Three novel observations came from this study: that Xnr3 expression is dependent on FoxH1 in a phospho-Smad2-independent fashion, that when ARF activity is severely reduced, the expression of mesodermal and endodermal genes including `nodal response genes' continues in animal caps over-expressing Xnr1 or stimulated by activin, and that Xnr5 and 6 are negatively regulated by FoxH1.
Xnr3 regulation
Xnr3 was first characterized as an axis-rescuing activity in a
Xenopus functional screen (Smith
et al., 1995), and is expressed in a very restricted
spatiotemporal pattern in the organizer region. Loss of function experiments
show that it is essential for normal head formation, the convergent extension
movements of gastrulation, and for the correct expression of several genes
including Xbra (Yokota et al.,
2003
). Xnr3 expression has been shown to be regulated by
the maternal ß-catenin/XTcf3 pathway, since interfering with this pathway
by expressing dominant negative gsk-3
(Dominguez et al., 1995
;
He et al., 1995
;
Pierce and Kimelman, 1995
) or
by depleting ß-catenin (Xanthos et
al., 2002
) blocks Xnr3 expression. The experiments
presented here suggest that, in wild-type embryos, the de-repression of XTcf3
by ß-catenin on the dorsal side is accompanied by transcriptional
activation by FoxH1. Transcriptional activation of Xnr3 by FoxH1 on
the ventral side is prevented by XTcf3. In zebrafish, another organizer gene,
goosecoid is dependent on FoxH1 for its expression
(Pogoda et al., 2000
). In
Xenopus, goosecoid expression is regulated primarily by VegT and
XTcf3/ß catenin (Houston et al.,
2002
) (Fig.
3A).
We do not demonstrate whether the effects of FoxH1 on Xnr3 are
direct or indirect. However, two potential FoxH1 consensus binding sites are
present in the published 257-base fragment of the published promoter sequence
for Xnr3 (at positions 238 to 245; 7/8 match and
173 to 180; 8/8 match; data not shown)
(McKendry et al., 1997), and
Xnr3 expression occurs immediately after MBT
(Xanthos et al., 2001
), making
it likely that maternal FoxH1 protein regulates Xnr3 directly.
Two lines of evidence suggest a novel aspect of FoxH1 activity; that the
transcriptional activation of Xnr3 by FoxH1 does not require the
interaction of FoxH1 with phospho-Smad2. Firstly, Xnr3 mRNA continues
to be expressed in VegT-depleted embryos
(Xanthos et al., 2001) and
(Fig. 6B), in which we have
shown there is no detectable phosphorylation of Smad2 protein
(Lee et al., 2001
). Secondly,
we show here that inactivation of nodal signaling by the expression of the
mutant, nodal binding fragment of Cerberus, CerS
(Agius et al., 2000
) also
blocks Smad2 phosphorylation, but does not prevent Xnr3 expression in
wild-type embryos, nor alter its inhibition by the depletion of FoxH1. We have
shown (Rex et al., 2002
) that
expression in whole embryos of a truncated form of the activin receptor, which
has dominant negative activity against a range of nodal-like signals, also has
no inhibitory effect upon Xnr3 expression. Previous studies have
focused on the activity of FoxH1 in a complex with phospho-Smad2 and 4, and
although its potential as a Smad-independent regulator was suggested by the
observation that it binds DNA in the absence of Smads
(Howell et al., 2002
;
Yeo et al., 1999
), this is, to
our knowledge, the first evidence for such a role. Further analysis of the
Xnr3 promoter is required to understand the complexity of its
regulation by FoxH1, XTcf3 and ß-catenin.
FoxH1 and nodal target gene expression
Previous loss of function studies on Xenopus FoxH1, using blocking
antibody or a Drosophila EngrailedFoxh1-DNA-binding domain
fusion construct, suggested a major role for FoxH1 in regulating the nodal
target genes, including Xlim1, Xbra, cerberus, Mix.2 and
goosecoid (Watanabe and Whitman,
1999). Here we show that, although the activity of a reporter that
consists of a triplet repeat of 50 bp of Mix.2 promoter containing
the FoxH1 binding site is much reduced by FoxH1 depletion, the responses to
nodal-type signaling are not correspondingly affected. Two pieces of evidence
show this. The expression of nodal target genes in response to Xnr1
mRNA injection or activin protein induction is unaffected by FoxH1 depletion
in animal caps, suggesting that FoxH1 and ARF are not required for this
activity. As a second test of the importance of FoxH1, we examined the
endogenous expression of nodal response genes including Xnr1, Mix.2,
goosecoid and Xlim1 in FoxH1-depleted embryos, siblings of which
developed with a headless phenotype. The level of expression of these genes
was reduced, suggesting that FoxH1 modulates their expression levels, but none
showed the extreme sensitivity of Xnr3. FoxH1 genetic mutants in
zebrafish have been shown to affect only a subset of nodal target genes, and
to cause reduction rather than complete inhibition of expression
(Pogoda et al., 2000
;
Sirotkin et al., 2000
). Our
studies support the evidence of Pogoda et al., in zebrafish, and Germain et
al., in Xenopus (Germain et al.,
2000
), suggesting that, although FoxH1/Smad2/4 is an important
complex in modulating nodal target genes, other transcription factors are also
involved. A likely second pathway involves the TAK1-NLK-STAT1 cascade
(Ohkawara et al., 2004
). The
more extreme effects observed using Engrailed repressor constructs and
blocking antibody may have been caused by interference with a broader spectrum
of genes containing fork-head domains or, in the case of the antibody,
Smad-interacting domains.
In Xenopus, the second Fox gene, Fast3 has been suggested
to act, like Foxh1, as a mediator of nodal signals. Fast3 has been
shown to bind to the same consensus sequence as FoxH1
(Howell et al., 2002). Here,
we show that Fast3 is expressed normally in FoxH1-depleted embryos.
Since these embryos lack heads and also lack the ability to activate
ARE-luciferase robustly, this suggests that Fast3 does not activate
ARE-luciferase or play a role in head formation, although it may regulate the
partial expression of nodal-target genes seen here in FoxH1-depleted embryos.
It is likely that the expression of each of the `nodal target genes' is in
fact complexly regulated by several transcription factors and co-activators,
and repressors, as has been shown recently for the cerberus gene
(Yamamoto et al., 2003
).
Xnr5 regulation
Forkhead genes have generally been shown to be transcriptional activators,
but in some contexts may also act as transcriptional repressors (reviewed in
Carlsson and Mahlapuu, 2002).
Here, depletion of FoxH1 causes an increase in the expression of the two
nodals Xnr5 and 6. Xnr5 and 6 were first described as novel Xnrs expressed
very early in development before the other family members, in dorsal vegetal
cells (Takahashi et al., 2000
;
Yang et al., 2002
). Expression
of Xnr5 has been shown to be unaffected by cycloheximide treatment, suggesting
that its expression is independent of new protein synthesis
(Rex et al., 2002
;
Takahashi et al., 2000
).
Previous studies determined that the transcription factors XTcf3 and VegT
regulate Xnr5 expression, and binding sites for these factors were
identified in the Xnr5 promoter. The activity of this promoter has
been shown to depend on derepression of XTcf3 by ß catenin together with
VegT activation (Hilton et al.,
2003
). Here we show that Xnr5 mRNA is also prevented from
ectopic expression by FoxH1, since FoxH1-depleted embryos express
Xnr5 mRNA in ventral vegetal cells. The effect of FoxH1 on
Xnr5 expression could be direct or indirect. Four potential FoxH1
binding sites were identified in the 785 bp fragment upstream of the TCF
binding site in the Xnr5 promoter
(Hilton et al., 2003
). A fifth
potential site lies in the first intron and this sequence is conserved in both
the Xenopus laevis and Xenopus tropicalis genomic sequence.
We show here that while depleting FoxH1 or XTcf3 enhances Xnr5
expression, the effects of depleting both XTcf3 and FoxH1 are not additive.
This may suggest that the two transcription factors interact to regulate Xnr5,
and both are essential to form one repressive complex. We confirm here that
VegT is essential for the activation of Xnr5 expression in
FoxH1-depleted embryos, just as it is in XTcf3-depleted embryos. We further
suggest that this repressive activity of FoxH1 is nodal signalling-dependent.
It has been shown that the pattern of Xnr5 expression in the deep endoderm is
dynamic at stages 8.5 and 9 (Takahashi et
al., 2000
). It seems likely, but as yet remains unconfirmed, that
this dynamic pattern of Xnr5 expression relates directly to the
dynamic changes in Smad2 phosphorylation known to be taking place at this time
(Lee et al., 2001
). Further
work is required to determine whether FoxH1 binds directly to either of the
putative FoxH1-binding sites in Xnr5 and, if so, to show how it acts in an
inhibitory fashion when bound.
These results add to our understanding of the maternal regulation of early zygotic gene expression in Xenopus. While FoxH1 has been considered as an activator of nodal target gene expression, we show here that nodal responsiveness is not lost in FoxH1-depleted embryos. We find that FoxH1 has specific, non-redundant roles, acting as a co-activator of Xnr3 together with XTcf3-ß catenin, and as a repressor of Xnr5. We propose that FoxH1 participates in patterning the mesendoderm by simultaneously repressing Xnr5 in the ventral region and activating Xnr3 in the dorsal region. We suggest that these roles of FoxH1 depend on its participating with different transcription factors and co-factors to form different regulatory modules controlling Xnr5 and Xnr3 expression. The challenge is to define, for each of the mes-endodermal genes transcribed after MBT, the different combination of maternal and early zygotic transcription factors and co-regulators forming these modules.
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ACKNOWLEDGMENTS |
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REFERENCES |
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