1 Developmental Genetics Program, Skirball Institute of Biomolecular Medicine,
Department of Cell Biology, New York University School of Medicine, New York,
NY 10016, USA
2 Department of Cell Biology, Harvard Medical School, Boston, MA 02115,
USA
Author for correspondence (e-mail:
schier{at}saturn.med.nyu.edu)
Accepted 12 August 2003
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SUMMARY |
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Key words: Nodal, Smad, Mesoderm, Endoderm, FoxH1, Mix, Zebrafish
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Introduction |
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Members of the FoxH1 (Fast1, Fast3) and Mix/Bix (Mixer, Milk, Bix3)
families are the best characterized partners of phosphorylated Smad2 during
embryogenesis (Hill, 2001;
Whitman, 2001
). FoxH1 proteins
are forkhead/winged helix transcription factors that can recruit active Smad
complexes to activin responsive elements (AREs) in Xenopus mix.2,
xnr1 and other genes (Chen et al.,
1996
; Watanabe and Whitman,
1999
; Osada et al.,
2000
). Mixer and related Mix/Bix proteins are paired-like
homeodomain proteins and can recruit active Smad complexes to the distal
element (DE) of the Xenopus goosecoid (gsc) promoter
(Germain et al., 2000
;
Randall et al., 2002
). The
interaction of these transcription factors with the activated Smad complex is
mediated through a Smad interaction motif (SID in Fast1, SIM in Mixer)
(Chen et al., 1997
;
Randall et al., 2002
).
The in vivo roles of FoxH1 have been analyzed genetically in mouse and
zebrafish, and through the use of interference approaches in Xenopus.
FoxH1 mutant mice have variable but severe phenotypes, including loss of
anterior structures, failure to form the node and its midline derivatives, and
defects in definitive endoderm formation
(Hoodless et al., 2001;
Yamamoto et al., 2001
). In
contrast to nodal mutants, however, foxH1 mutants develop
most mesoderm. Blocking antibodies against Xenopus Fast1 led to
defects in mesoderm formation, including the inhibition of the mesodermal
marker T/Xbra and the dorsal marker gsc
(Watanabe and Whitman, 1999
).
In addition, Activin-mediated induction of mix2, lim1 and
gsc is blocked by anti-Fast1 antiserum
(Watanabe and Whitman, 1999
).
Somewhat conflicting results have been reported when Xenopus Fast1
and Fast3 activity was knocked down using morpholinos: although gastrulation
movements were inhibited in these embryos, most marker genes (including
gsc, mix2 and lim1) seem to be expressed normally
(Howell et al., 2002
). Taken
together, these results suggest that Xenopus FoxH1 might mediate the
activation of gastrulation movements and/or mesoderm induction.
Genetic screens in zebrafish have identified mutations in FoxH1
[schmalspur (sur)] (Brand
et al., 1996; Schier et al.,
1996
; Solnica-Krezel et al.,
1996
; Pogoda et al.,
2000
; Sirotkin et al.,
2000
). Mutants that lack zygotic sur activity
(Zsur) have variable, relatively mild phenotypes, ranging from
randomization of left-right asymmetry but normal early patterning to reduction
of prechordal plate and floor plate (Brand
et al., 1996
; Schier et al.,
1996
; Solnica-Krezel et al.,
1996
; Pogoda et al.,
2000
; Sirotkin et al.,
2000
). Embryos lacking both maternal and zygotic sur
function (MZsur) can have more severe but variable phenotypes,
including reduction of axial midline structures
(Pogoda et al., 2000
;
Sirotkin et al., 2000
) (this
report). This phenotype is much milder than the one observed upon complete
loss of Nodal signaling, which leads to a lack of all endoderm, head and trunk
mesoderm, and ventral neuroectoderm
(Feldman et al., 1998
;
Gritsman et al., 1999
;
Meno et al., 1999
;
Thisse and Thisse, 1999
).
In the case of Mix/Bix genes, misexpression studies in
Xenopus have shown that members of this family can induce endoderm
development but their individual requirements in this process have not been
resolved (Ecochard et al.,
1998; Henry and Melton,
1998
; Lemaire et al.,
1998
). Supporting a role for Mix/Bix genes in endoderm
formation, zebrafish mutants for the mixer-like gene bonnie and
clyde (bon) (Chen et al.,
1996
; Stainier et al.,
1996
; Alexander et al.,
1999
; Kikuchi et al.,
2000
) or embryos lacking bon activity because of
morpholino (MO) injection (Kikuchi et al.,
2000
) have a dramatic reduction of endoderm. Additional phenotypes
include cardia bifida and pericardial edema, but mesoderm induction appears
largely normal in these embryos (Stainier
et al., 1996
; Chen et al.,
1996
; Kikuchi et al.,
2000
). In contrast to the sur phenotypes, the
bon phenotype is fully penetrant and largely invariable.
The role of Mix/Bix genes in Nodal signaling is complicated by the
observation that some of these genes are regulated transcriptionally by Nodal
signals. For instance, biochemical studies and sequence analysis indicate that
Bon can serve as a binding partner of phosphorylated Smad2
(Randall et al., 2002),
suggesting that Bon is a component of the Nodal signaling pathway. Other
studies have emphasized that bon is a transcriptional target of Nodal
signaling (Alexander and Stainier,
1999
). In particular, bon expression is absent or barely
detectable at the onset of gastrulation in the absence of Nodal signaling,
suggesting that bon is primarily a target of the Nodal signaling
pathway rather than a necessary transducer of Nodal signals
(Alexander and Stainier, 1999
).
This raises the question of whether Bon is a Smad-associated component and/or
a downstream gene of the Nodal signaling pathway. An additional level of
complexity derives from the observation that some members of the Mix/Bix
family, such as mouse Mixl1, appear not to interact with phosphorylated Smad2
(Germain et al., 2000
;
Randall et al., 2002
) but are
involved in processes that are regulated by Nodal signaling. For instance,
mouse Mixl1 mutant embryos display complex gastrulation defects and,
in chimeras, Mixl1 mutant cells are largely excluded from endoderm
and heart (Hart et al.,
2002
).
Here, we analyze the regulation of bon and sur by Nodal signaling, determine whether bon and sur have overlapping, additive or antagonistic functions, and test whether Nodal signaling is mediated exclusively by bon and sur. We find that sur expression is independent of Nodal signaling, whereas bon is initially expressed in the absence of Nodal signaling but requires Nodal signaling and sur for full and maintained expression. We find that MZsur;bon double mutants and MZsur;bonMO embryos have severe phenotypes not observed upon loss of either bon or sur. Double mutants lack heart, prechordal plate and ventral neuroectoderm, a subset of the phenotypes seen upon complete loss of Nodal signaling. Analysis of Nodal downstream genes indicates that bon and sur have both divergent and overlapping functions in gene regulation, and reveals that some Nodal-dependent genes do not require bon and sur activity. Overall, our study establishes that sur and bon have both independent and overlapping roles as components of the Nodal signaling pathway but do not account for all effects of Nodal signaling during mesendoderm induction.
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Materials and methods |
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In situ hybridization and phosphorylated-Smad2 detection
In situ hybridization and preparation of RNA probes were performed as
described (Schier et al.,
1997). Phosphorylated-Smad2 detection was as described
(Mintzer et al., 2001
).
Microinjection of mRNA and bon MO
Synthetic capped squint RNA was synthesized and injected as
described (Chen and Schier,
2001). Approximately 3 ng of bon MO
(Kikuchi et al., 2000
)
dissolved in phenol red buffer was injected into the yolk of one- to
two-cell-stage wild-type or MZsur embryos.
Genotyping of bon and sur fish
Fish were genotyped as described (Chen
and Schier, 2001). Primers for bon are described in
Kikuchi et al. (Kikuchi et al.,
2000
). Primers for sur are
5'-TCACCTTGACTGCAGAATCGG-3' [fast 330 f2
(Sirotkin et al., 2000
)] and
5'-GCCAGGTAAGAGTACGGTGGTTTGGGATAT-3' (SurDCWTR2). SurDCWTR2, a
dCAP (derived cleaved amplified polymorphic sequence) primer
(Neff et al., 1998
),
introduces an EcoRV restriction site into the wild type to give a 205
base pair band but does not introduce this site into
surm768, resulting in a 235 base pair band. The conditions
for genotyping of both bon and sur were: 94°C for 3
minutes (1 cycle), followed by 45 cycles of 94°C for 30 seconds, 55°C
for 30 seconds and 72°C for 30 seconds, and finally 72°C for 5
minutes. The amplified sur product was digested with EcoRV
and resolved on 2% agarose gels.
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Results |
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Generation of embryos that lack both bon and sur
activity
The findings that both bon and sur are expressed in the
absence of Nodal signaling, that both Mixer and FoxH1 can interact with
phosphorylated Smad2, and that bon and sur have different
phenotypes suggested that bon and sur might have either
additive or overlapping roles during mesendoderm induction and Nodal
signaling. To distinguish between these possibilities, we generated embryos
that lack both bon and sur activity using two different
approaches (Fig. 2). In a
genetic approach (Fig. 2A), we
generated embryos that lack both maternal and zygotic sur
(sur is expressed maternally and zygotically) and also lacked zygotic
bon [bon is only expressed zygotically
(Kikuchi et al., 2000)]. We
crossed bon/+ and sur/+ heterozygous fish to generate
bon/+; sur/+ double heterozygotes. These were crossed to
sur/sur fish and resulting embryos were injected with
wild-type sur mRNA to rescue sur/sur mutants. This
allowed us to raise sur/sur;bon/+ fish to adulthood.
Intercrosses of these fish result in embryos that lack both maternal and
zygotic sur activity (MZsur), and one-quarter of embryos are also
homozygous for bon. These MZsur;bon mutants lack all
sur and bon activity. In a second approach
(Fig. 2B), we used a MO
antisense oligonucleotide against bon
(Summerton and Weller, 1997
;
Heasman et al., 2000
;
Nasevicius and Ekker, 2000
).
Previous studies have shown that this MO efficiently phenocopies the
bon phenotype (Kikuchi et al.,
2000
). Injection of bonMO into MZsur mutants at
the one or two cell stage results in MZsur;bonMO embryos.
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Ectopic activation of Nodal target genes is regulated by Bon and
Sur
As an additional test of the requirement for Bon and Sur in mediating Nodal
signaling, we determined the response of ntl, cas, gsc and
bik to the ectopic activation of the Nodal signaling pathway
(Fig. 8). RNA for the Nodal
signal Squint was injected at the one- to two-cell stage and gene response was
assayed at 50% epiboly. The expression of ntl was induced in
bon, MZsur and MZsur;bon embryos (data not shown).
The expression of cas was induced in MZsur but not in
bon or MZsur;bon embryos
(Fig. 8E-H). The expression of
bik was induced in bon but not in MZsur embryos
(Fig. 8I-L). Surprisingly, the
expression of bik was weakly induced in MZsur;bon and
MZsur;bon/+ (data not shown) embryos, suggesting that bon
might act as a repressor of bik at high levels of Nodal ligand and in
the absence of sur. Finally, the expression of gsc was not
induced in MZsur;bon mutants but was activated in bon and
MZsur mutant embryos (Fig.
8A-D). These results provide further evidence for independent and
overlapping functions of Bon and Sur in the regulation of Nodal downstream
genes.
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Discussion |
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Although not required for the initiation of bon expression, Nodal
signaling is essential for normal bon expression
(Alexander and Stainier, 1999)
(Fig. 1). At the onset of
gastrulation, bon expression is lost or barely detectable in the
absence of Nodal signaling. The enhancement and maintenance of bon
expression is in part mediated by sur, because MZsur mutants
display reduced bon expression. The downregulation of bon
might explain the reduced expression of the endodermal markers axial
and sox17 in MZsur mutants
(Pogoda et al., 2000
;
Sirotkin et al., 2000
)
(Fig. 6), because bon
is required for axial and sox17 expression
(Alexander and Stainier, 1999
;
Kikuchi et al., 2000
). The low
levels of bon in MZsur embryos are apparently sufficient for
many processes that are disrupted upon complete loss of both sur and
bon in MZsur;bon embryos. For instance, cardiac mesoderm,
endoderm, prechordal plate and ventral neuroectoderm form in MZsur
embryos despite the lower levels of bon. It is conceivable that the
phenotypic variability observed in MZsur mutants
(Pogoda et al., 2000
;
Sirotkin et al., 2000
)
(Fig. 3) is in part caused by
slightly varying levels of bon in these mutants. Reduction of
bon expression is not as severe in MZsur or
MZsur;bon mutants as in MZoep mutants
(Fig. 1), indicating that
factors other than Sur and Bon are also involved in Nodal signaling to enhance
bon expression (see below). In contrast to bon, sur
expression is not affected by loss of Nodal signaling. Taken together, these
results indicate that both Sur and Bon are initially expressed in responsive
cells independently of Nodal signaling and can thus serve as components of the
Nodal signaling pathway. Nodal signaling, in part mediated by Sur but not Bon,
then further enhances and maintains bon expression, allowing
efficient activation of Bon target genes.
The finding that Bon can associate with phosphorylated Smad2
(Randall et al., 2002) and is
initially expressed independently of Nodal signaling also offers an
explanation for the finding that Bon is not able to activate Nodal target
genes such as cas in the animal region of the blastula
(Kikuchi et al., 2000
). We
suggest that Bon is only active upon association with phosphorylated Smad2,
and this association is Nodal dependent. In turn, Bon might restrict the
expression domain of some targets of Nodal signaling, because bon is
expressed only in cells at the margin. For example, cas and
sox17 are only expressed in the domain where high levels of Nodal
signaling overlap with and induce bon expression. Ectopic expression
of bon extends the territory of cas and sox17
expression, but this domain is still within the normal range of Nodal signals
(Kikuchi et al., 2000
;
Chen and Schier, 2001
).
Interestingly, these observations are reminiscent of the Dorsal-dependent
regulation of a subset of target genes in the Drosophila embryo. High
levels of Dorsal induce expression of the transcription factor Twist in the
ventral-most region of the embryo. Dorsal and Twist then act together to
activate a group of ventrally expressed target genes such as snail
(Ip et al., 1992
;
Stathopoulos and Levine,
2002
). Analogously, phosphorylated Smad2 might activate
bon in the margin region of the zebrafish embryo. Phosphorylated
Smad2 and Bon would then associate in marginal-most cells where phosphorylated
Smad2 levels are high and specifically regulate vegetally expressed target
genes. Hence, both Dorsal and phosphorylated Smad2 appear to induce
transcriptional activators to regulate a specific set of target genes. It is
tempting to speculate that this strategy is a general mechanism to translate
the graded activity of a transcription factor into discrete downstream
responses.
Bon and Sur have overlapping roles in prechordal plate, heart and
endoderm formation
Although a plethora of factors has been identified that interact with
regulatory Smads, an in vivo requirement for these factors during vertebrate
development has been established in only a few cases
(Brand et al., 1996;
Chen et al., 1996
;
Schier et al., 1996
;
Solnica-Krezel et al., 1996
;
Stainier et al., 1996
;
Kikuchi et al., 2000
;
Pogoda et al., 2000
;
Sirotkin et al., 2000
;
Hoodless et al., 2001
;
Yamamoto et al., 2001
). Our
double mutant analysis now provides evidence that partners of regulatory Smads
have overlapping roles in vivo (Figs
3,
4,
5,
6,
7,
8). Formation of heart,
prechordal plate and ventral neuroectoderm are only mildly affected in
bon or MZsur embryos but are severely disrupted in embryos
lacking both sur and bon activity. Moreover, although the
penetrance and expressivity of MZsur mutants are variable, loss of
sur and bon leads to fully penetrant and expressive
phenotypes.
The wider roles for bon revealed in MZsur;bon mutants are
also supported by the phenotypes of embryos lacking both bon and
mezzo (Poulain and Lepage,
2002) or bon and spadetail
(Griffin and Kimelman, 2002
)
activity. Loss of the T-box transcription factor spadetail and
bon results in loss of myocardium, indicating overlapping roles of
these genes during cardiac development
(Griffin and Kimelman, 2002
).
Mezzo is another member of the Mix family but, in contrast to Bon, does not
contain phosphorylated Smad interaction motifs and thus appears to act
exclusively downstream of Nodal signaling
(Poulain and Lepage, 2002
).
Loss of mezzo and bon results in heart and prechordal plate
defects, suggesting overlapping roles of these two genes in the formation of
these structures (Poulain and Lepage,
2002
). Although removal of Mezzo enhances the bon
phenotype, we have found no enhancement of MZsur;bon embryos upon
depletion of Mezzo (S.Z. and A.F.S., unpublished).
Bon and Sur regulate separate and common target genes
The requirements for sur and bon are already reflected
before gastrulation in the regulation of downstream genes (Figs
7,
8). We found Nodal-regulated
genes whose expression requires bon but not sur
(cas), sur but not bon (bhikhari, bon,
mezzo), bon or sur (gsc), or neither
bon nor sur (flh, ntl, snail1). It is as yet
unclear whether all these genes are directly regulated by Nodal signaling, but
studies in Xenopus indicate that at least some of these genes might
be direct targets. Experiments involving cycloheximide and/or VP16 fusion
constructs suggest that Mixer-like proteins can directly regulate gsc
(Germain et al., 2000) and
FoxH1 can directly activate mix.2, Xbra, lim-1 and gsc
(Chen et al., 1996
;
Watanabe and Whitman, 1999
;
Osada et al., 2000
).
Similarly, zebrafish cas, mezzo and ntl appear to be
directly regulated by Nodal signaling
(Poulain and Lepage, 2002
).
Moreover, bik elements contain binding sites for FoxH1
(Vogel and Gerster, 1999
) and
the zebrafish gsc promoter contains sequences resembling Mixer
binding sites (McKendry et al.,
1998
). These observations suggest that Nodal signaling leads to
the activation of genes regulated by Bon or Sur, Bon and Sur, or neither Bon
nor Sur.
The use of different transcription factors, such as Sur and Bon,
associating with phosphorylated Smad2 allows Nodal signaling to diverge
downstream of receptor activation. For instance, and as outlined above, the
restricted expression of bon might contribute to the restricted
expression of Nodal-regulated genes implicated in endoderm formation. Indeed,
we might speculate that, during evolution, specific genes have come under the
control of Nodal signaling by the phosphorylated-Smad2-mediated recruitment of
different transcription factors. In this scenario, subsets of genes were
initially regulated by transcription factors independently of phosphorylated
Smad2. Interaction with and eventual dependence on phosphorylated Smad2 would
then usurp these factors into the Nodal signaling pathway. Intriguingly, some
members of the mix family are independent of phosphorylated Smad2,
whereas others interact with phosphorylated Smad2
(Rosa, 1989;
Vize, 1996
;
Chen et al., 1996
;
Ecochard et al., 1998
;
Henry and Melton, 1998
;
Alexander and Stainier, 1999
;
Germain et al., 2000
;
Hill, 2001
;
Whitman, 2001
;
Randall et al., 2002
).
Moreover, FoxH1-VP16 fusion proteins can regulate Nodal targets in the absence
of Nodal signaling (Watanabe and Whitman,
1999
; Pogoda et al.,
2000
). It is thus conceivable that ancestral Mixer- and FoxH1-like
proteins were active independently of phosphorylated Smad2 and have only
recently been recruited into the Nodal signaling pathway. Support for this
model is also provided by the observation that Forkhead transcription factors,
but not Activin/Nodal signals, are involved in endoderm formation in
Caenorhabditis elegans and Drosophila
(Gaudet and Mango, 2002
;
Stainier, 2002
).
Limited roles for Sur and Bon in Nodal autoregulation
It has been speculated that Sur might exclusively enhance the expression of
Nodal signals (Pogoda et al.,
2000). This suggestion was based on the observation that the
expression of the Nodal genes cyclops and squint appears to
be downregulated in MZsur mutants. In apparent contradiction to this,
however, a Sur-VP16 fusion can rescue aspects of the MZoep mutant
phenotype (Pogoda et al.,
2000
). These mutants are unable to transmit Nodal signals
(Gritsman et al., 1999
) and so
Sur-VP16-mediated activation of cyclops and squint would not
have any effect, indicating that, in this context, Sur must regulate other
genes to rescue MZoep mutants. Our results also indicate that a
purely autoregulatory role of Sur is unlikely. In particular, we find that
phosphorylated-Smad2 levels are reduced at dome but not shield stage in
MZsur embryos (Fig.
9). These observations are consistent with results in
Xenopus, in which a FoxH1-Engrailed fusion construct has no effect on
overall phosphorylated Smad2 levels during gastrulation
(Lee et al., 2001
). It is
possible that the effects on phosphorylated Smad2 levels are relatively minor,
because FoxH1 does not only regulate the expression of Nodal ligands but also
feedback inhibitors of Nodal signaling, such as Lefty and Cerberus
(Whitman, 2001
;
Hamada et al., 2002
;
Schier, 2003
). Therefore, the
net effect of elimination of FoxH1 on phosphorylated Smad2 levels might be
quite limited (Fig. 9). In
addition, no change in phosphorylated Smad2 levels is seen upon blocking
bon in wild-type or MZsur mutants. This indicates that the
much more severe phenotype of MZsur;bon mutants is unlikely to be due
to the reduced activity of cyclops, squint or other components of the
Nodal signaling pathway upstream of Smad2 phosphorylation.
Multiple aspects of Nodal signaling are independent of Bon and
Sur
Previous studies have identified several transcription factors that
interact with Smad proteins to regulate the expression of specific genes
(Massague and Wotton, 2000;
Whitman, 2001
;
Hill, 2001
). It is unclear how
many of these factors are required or sufficient to mediate a particular
TGFß signaling process in vivo. We find that the defects in morphology
and gene regulation observed in MZsur;bon double mutants represent
only a subset of the phenotypes observed upon complete block of Nodal
signaling. In particular, Nodal mutants lack all trunk mesoderm, including
blood, pronephros, somites and notochord, and display disrupted expression of
genes such as snail1, flh and ntl
(Feldman et al., 1998
;
Gritsman et al., 1999
). These
defects are not observed in MZsur;bon double mutants, establishing
that Bon and Sur cannot account for all Nodal signaling during mesendoderm
induction. The p53 tumor suppressor has recently been implicated in the
regulation of a subset of Nodal target genes
(Cordenonsi et al., 2003
).
However, blocking p53 in wild type does not lead to mesendoderm
defects in zebrafish (Langheinrich et al.,
2002
) and depletion of p53 in MZsur;bon embryos does not
enhance the phenotype (J.T.B. and A.F.S., unpublished). Our results thus
indicate that at least one additional Smad-associated transcription factor
remains to be identified as a component of the Nodal signaling pathway.
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ACKNOWLEDGMENTS |
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Footnotes |
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