Cincinnati Childrens Hospital Medical Center, Division of Developmental Biology and The Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
Author for correspondence (e-mail:
aaron.zorn{at}chmcc.org)
Accepted 18 March 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Endoderm, Transcription, Sox17, ß-catenin, Foxa, Gata, Xenopus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Xenopus, endoderm development is initiated by the maternal
T-box transcription factor VegT, which is localized to vegetal region of the
egg and early embryo. VegT starts the cascade of endoderm specification in the
vegetal cells by activating the transcription of zygotic endodermal genes
(Clements et al., 1999;
Xanthos et al., 2001
;
Zhang et al., 1998
), which
encode nodal-related proteins (Xnr1,2,4,5,6) and Derriere members of
the TGFß growth factor family (Jones
et al., 1995
; Joseph and
Melton, 1997
; Sun et al.,
1999
; Takahashi et al.,
2000
), homeodomain proteins of the Mixer/Mix/Bix family
(Casey et al., 1999
;
Henry and Melton, 1998
;
Rosa, 1989
;
Tada et al., 1998
;
Vize, 1996
), the zinc-finger
factors Gata4, Gata5 and Gata6 (Jiang and
Evans, 1996
; Weber et al.,
2000
; Xanthos et al.,
2001
), and two closely related HMG domain transcription factors,
Sox17
and Sox17ß (Hudson et
al., 1997
).
Several of these zygotic factors, including Xnr1, Xnr5 and Sox17 are
known to be direct targets of VegT (Hyde
and Old, 2000
; Hilton et al.,
2003
; Engleka et al.,
2001
). In the case of Sox17 it is thought that VegT initially
activates Sox17 transcription but then nodal signaling is required to maintain
its full expression (Engleka et al.,
2001
; Yasuo and Lemaire,
1999
; Clements et al.,
1999
). Indeed, the available data indicates that nodals are the
primary target of VegT and that nodal signaling acts up stream of and is
required to maintain the expression of the Mixer/Mix/Bix, Gata4,
Gata5 and Gata6, and Sox17 transcription factors
(Alexander and Stainier, 1999
;
Clements et al., 1999
;
Kofron et al., 1999
;
Xanthos et al., 2001
;
Yasuo and Lemaire, 1999
).
The epistatic relationships between Mixer, Mix1, Mix2, Bix1, Bix2, Bix3,
Bix4, Gata4, Gata5, Gata6 and Sox17/ß are unresolved, but the
limited data indicate that Mixer and Gata function upstream of Sox17. In both
frog and fish, overexpression of Mixer and Gata4/5 can induce Sox17
transcription but Sox17 cannot induce the expression of Mixer
(Alexander and Stainier, 1999
;
Henry and Melton, 1998
;
Weber et al., 2000
;
Xanthos et al., 2001
).
Furthermore mutations in the zebrafish Mixer/Mix family member
(bon) and Gata5 (faust) indicate that they are both
required for Sox17 expression
(Kikuchi et al., 2000
;
Reiter et al., 2001
). In
Xenopus, a dominant-negative version of Sox17 can inhibit Mixer
function, but dominant-negative Mixer cannot inhibit Sox17 function
(Henry and Melton, 1998
),
suggesting that Mixer acts via Sox17. Thus, although Sox17 is
initially transactivated by the maternal VegT, these data suggests that Sox17
functions as one of the most downstream component of the pathway leading to
endoderm differentiation. However, this model of the endoderm specification
pathway is likely to be an over simplification and the exact relationships
between Sox17, Gata4, Gata5, Gata6 and the Mixer/Mix/Bix family still need to
be carefully resolved.
In Xenopus Sox17/ß are specifically expressed
in the presumptive endoderm at late blastula and gastrula stages and they can
induce endoderm differentiation when ectopically expressed in naïve
ectoderm (Clements and Woodland,
2000
; Hudson et al.,
1997
). Blocking endogenous Sox17 function with a dominant-negative
Sox17 engrailed transcriptional repressor (Sox17:EnR) construct
(Hudson et al., 1997
) or by
antisense oligos (Clements et al.,
2003
) disrupts endogenous endoderm development. Similarly,
targeted deletion of Sox17 in mouse causes severe definitive endoderm defects
and embryonic lethality (Kanai-Azuma et
al., 2002
). Although Sox17 is clearly crucial for endoderm
formation, the downstream transcriptional targets of Sox17, which subsequently
direct endodermal differentiation, are largely unknown.
How Sox17 regulates the transcription of its targets is also an important
unresolved issue. Although all Sox proteins, 30 in the vertebrate genome,
have remarkably similar DNA-binding properties
(Bowles et al., 2000
;
Kamachi et al., 2000
),
distinct Sox proteins none-the-less regulate unique target genes. The
prevailing idea is that interacting protein partners are key determinants of
Sox protein specificity and activity
(Kamachi et al., 2000
;
Wilson and Koopman, 2002
), but
in the case of Sox17, its interacting transcriptional co-factors were
previously unknown.
We have sought to extend our understanding of endoderm development by
focusing on the targets of Sox17 and the mechanism by which Sox17 regulates
their transcription. We have identified a number of transcriptional targets of
Sox17 and provide evidence that ß-catenin is an essential transcriptional
co-factor of Sox17. ß-catenin is best known as a mediator of Wnt
responsive transcription (Wodarz and
Nusse, 1998), and in the Xenopus blastula ß-catenin
interacts with Tcf/Lef HMG transcription factors to activate dorsal organizer
gene expression (Heasman,
1997
; Moon and Kimelman,
1998
). We had previously shown that ß-catenin can also
physically interact with Sox17 (Zorn et
al., 1999a
) but the biological relevance of this interaction was
unclear. We now show that the transactivation domain of Sox17 mediates this
interaction, suggesting that ß-catenin binding is important for Sox17
activity. In animal cap experiments, Sox17 and ß-catenin cooperate to
activate Sox17 target genes, while depletion of ß-catenin from embryos
results in a repression of Sox17 target gene expression. These results extend
our understanding of early endoderm development and suggest that Sox17 and
ß-catenin cooperate to regulate endodermal gene expression. Our findings
also suggest that, like the Tcf/Lef family of HMG box transcription factors,
Sox proteins may act as Wnt/ß-catenin effectors.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RT-PCR analysis
Each experiment was repeated at least three times and a representative
example is shown. Total RNA was extracted from embryonic tissue and RT-PCR
analysis was preformed as previously described
(Wilson and Melton, 1994). The
primers in this study are shown in the supplementary data (see Table S1 at
http://dev.biologists.org/supplemental).
For RT-PCR analysis by gel electrophoresis the number of cycles required for
each primer set was empirically determined and a dilution series of whole
embryo cDNA was included in every assay to ensure that the PCR reaction was in
the log-linear range. Controls without reverse transcriptase (RT) were
always included. Owing to space constraints these linearity and RT
controls are not shown.
An Opticon machine (MJ Research) was used for semi-quantitative analysis.
The only change to our PCR reaction conditions was the inclusion of SYBR green
dye in the PCR mix, for convenience we used Qiagen SYBR green PCR mix. For
each experiment and primer pair a serial dilution of whole embryo cDNA was
used to generate a standard curve from which the amount of product in the
experimental samples was determined at the log-linear amplification phase. The
data for each sample is normalized to the expression level of the ubiquitously
expressed gene ornithine decarboxylase (ODC) and presented
as a ratio of ODC expression as previously described
(Xanthos et al., 2001).
DNA constructs and synthetic mRNA
The following DNA constructs and details of RNA synthesis have been
previously described: pT7TS-HA-Sox17ß, pT7TS-HA-Sox17-deletions
constructs, pGEX-ß-catenin, UAS:luciferase reporter, pcDNA6
Sox17ß-V5, pcDNA6 Sox17-V5 and pcDNA6 d1-315-V5
(Zorn et al., 1999a
); and
pCS2+MT-pt-ß-catenin, pCS2+MT-
N-ß-catenin, pCS2+GSK3ß
and pCS2+kdGSK3ß (Yost et al.,
1996
). pT7TS-GR:Sox17ß (Pst1 and T7) was constructed
by inserting the hormone-binding domain of the human glucocorticoid receptor
(a gift from Paul Krieg) in frame into pT7TS-HA-Sox17ß. The Gal4:Sox17
deletion constructs were generated by PCR amplifying indicated fragments of
Sox17ß (Fig. 3A) with
Pfu polymerase and these were cloned in frame with the Gal4
DNA-binding domain in pCMVGT (Zorn et al.,
1999a
). The 3G and
TA mutations were made from
pT7TS-HA-Sox17ß, pT7TS-HA-Sox17
, pcDNA6 Sox17ß-V5 and pcDNA6
Sox17
-V5 parent plasmids using a GeneTailor mutagenesis kit
(Invitrogen).
|
Luciferase assays
COS-1 cells in 24 well plates were co-transfected with 100 ng of
5xGal4 luciferase reporter, 50 ng of pTK:Renilla, and 300 ng of
Gal4DBD:Sox17ß fusion constructs using Fugene (Roche). Cells were
harvested, after 36 hours, extracts prepared, and luciferase activity was
measured and normalized for transfection efficiency with Renilla luciferase
activity using a Promega Luciferase/Renilla Assay System. Experiments were
carried out in triplicate and the average result is shown.
Western blots and protein binding assays
Standard western immunoblotting procedures were used with the following
antibodies: anti-V5-HRP (1:4000, Invitrogen), rabbit anti-ß-catenin
(1:1000, Santa Cruz Biotechnology, #sc-7199), goat anti-rabbit:HRP (1:20,000,
Jackson ImmunoResearch), mouse anti-tubulin (1:1000, Sigma), mouse
anti-histone H1 (1:500, AE-4, Santa Cruz) and goat anti-mouse:HRP (1:10,000,
Jackson ImmunoResearch). Affinity-purified rabbit anti-XSox17ß antibodies
or rabbit anti-humanSox17 antibodies were raised to
IYTIDQDSGAYSTNLLPSLI and CKPEMGLPYQGHDCGVNLSDS
peptides respectively (1:1000, produced by Bethyl Laboratories).
For embryos extracts tissue was homogenized on ice in 10-20 µl per
animal cap or embryo in 250 mM sucrose, 10 mM HEPES (pH 6.8), 1 mM EDTA, 0.5
mM EGTA, 2 mM Na3VO4, 0.2 mM NaF with protease
inhibitors. If the sample was also to be assayed by RT-PCR, half of the
extract was removed and processed to isolate total RNA. The remaining extracts
were cleared by centrifugation (14,000 g, 45 minutes at
4°C), which pellets most of the cytoskeleton and membrane bound
ß-catenin, allowing preferential analysis of the cytosolic signaling pool
of ß-catenin (Heasman et al.,
2000).
For ß-catenin binding experiments, COS-1 cells were transfected with the indicated constructs in pcDNA6-V5. Thirty-six hours after transfection, cells were lysed on ice with 1 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5% NP-40 and protease inhibitors) and centrifuged at 14,000 g for 10 minutes at 4°C. Equal amounts of lysate were incubated for 3 hours at 4°C, with 5 µg of either purified GST or GST-ß-catenin bound to agarose beads in lysis buffer adjusted to 20% glycerol and 1 mM DTT. Agarose beads were washed five times with lysis buffer containing 500 mM NaCl and bound proteins were eluted in SDS sample buffer.
SW480 cell fractionation and co-immunoprecipitation
Approximately 109 SW480 cells were homogenized in 3.5 ml of cell
lysis buffer (250 mM sucrose, 30 mM KCl, 6 mM MgCl2, 20 mM HEPES pH
7.9, 0.5 mM EDTA, 0.2 mM NaF, 2 mM Na3VO4 and 0.1%
Triton X-100 with protease inhibitors). Nuclei were liberated from the cells
by 15 gentle strokes of a tight fitting pestle in a dounce homogenizer. The
extract was centrifuged at 500 g for 10 minutes at 4°C.
The supernatant was used as the cytosolic fraction. The pellet was resuspended
in nuclear lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% NP-40 and 0.5%
Triton X 100 with protease inhibitors) and sonicated to lyse the nuclei and
thoroughly sheer the genomic DNA. The resulting extract was centrifuged at
12,000 g for 15 minutes at 4°C and the supernatant was
used as the nuclear fraction.
For co-immunoprecipitation, equal amounts of nuclear extract were precleared with 1 µg of rabbit preimmune serum and Protein-A agarose for 1 hour at 4°C. The extracts were then incubated for 4 hours at 4°C with Protein-A agarose and either 1 µg of anti-Sox17 antibody or 1 µg of anti-HA as a negative control. In some samples, 2 µg of either a competing Sox17 peptide recognized by the Sox17 antibody or a negative control peptide, not recognized by the Sox17 antibody (from a different part of the Sox17 protein) were included in the incubations. Immunoprecipitates were washed four times in nuclear lysis buffer, resolved by SDS-page and subjected to anti-ß-catenin immunoblotting.
Immunocytochemistry and confocal microscopy
Embryos were fixed in 80 mM PIPES (pH 6.8), 5 mM EGTA (pH 8.0), 1 mM
MgCl2, 0.2% TritonX-100, 3.7% formaldehyde for 30 minutes; bisected
with a razor blade; re-fixed for 1 hour; rinsed in PBS; and then stored in 80%
methanol/20% DMSO at 20°C. Rehydrated embryos were blocked in 10%
lamb serum, 4% BSA, 2% DMSO, 0.2% Tween-20 in PBS overnight at 4°C,
incubated in rabbit anti-ß-catenin antibodies (1:200, H-102, Santa Cruz
Biotechnology, #sc-7199 or rabbit-anti-ß-catenin from Dr P McCrea) in
PBST (PBS + 0.2% Tween-20) for 30-36 hours at 4°C, washed in PBST three
times for 1 hour per wash and then overnight at 4°C, followed by
incubation with goat anti-rabbit Cy5, (1:250, Jackson ImmunoResearch,
#111-175-144) in PBST for 30-36 hours at 4°C. Embryos were again washed,
as above, dehydrated in methanol, and cleared in 2 volumes benzyl benzoate: 1
volume benzyl alcohol. Images were captured with LSM 510 software using a
Zeiss 510 Laser Scanning Confocal Microscope (10x objective; HeNe laser
scanning at 633 nm). A z-series projection of 16 serial scan 10 µM
sections is shown as an optical stack.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
It was important to determine if these genes are direct Sox17
transcriptional targets or not. Therefore we repeated the animal cap
experiments with a hormone inducible form of Sox17ß consisting of the
hormone-binding domain of the human glucocorticoid receptor fused to the
N-terminus of Sox17ß (GR:Sox17ß;
Fig. 2A). The resulting
GR:Sox17ß protein is translated immediately but remains inactive in the
cytoplasm until dexamethasone is added to the medium, at which time GR:fusion
enters the nucleus and becomes active
(Hollenberg et al., 1993).
This allowed us to control the time of Sox17 activity and repeat the induction
of Sox17 target genes in the presence of 10 µg/ml cycloheximide, which
blocks translation (Cascio and Gurdon,
1987
). Genes transcribed in response to GR:Sox17ß when
translation was blocked must be direct targets. As a control, we treated
animal caps with activin protein, a Nodal-like TFGß ligand, which is
known to induce ectopic endoderm and mesoderm in animal cap tissue
(Hudson et al., 1997
).
Chordin is an indirect mesodermal target of activin
(Crease et al., 1998
) and as
expected it was not strongly induced by activin in the presence of
cycloheximide, indicating that translation was effectively inhibited.
|
Whole-mount in situ hybridization to bisected gastrulae confirmed that the
target genes are co-expressed with Sox17 in the deep endoderm
(Fig. 2C). In summary, the data
indicate that Hnf1ß, Foxa1, Foxa2, Sox17 and
Edd (this study) (Clements et
al., 2003
) are direct Sox17 targets, whereas 26D10 is an
indirect target, and we could not determine if Gata4, Gata5, Gata6,
Gsc and Xnr4 are direct Sox17 targets or not.
The transactivation domain of Sox17ß
To better understand how Sox17ß regulates the transcription of its
targets, we performed a structure-function analysis to localize its
transactivation domain. Similar studies have been useful for identifying
potential co-factor interaction sites in other Sox proteins
(Kamachi et al., 1999;
Kamachi et al., 2000
;
Nowling et al., 2000
).
Different parts of Sox17ß were fused to the GAL4 DNA-binding domain and
assayed for transactivation capacity on a UAS:Luciferase reporter in mammalian
COS-1 cells (Fig. 3A). GAL4
fused to the entire Sox17ß had little activity, but a latent
transactivation domain in the C terminus was revealed when regions of the N
terminus and HMG box were removed. This suggests that transactivation domain
may be regulated somehow by inhibition from other regions of the protein.
Testing further deletion construct allowed us to map the minimal
transactivation domain to 25 amino acids between residues 315-340.
To determine if this transactivation domain was functionally important in
embryos, we tested a similar set of Sox17ß deletion constructs for the
ability to induce the transcription of endogenous Sox17 target genes in animal
cap assays. Consistent with the tissue culture experiments, C-terminal amino
acids 315-373 were required to activate target gene transcription and deletion
of the N terminus (d56-373) resulted in a more potent transcriptional
activator (Fig. 3B). We noticed
that deletion d1-340 (which based on the tissue culture experiments, still
contains the putative activation domain) could not transactivate
Hnf1ß, Edd and Foxa2, whereas Foxa1
and Sox17 were still induced, suggesting that in vivo the
activation domain is compromised in the d1-340 mutant. The deletion mutant
d1-315, which lacks the activation motif, was unable to stimulate
transcription of any Sox17 targets, except Foxa1, at a reduced level.
This indicates that the transactivation motif is essential in vivo and also
suggests that different Sox17 targets have a differential requirement for the
transactivation domain. Western blot analysis indicated that all of the
truncated Sox proteins were expressed to similar levels in COS-1 and animal
cap cells (data not shown).
Close examination of the transactivation domain sequence revealed a short
motif conserved in all the Sox F subclass of Sox proteins
(Fig. 3C), which includes
Sox17, Sox18 and Sox7 (Bowles et al.,
2000). Interestingly, zebrafish Sox17 does not have this motif,
but it is present in Casanova, a zebrafish Sox17-like protein essential for
endoderm formation (Dickmeis et al.,
2001
; Kikuchi et al.,
2001
). Outside of the highly conserved HMG domain, the sequences
of Sox17, Sox18, Sox7 and Casanova are very divergent, only 5-10% identical
amino acids, with the exception of this short conserved motif. To test if this
conserved motif is essential for transactivation, we mutated the conserved the
amino acids EQY or DQY to GGG (referred to as 3G mutant) or we deleted the
amino acids EFDQY or EFEQY (referred to as
TA mutant) in Sox17
and Sox17ß respectively (Fig.
3C). In GAL4:fusions-reporter assays
(Fig. 3A; compare 200-340 with
200-340-3G and 200-340-
TA) and in Xenopus animal cap
experiments (Fig. 3D), the
mutant Sox17 proteins had significantly reduced transactivation capacity.
Again, we observed that the transcription of Hnf1ß and
Foxa2, was more sensitive to an intact transactivation motif than
Foxa1 and Edd.
In summary we have identified a conserved motif in SoxF class proteins,
which is essential for both Sox17 and Sox17ß transcriptional
activity and which may mediate interactions with important protein
co-factors.
Sox17 associates with ß-catenin
Sox proteins generally require interacting protein partners to function
(Kamachi et al., 2000;
Wilson and Koopman, 2002
) and
we had previously shown in overexpression experiments that Sox17
and
Sox17ß can directly bind the armadillo repeats of ß-catenin
(Zorn et al., 1999a
), but the
biological relevance of this Sox17/ß-catenin interaction was unclear. We
hypothesize that analogous to Tcf/Lef, ß-catenin may be an important
transcriptional co-factor of Sox17.
If Sox17 and ß-catenin interact to regulate endodermal gene
expression, then ß-catenin must be present in the nuclei of gastrula
endoderm cells. We therefore closely examined the subcellular distribution of
ß-catenin protein by immunostaining and confocal microscopy and observed
obvious nuclear ß-catenin throughout the Sox17 expressing deep endoderm
cells of the gastrula (Fig.
4A), as previously reported
(Schohl and Fagotto,
2002).
|
If the interaction with ß-catenin influences the ability of Sox17 to
regulate the transcription of its target genes, we predicted that the
conserved transactivation motif would be involved in ß-catenin binding.
To test this, we transfected COS-1 cells with V5-epitope tagged versions of
either wild-type Sox17ß, Sox17 or various versions with mutated
transactivation domains: Sox17ß-3G, Sox17ß-
TA,
Sox17ß-d1-315, Sox17
-3G, Sox17
-
TA (details of these
mutants are shown in Fig. 3). As a control, we also tested a Sox17ß construct with a mutation in the
HMG box where Gly93 was changed to arginine (Sox17ß-G93R), which disrupts
the HMG domain structure and DNA-binding activity
(Love et al., 1995
) (data not
shown) but which should not effect ß-catenin binding. Extracts were
prepared from the resulting cells and incubated with either GST-agarose or
GST-ß-catenin-agarose beads. After washing, the bound proteins were
visualized by anti-V5 western blotting.
We found that mutations or deletion of the conserved transactivation motif
in either Sox17 or Sox17ß impaired or abolished the ability to
bind to ß-catenin, while the mutation in the HMG box had no effect on
ß-catenin binding (Fig.
5). Thus, the ability of Sox17 to activate transcription of its
target genes (Fig. 3)
correlates with ß-catenin binding, suggesting that ß-catenin may
facilitate the ability of Sox17 to activate transcription.
|
|
We next asked if Sox17 required ß-catenin to activate transcription of
its targets. It is possible that the low level of nuclear ß-catenin found
in the animal cap cells (Schohl and
Fagotto, 2002) (Fig.
4) facilitates the ability of Sox17 to activate its targets in
this non-endodermal tissue. To test this, we expressed Sox17ß in animal
cap tissue where endogenous ß-catenin had been depleted by antisense
morpholino oligos (Heasman et al.,
2000
) and assayed for the expression of Sox17 target genes by
real-time RT-PCR (Fig. 7A).
Endogenous ß-catenin protein levels and the expression of injected
Sox17ß were monitored by western blotting
(Fig. 7B).
|
ß-Catenin is required for normal endoderm formation
If Sox17 and ß-catenin interact to regulate expression of endodermal
genes during normal development, then ß-catenin should be required for
the expression of Sox17 target genes. To test this prediction, we depleted
endogenous ß-catenin protein from embryos by microinjecting antisense
ß-catenin morpholino oligos into 2-cell stage embryos. The resulting
embryos were assayed at gastrula stage by real-time RT-PCR for endodermal and
organizer gene expression (Fig.
8A). A proportion of the same sample was assayed by western
blotting to monitor the levels of ß-catenin protein
(Fig. 8B).
|
We also assayed the expression of components of the endoderm specification
pathway such as nodals and Mixer to determine if all endoderm development was
compromised or just Sox17 targets. We observed that Xnr1 and
Xnr2 were moderately downregulated while the expression of
Derriere was unchanged and Xnr4 was moderately increased in
ß-catenin-depleted embryos (Fig.
8A) (Xanthos et al.,
2002). The reduction in Xnr1 and Xnr2 mRNA
levels is unlikely to account for the reduced expression of Sox17 targets for
several reasons. First, the levels of Sox17 and Mixer RNA,
both of which are nodal targets (Hudson et
al., 1997
; Henry and Melton,
1998
) (Fig. 2),
were changed only modestly by depletion of ß-catenin
(Fig. 8)
(Xanthos et al., 2002
).
Sox17
transcripts were only reduced to
60% of wild-type
levels and Sox17ß mRNA levels were actually increased to
160% wild-type levels. Furthermore, nodal signaling regulates
Hnf1ß, Edd and Foxa2 transcription indirectly,
via Sox17. Blocking Sox17 function either with a dominant negative Sox17 or by
depleting Sox17 with antisense oligos, inhibits activin (nodal) induction of
Hnf1ß, Edd and Foxa2 in animal caps
(Hudson et al., 1997
;
Clements et al., 2003
). This
is consistent with our results indicating Hnf1ß, Edd
and Foxa2 transcription is indirectly regulated by activin but
directly activated by Sox17. Therefore it is unlikely that the modest decrease
in Xnr1, Xnr2 could account for the reduced expression of Sox17 targets.
In summary, these experiments show that ß-catenin is essential for endoderm formation downstream of Sox17. Furthermore, our results suggest that ß-catenin is an important co-factor of Sox17, assisting in the transcription of some of its downstream target genes.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endodermal genes reported to be downregulated in Sox17 loss-of-function
studies are largely consistent with the Sox17 targets we have identified. In a
recent study by Clements et al. (Clements
et al., 2003) depletion of Sox17 in Xenopus embryos by
antisense morpholino oligos resulted in a reduction of Gata5 and
Edd expression. In addition, analysis of Sox17 null mutant mice found
that Foxa1 and Foxa2 expression was dramatically reduced
(Kanai-Azuma et al., 2002
).
However, Foxa2 was largely unaffected in Sox17-depleted
Xenopus embryos (Clements et al.,
2003
), suggesting that other factors also regulate its
expression.
The identification of Foxa1 and Foxa2 as direct Sox17
targets is particularly important as these hepatic nuclear factors are known
to be involved in endodermal organ differentiation and tissue-specific gene
expression (Duncan et al.,
1998; Kaestner et al.,
1999
). Furthermore, Foxa2 is essential for definitive
endoderm development in mice (Ang et al.,
1993
; Hallonet et al.,
2002
), but its epistatic position in the endoderm specification
pathway was unclear.
ß-catenin is a Sox17 co-factor
Sox proteins generally require interacting protein partners in order to
regulate the transcription of their target genes
(Wilson and Koopman, 2002). We
had previously shown that Sox17
and Sox17ß could physically
interact with the armadillo repeats of ß-catenin
(Zorn et al., 1999a
), but at
that time the biological significance was previously unclear. Our data now
suggests that ß-catenin is a transcriptional co-factor of Sox17.
It is interesting that the transactivation motif and ß-catenin binding
are not absolutely required for Sox17 to activate the transcription of
Foxa1, whereas Sox17-induced transcription of Hnf1ß and
Foxa2 is much more dependent on the transactivation motif and
ß-catenin. The basis of this difference and how ß-catenin
potentiates the ability of Sox17 to activate the transcription remains to be
determined. One possibility is that ß-catenin recruits the co-activator
CBP/p300 (Hecht et al., 2000;
Takemaru and Moon, 2000
) to
Sox17 target gene promoters.
The role of ß-catenin in normal endoderm development
ß-Catenin is best known in Xenopus development for activating
the expression of organizer genes through Tcf/Lef complexes. We suggest that
the same dorsoanterior ß-catenin activity that regulates organizer gene
expression, promotes high levels of Sox17 target gene transcription when
complexed with Sox17 in the anterior endoderm. If fact, we have observed that
most of the Sox17 target genes are first more strongly activated in the
dorsoanterior endoderm relative to the ventroposterior (data not shown). It is
unlikely that ß-catenin/Tcf complexes directly regulate the transcription
of the Sox17 targets, because even though Tcf1, Tcf3, Tcf4 and Lef1 are
endogenously expressed in animal cap cells
(Roel et al., 2003;
Molenaar et al., 1998
;
Houston et al., 2002
),
overexpression of activated ß-catenin or Tcf1, Tcf3, Tcf4 and Lef1 did
not activate the transcription of Sox17 targets (see Figs S1-S3 at
http://dev.biologists.org/supplemental).
In addition, in Xenopus embryos depleted of Tcf3, the only Tcf/Lef
protein shown to regulate organizer gene expression
(Houston et al., 2002
), we
observed no changes in Sox17 target gene expression (see Figs S1-S3 at
http://dev.biologists.org/supplemental).
Together, these data suggest that ß-catenin/Tcf complexes are unlikely to
regulate Sox17 target genes in vivo.
Considering all of our data, the simplest interpretation is that Sox17
requires ß-catenin to robustly activate the transcription of its target
genes. Emerging evidence from other model systems also indicates that
ß-catenin is required for endoderm formation in C. elegans, sea
urchin, ascidian and the mouse (Imai et
al., 2000; Logan et al.,
1999
; Rocheleau et al.,
1997
; Stainier,
2002
).
Sox proteins as effectors of ß-catenin signaling
Our findings have broader implications for how Sox proteins may act as
Wnt/ß-catenin transcriptional effectors. Our data suggest that
Sox17ß and ß-catenin interact to regulate endodermal gene
transcription in a manner analogous to ß-catenin/Tcf regulation of Wnt
responsive transcription (Behrens et al.,
1996; Molenaar et al.,
1996
; Wodarz and Nusse,
1998
). The fact that the transactivation and
ß-catenin-binding motif in Sox17 is conserved in most SoxF subgroup
members, suggests that other SoxF proteins may similarly interact with
ß-catenin. Indeed, human Sox7 was recently shown to interact with
ß-catenin in tissue culture reporter assays
(Takash et al., 2001
).
Furthermore, in other contexts, Sox proteins (Sox17, Sox3 and Sox7) can
antagonize ß-catenin/Tcf-mediated transcription
(Zorn et al., 1999a
;
Takash et al., 2001
;
Zhang et al., 2003
). Thus, the
interaction of ß-catenin with different Tcf or Sox proteins may explain,
in part, how the Wnt signaling pathway can elicit diverse transcriptional
responses in different cellular contexts.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* These authors contributed equally to this work
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexander, J. and Stainier, D. Y. (1999). A molecular pathway leading to endoderm formation in zebrafish. Curr. Biol. 9,1147 -1157.[CrossRef][Medline]
Ang, S. L., Wierda, A., Wong, D., Stevens, K. A., Cascio, S.,
Rossant, J. and Zaret, K. S. (1993). The formation and
maintenance of the definitive endoderm lineage in the mouse: involvement of
HNF3/forkhead proteins. Development
119,1301
-1315.
Behrens, J., von Kries, J. P., Kühl, M., Bruhn, L., Wedlich, D. R. G. and Birchmeier, W. (1996). Functional interaction of b-catenin with the transcription factor LEF-1. Nature 382,638 -642.[CrossRef][Medline]
Bolce, M. E., Hemmati-Brivanlou, A. and Harland, R. M. (1993). XFKH2, a Xenopus HNF-3 alpha homologue, exhibits both activin-inducible and autonomous phases of expression in early embryos. Dev. Biol. 160,413 -423.[CrossRef][Medline]
Bowles, J., Schepers, G. and Koopman, P. (2000). Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev. Biol. 227,239 -255.[CrossRef][Medline]
Cascio, S. and Gurdon, J. B. (1987). The initiation of new gene transcription during Xenopus gastrulation requires immediately preceding protein synthesis. Development 100,297 -305.[Abstract]
Casey, E. S., Tada, M., Fairclough, L., Wylie, C. C., Heasman,
J. and Smith, J. C. (1999). Bix4 is activated directly by
VegT and mediates endoderm formation in Xenopus development.
Development 126,4193
-4200.
Cho, K. H. Y., Blumberg, B., Steinbeisser, H. and de Robertis, E. M. (1991). Molecular nature of Spemanns organizer: the role of the Xenopus homeobox gene goosecoid. Cell 67,1111 -1120.[Medline]
Clements, D. and Woodland, H. R. (2000). Changes in embryonic cell fate produced by expression of an endodermal transcription factor, Xsox17. Mech. Dev. 99, 65-70.[CrossRef][Medline]
Clements, D., Cameleyre, I. and Woodland, H. R. (2003). Redundant early and overlapping larval roles of Xsox17 subgroup genes in Xenopus endoderm development. Mech. Dev. 120,337 -348.[CrossRef][Medline]
Clements, D., Friday, R. V. and Woodland, H. R.
(1999). Mode of action of VegT in mesoderm and endoderm
formation. Development
126,4903
-4911.
Crease, D. J., Dyson, S. and Gurdon, J. B.
(1998). Cooperation between the activin and Wnt pathways in the
spatial control of organizer gene expression. Proc. Natl. Acad.
Sci. USA 95,4398
-4403.
Dickmeis, T., Mourrain, P., Saint-Etienne, L., Fischer, N.,
Aanstad, P., Clark, M., Strahle, U. and Rosa, F. (2001). A
crucial component of the endoderm formation pathway, CASANOVA, is encoded by a
novel sox-related gene. Genes Dev.
15,1487
-1492.
Duncan, S. A., Navas, M. A., Dufort, D., Rossant, J. and
Stoffel, M. (1998). Regulation of a transcription factor
network required for differentiation and metabolism.
Science 281,692
-695.
Engleka, M. J., Craig, E. J. and Kessler, D. S. (2001). VegT Activation of Sox17 at the midblastula transition alters the response to nodal signals in the vegetal endoderm domain. Dev. Biol. 237,159 -172.[CrossRef][Medline]
Gawantka, V., Pollet, N., Delius, H., Vingron, M., Pfister, R., Nitsch, R., Blumenstock, C. and Niehrs, C. (1998). Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning. Mech. Dev. 77,95 -141.[CrossRef][Medline]
Hallonet, M., Kaestner, K. H., Martin-Parras, L., Sasaki, H., Betz, U. A. K. and Ang, S. L. (2002). Maintenance of the specification of the anterio definitive endoderm and forebrain depends on the axial mesendoderm: a study using HNF3ß/Foxa2 conditional mutants. Dev. Biol. 243,20 -33.[CrossRef][Medline]
Heasman, J. (1997). Patterning the Xenopus
blastula. Development
124,4179
-4191.
Heasman, J., Kofron, M. and Wylie, C. (2000). Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222,124 -134.[CrossRef][Medline]
Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F. and
Kemler, R. (2000). The p300/CBP acetyltransferases function
as transcriptional coactivators of beta-catenin in vertebrates.
EMBO J. 19,1839
-1850.
Henry, G. L. and Melton, D. A. (1998). Mixer, a homeobox gene required for endoderm development. Nature 281,91 -96.[CrossRef]
Hilton, E., Rex, M. and Old, R. (2003). VegT activation of the early zygotic gene Xnr5 requires lifting of Tcf-mediated repression in the Xenopus blastula. Mech. Dev. 120,1127 -1138.[CrossRef][Medline]
Hollenberg, S. M., Cheng, P. F. and Weintraub, H.
(1993). Use of a conditional MyoD transcription factor in studies
of MyoD trans-activation and muscle determination. Proc. Natl.
Acad. Sci. USA 90,8028
-8032.
Houston, D. W., Kofron, M., Resnik, E., Langland, R., Destree,
O., Wylie, C. and Heasman, J. (2002). Repression of organizer
genes in dorsal and ventral Xenopus cells mediated by maternal XTcf3.
Development 129,4015
-4025.
Hudson, C., Clements, D., Friday, R. V., Scott, D. and Woodland, H. R. (1997). XSox17 alpha and -beta mediate endoderm formation in Xenopus. Cell 91,397 -405.[Medline]
Hyde, C. E. and Old, R. W. (2000). Regulation
of the early expression of the Xenopus nodal-related 1 gene, Xnr1.
Development 127,1221
-1229.
Imai, K., Takada, N., Satoh, N. and Satou, Y.
(2000). ß-Catenin mediates the specification of endoderm
cells in ascidian embryos. Development
127,3009
-3020.
Jiang, Y. and Evans, T. (1996). The Xenopus GATA-4/5/6 genes are associated with cardiac specification and can regulate cardiac-specific transcription during embryogenesis. Dev. Biol. 15,258 -270.
Jones, C. M., Kuehn, M. R., Hogan, B. L. M., Smith, J. C. and
Wright, C. V. E. (1995). Nodal-related signals induce axial
mesoderm and dorsalize mesoderm during gastrulation.
Development 121,3651
-3662.
Joseph, E. M. and Melton, D. A. (1997). Xnr4: a Xenopus nodal-related gene expressed in the Spemann organizer. Dev. Biol. 184,367 -372.[CrossRef][Medline]
Kaestner, K. H., Katz, J., Liu, Y., Drucker, D. J. and Schutz,
G. (1999). Inactivation of the winged helix transcription
factor HNF3alpha affects glucose homeostasis and islet glucagon gene
expression in vivo. Genes Dev.
13,495
-504.
Kamachi, Y., Cheah, K. S. and Kondoh, H.
(1999). Mechanism of regulatory target selection by the SOX
high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and
SOX9. Mol. Cell Biol.
19,107
-120.
Kamachi, Y., Uchikawa, M. and Kondoh, H. (2000). Pairing SOX off: with partners in the regulation of embryonic development. Trends Genet. 16,182 -187.[CrossRef][Medline]
Kanai-Azuma, M., Kanai, Y., Gad, J. M., Tajima, Y., Taya, C., Kurohmaru, M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P. P. et al. (2002). Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 129,2367 -2379.[Medline]
Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron,
S., Yelon, D., Thisse, B. and Stainier, D. Y. (2001).
casanova encodes a novel Sox-related protein necessary and sufficient for
early endoderm formation in zebrafish. Genes Dev.
15,1493
-1505.
Kikuchi, Y., Trinh, L. A., Reiter, J. F., Alexander, J., Yelon,
D. and Stainier, D. Y. (2000). The zebrafish bonnie and clyde
gene encodes a Mix family homeodomain protein that regulates the generation of
endodermal precursors. Genes Dev.
14,1279
-1289.
Kofron, M., Demel, T., Xanthos, J., Lohr, J., Sun, B., Sive, H.,
Osada, S., Wright, C., Wylie, C. and Heasman, J. (1999).
Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT
via TGFbeta growth factors. Development
126,5759
-5770.
Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, M. M. and Kemler, R. (2002). Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm. Dev. Cell 3,171 -181.[Medline]
Logan, C. Y., Miller, J. R., Ferkowicz, M. J. and McClay, D.
R. (1999). Nuclear beta-catenin is required to specify
vegetal cell fates in the sea urchin embryo.
Development 126,345
-357.
Love, J. J., Li, X., Case, D. A., Giese, K., Grosschedl, R. and Wright, P. E. (1995). Structural basis for DNA bending by the architectural transcription factor LEF-1. Nature 376,791 -795.[CrossRef][Medline]
Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Marduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O. and Clevers, H. (1996). XTcf-3 transcription factor mediates ß-catenin-induced axis formation in Xenopus embryos. Cell 86,391 -399.[Medline]
Molenaar, M., Roose, J., Peterson, J., Venanzi, S., Clevers, H. and Destree, O. (1998). Differential expression of the HMG box transcription factors XTcf-3 and XLef-1 during early xenopus development. Mech. Dev. 75,151 -154.[CrossRef][Medline]
Moon, R. T. and Kimelman, D. (1998). From cortical rotation to organizer gene expression: towards a molecular explanation of axis specification in Xenopus. BioEssays 20,536 -545.[CrossRef][Medline]
Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B. and Polakis, P. (1995). Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc. Natl. Acad. Sci. USA 92,3046 -3050.[Abstract]
Nieuwkoop, P. D. and Faber, J. (1994).Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis . New York: Garland Publishing.
Nowling, T. K., Johnson, L. R., Wiebe, M. S. and Rizzino, A.
(2000). Identification of the transactivation domain of the
transcription factor Sox-2 and an associated co-activator. J. Biol.
Chem. 275,3810
-3818.
Reiter, J. F., Kikuchi, Y. and Stainier, D. Y.
(2001). Multiple roles for Gata5 in zebrafish endoderm formation.
Development 128,125
-135.
Rocheleau, C. E., Downs, W. D., Lin, R., Wittmann, C., Bei, Y., Cha, Y. H., Ali, M., Priess, J. R. and Mello, C. C. (1997). Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 90,707 -716.[CrossRef][Medline]
Roel, G., van den Broek, O., Spieker, N., Peterson-Maduro, J. and Destree, O. (2003). Tcf-1 expression during Xenopus development. Gene Expr. Patt. 3, 123-126.[CrossRef]
Rosa, F. M. (1989). Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumtive endoderm cells of Xenopus embryos. Cell 57,965 -974.[Medline]
Ruiz i Altaba, A., Prezioso, V. R., Darnell, J. E. and Jessell, T. M. (1993). Sequential expression of HNF-3 beta and HNF-3 alpha by embryonic organizing centers: the dorsal lip/node, notochord and floor plate. Mech. Dev. 44, 91-108.[CrossRef][Medline]
Sasai, Y., Lu, B., Piccolo, S. and De Robertis, E. M. (1996). Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps. EMBO J. 15,4547 -4555.[Abstract]
Schohl, A. and Fagotto, F. (2002).
ß-catenin, MAPK and Smad signaling during early Xenopus development.
Development 129,37
-52.
Sive, H. L., Grainger, R. M. and Harland, R. M. (2000). Early Development of Xenopus laevis: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Stainier, D. Y. (2002). A glimpse into the
molecular entrails of endoderm formation. Genes Dev.
16,893
-907.
Sun, B. I., Bush, S. M., Collins-Racie, L. A., LaVallie, E. R.,
DiBlasio-Smith, E. A., Wolfman, N. M., McCoy, J. M. and Sive, H. L.
(1999). derriere: a TGF-beta family member required for posterior
development in Xenopus. Development
126,1467
-1482.
Tada, M., Casey, E. S., Fairclough, L. and Smith, J. C.
(1998). Bix1, a direct target of Xenopus T-box genes, causes
formation of ventral mesoderm and endoderm.
Development 125,3997
-4006.
Takahashi, S., Yokota, C., Takano, K., Tanegashima, K., Onuma,
Y., Goto, J. and Asashima, M. (2000). Two novel nodal-related
genes initiate early inductive events in Xenopus Nieuwkoop center.
Development 127,5319
-5329.
Takash, W., Canizares, J., Bonneaud, N., Poulat, F., Mattei, M.
G., Jay, P. and Berta, P. (2001). SOX7 transcription factor:
sequence, chromosomal localisation, expression, transactivation and
interference with Wnt signalling. Nucleic Acids Res.
29,4274
-4283.
Takemaru, K. I. and Moon, R. T. (2000). The
transcriptional coactivator CBP interacts with beta-catenin to activate gene
expression. J. Cell Biol.
149,249
-254.
Vize, P. D. (1996). DNA sequences mediating the transcriptional response of the Mix.2 homeobox gene to mesoderm induction. Dev. Biol. 177,226 -231.[CrossRef][Medline]
Weber, H., Symes, C. E., Walmsley, M. E., Rodaway, A. R. and
Patient, R. K. (2000). A role for GATA5 in Xenopus endoderm
specification. Development
127,4345
-4360.
Wells, J. M. and Melton, D. A. (1999). Vertebrate endoderm development. Annu. Rev. Cell Dev. Biol. 15,393 -410.[CrossRef][Medline]
Wilson, M. and Koopman, P. (2002). Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators. Curr. Opin. Genet. Dev. 12,441 -446.[CrossRef][Medline]
Wilson, P. A. and Melton, D. A. (1994). Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Curr. Biol. 4, 676-686.[Medline]
Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14,59 -88.[CrossRef][Medline]
Xanthos, J. B., Kofron, M., Wylie, C. and Heasman, J.
(2001). Maternal VegT is the initiator of a molecular network
specifying endoderm in Xenopus laevis. Development
128,167
-180.
Xanthos, J. B., Kofron, M., Tao, Q., Schaible, K., Wylie, C. and
Heasman, J. (2002). The roles of three signaling pathways in
the formation and function of the Spemann Organizer.
Development 129,4027
-4043.
Yasuo, H. and Lemaire, P. (1999). A two-step model for the fate determination of presumptive endodermal blastomeres in Xenopus embryos. Curr. Biol. 9, 869-879.[CrossRef][Medline]
Yost, C., Torres, M., Miller, J. R., Huang, E., Kimelman, D. and Moon, R. T. (1996). The axis-inducing activity, stability and subcellular distribution of ß-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev. 10,1443 -1454.[Abstract]
Zhang, C., Basta, T., Jensen, E. D. and Klymkowsky, M. W.
(2003). The ß-catenin/VegT-regulated early zygotic gene Xnr5
is a direct target of Sox3 regulation. Development
130,5609
-5624.
Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998). The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94,515 -524.[Medline]
Zorn, A. M., Barish, G. D., Williams, B. O., Lavender, P.,
Klymkowsky, M. W. and Varmus, H. E. (1999a). Regulation of
Wnt signaling by Sox proteins: XSox17/ß and XSox3 physically
interact with ß-catenin. Mol. Cell
4, 487-498.[Medline]
Zorn, A. M., Butler, K. and Gurdon, J. B. (1999b). Anterior endomesoderm specification in Xenopus by Wnt/beta-catenin and TGF-beta signalling pathways. Dev. Biol. 209,282 -297.[CrossRef][Medline]