Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK
Author for correspondence (e-mail:
roger.patient{at}imm.ox.ac.uk)
Accepted 10 December 2004
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SUMMARY |
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Key words: Activin, Animal caps, Cycloheximide, Emetine, Endoderm, GATA factors, Gut, HNF1ß, HNF3ß, Liver, Nodal-related, Sox17, TGFß, Transcription, VegT, Xenopus
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Introduction |
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The roles played by individual GATA factors in vertebrate endoderm
formation are currently unclear (Rossant
et al., 2003; Soudais et al.,
1995
; Weber et al.,
2000
; Yasuo and Lemaire,
1999
). Over-expression of either GATA4 or 5 in Xenopus
presumptive ectoderm can induce endoderm markers, and mutations in GATA5 in
zebrafish, known as faust, lead to their reduced expression in
embryos (Reiter et al., 1999
;
Reiter et al., 2001
;
Shoichet et al., 2000
;
Weber et al., 2000
). However,
while GATA4/ mice die by day 9.5 dpc and exhibit
severe defects in the closure of the gut, and embryoid bodies differentiated
from GATA4/ ES cells display a defect in the
differentiation of visceral yolk sac endoderm, the GATA5 null mutation is not
an early embryonic lethal mutation in the mouse, giving rise only to
abnormalities in the female genitourinary tract
(Kuo et al., 1997
;
Molkentin et al., 1997
;
Molkentin et al., 2000
;
Narita et al., 1997
). The
other GATA factor associated with endoderm formation is GATA6: null mice die
at day 5.5-6.5 dpc from defects in extra-embyonic endoderm, and recent
evidence suggests that the expression of GATA6 seen in the inner cell mass
(ICM) of the blastocyst as early as 3.5 dpc may represent future primitive
endoderm, identifying GATA6 as a very early determinant of this germ layer
(Koutsourakis et al., 1999
;
Morrisey et al., 1998
;
Rossant et al., 2003
).
Consistent with important roles for GATA4 and 6 in specifying primitive
endoderm, ectopic expression in mouse ES cells of GATA4 or 6, but not
downstream genes like HNF4, is sufficient to drive primitive endoderm
differentiation (Fujikura et al.,
2002
).
The observation that activins are expressed in the ICM of mouse blastocysts
at the same time as GATA6, and the reported induction of GATA4 and GATA5 by
activin in Xenopus presumptive ectoderm, suggest that GATA factors
may mediate TGFß/Nodal signalling during endoderm formation
(Albano et al., 1993;
Ariizumi et al., 2003
;
Hudson et al., 1997
;
Rossant et al., 2003
;
Weber et al., 2000
). This has
been suggested previously in Xenopus, but their precise roles remain
unclear (Clements et al., 1999
;
Clements and Woodland, 2003
;
Yasuo and Lemaire, 1999
). For
example it is unclear whether each GATA factor has a distinct function or
whether they represent a family of redundant genes. A knowledge of their
direct targets would be informative in this regard.
In this study, we explore the roles of Xenopus GATA4, 5 and 6 in
endoderm formation using over-expression and antisense oligonucleotide
approaches. We demonstrate that, like Xenopus GATA5
(Weber et al., 2000), both
GATA4 and the long isoform of GATA6 are potent inducers of the early endoderm
markers, Sox17
and HNF1ß, and the later marker, HNF3ß, also
known as FoxA2, in Xenopus presumptive ectoderm. In addition, using
protein synthesis inhibitors, we show that GATA6 is a direct activator of both
Sox17
and HNF1ß, whereas induction of these genes by GATA4 and 5
is either only partly direct or, in the case of GATA4 on Sox17
,
dependent on GATA6. By blocking production of these GATA factors in vivo, or
in presumptive ectoderm induced to form endoderm by TGFß signalling, we
confirm that they are required for full expression of endodermal markers and
for proper formation of the gut and its outgrowths. These results suggest that
TGFß signaling works through these GATA factors to maintain endodermal
gene expression, and they identify GATA6 as a major player in gut development
and a direct activator of target genes.
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Materials and methods |
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GATA4-, 5- and 6-GR were made by fusing their coding regions to the region
encoding the hormone-binding domain of human glucocorticoid receptor in the
BglII site of the host vector, pSP64T-GR previously described
(Tada et al., 1997). The
sequences of primers for generating the coding regions were the following:
GATA4: 5' GAT CCA GAT CTA GCT AAG ACC AGG TTG TTC C 3' for the
reverse primer and 5' GGA TCA GAT CTA CCA GGA TGT ATC AGA GTA TAG C
3' for the forward primer, and for GATA5: 5' GAT CCA GAT CTG GCA
AGT GCC AGC GCG CAC C 3' for the reverse primer and 5' GGA TCA GAT
CTA CCA GGA TGC CCA GCC GGC CTA CTC C 3' for the forward primer and for
long GATA6: 5' AGC CAA GGC CAA AGC ACA 3' for the reverse primer
and 5' GGA TCA GAT CTA CCA GGA TGG ACC TGA GTG 3' for the forward
primer. All the fusion plasmids were SalI-linearised and
SP6-transcribed. All the constructs were checked by restriction digestion and
by sequencing. Injected RNAs were synthesised using T3 or SP6 polymerase
mMESSAGE mMACHINE kits (Ambion) according to the manufacturer's
instructions.
Embryos and explants
Xenopus embryos were obtained and cultured as previously described
(Weber et al., 2000). They
were injected at the one-cell stage into the animal pole for animal cap assays
or into the vegetal pole for whole embryo assays. RNAs and morpholinos (MOs)
were injected in water (4 nl). In cap explants, the amount of each MO injected
was 5 ng per embryo (determined from titration experiments), or 10 ng in the
presence of activin. In whole embryos, 10-40 ng of each MO was injected per
embryo. Animal cap explants were dissected and cultured as previously
described (Weber et al.,
2000
). Cycloheximide treatment was as previously described
(Tada et al., 1997
). Emetine
was dissolved in water and treatment was similar to cycloheximide but used at
100 µg/ml final concentration. Incorporation of radiolabelled methionine
was measured by spotting protein extracts onto Whatman 3MM paper and
precipitating with 10% trichloroacetic acid (TCA). Unincorporated
radiolabelled methionine was removed by boiling in 5% TCA and
chemiluminescence was counted.
Protein analysis
Protein extraction and western blot analysis were as previously described
(Peterkin et al., 2003),
except that the cap explants were collected when the sibling whole embryos
reached stage 12.
RNA analysis
Whole-mount in situ hybridisation and in situ hybridisation on embryo
sections were conducted as previously described
(Ciau-Uitz et al., 2000). The
abundance of RNAs was determined using quantitative real-time RT-PCR as
described (Peterkin et al.,
2003
). Amounts relative to the housekeeping RNA, ornithine
decarboxylase (ODC), were expressed as a ratio either to stage 6.5
(Fig. 1) or to uninjected
animal caps or embryos (Figs 2,
3,
4,
5). For the sequences of
primers and probes used see Table
1.
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Results |
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Using in situ hybridisation on sections, we previously showed that GATA5 is
expressed in the presumptive sub-blastoporal endoderm in Xenopus
embryos (Weber et al., 2000).
Using the same technique to investigate the spatial expression profiles of
GATA4 and 6, we show that at stage 10, before the onset of gastrulation, both
GATA4 and 6 are expressed in the yolky vegetal cells
(Fig. 1C), which are fated to
form endoderm. The expression pattern is very similar to that seen for GATA5
(Weber et al., 2000
). However,
although we could not detect expression of GATA5 in the supra-blastoporal
endoderm, both GATA4 and 6 are expressed there
(Fig. 1C, immediately above the
arrowheads). We therefore conclude that, while GATA4, 5 and 6 are all
expressed in the non-involuting endoderm, only GATA4 and 6 are expressed in
the involuting endoderm. Thus, the pattern of expression of GATA4 and 6 at
stage 10 is similar to the pan-endodermal marker, Sox17
(Engleka et al., 2001
;
Hudson et al., 1997
). After
gastrulation has begun (stages 10.5-11), the supra-blastoporal endoderm is
negative for both GATA4 and 6 (Fig.
1C, immediately above the arrowheads). In contrast, Sox17
expression in these cells continues
(Engleka et al., 2001
;
Hudson et al., 1997
). It
therefore appears that GATA4 and 6 are expressed in the pre-involuting
endoderm early in gastrulation but not later.
Differences in intensity of expression do exist between the GATA factors in
the presumptive sub-blastoporal endoderm [compare
Fig. 1C with the work of Weber
et al. (Weber et al., 2000)].
Thus, while GATA5 is strongest in the centre of the vegetal hemisphere all the
way from the pole to the floor of the blastocoel, GATA4 and 6 are stronger
towards the edges of this group of cells. Similarly, whereas GATA5 expression
was not seen in any cells in the animal cap, GATA6 and to a lesser extent
GATA4 expression was detected there at stage 10, albeit weakly. The
significance of these observations is however unclear at the present time.
Overall, we conclude that GATA4, 5 and 6 are expressed in Xenopus
embryos early enough to play roles in endoderm formation. Although the
pan-endodermal gene, Sox17, is expressed before GATA4 and 6,
their expression coincides with the major increase in Sox17
expression
seen later and the maintenance of its expression. Their lack of expression in
the supra-blastoporal endoderm after the onset of gastrulation suggests that
they are only transiently involved in such maintenance in the posterior
involuting endoderm.
Differential induction of early endoderm markers by GATA4, 5 and 6 in Xenopus presumptive ectoderm
We have shown previously that Xenopus GATA4 and 5 can induce
endoderm markers in the presumptive ectoderm of Xenopus animal caps
using a semi-quantitative RT-PCR assay
(Weber et al., 2000). In these
experiments, only the short isoform of Xenopus GATA6 was available
and this was unable to induce endodermal markers in this assay. We therefore
asked if the long isoform of Xenopus GATA6
(Brewer et al., 1999
;
Peterkin et al., 2003
) has
this activity. Equal concentrations of mRNAs encoding GATA4, 5 and 6 were
injected into the animal poles of one-cell embryos. Animal cap explants were
removed from injected embryos when they reached stage 8.5-9 and cultured until
the sibling embryos reached stage 12. Harvested animal caps were divided into
two for determination of protein expression by Western blot and endodermal
gene induction by quantitative, real-time RT-PCR. Both short and long isoforms
of GATA6 were synthesised, with the short isoform representing roughly
one-third of the total GATA6 protein present
(Fig. 1D). However, when gel
loading was taken into account, the long isoform was judged to be present at
equivalent levels to GATA4 and 5, enabling a comparison to be made between the
GATA factors for their capacities to induce endodermal gene expression.
The extracted RNA was probed for the expression of three genes,
Sox17, HNF1ß and HNF3ß, expressed
in endoderm at stage 12. Expression of Sox17
is always restricted to
endoderm, while HNF1ß is also expressed in mesodermal derivatives from
gastrula stages (Demartis et al.,
1994
; Hudson et al.,
1997
) (see Fig. 5).
HNF3ß expression is restricted to the anterior and posterior endoderm
from early gastrula stages (Suri et al.,
2004
). Differences in the strength of induction of these
endodermal genes were observed between the GATA factors
(Fig. 1E,F). GATA4 and 6
induced both Sox17
and HNF3ß with similar efficiencies and
slightly more effectively than GATA5, already shown to be a potent inducer of
endoderm markers and used here as a positive control
(Weber et al., 2000
). For
HNF1ß, while all three GATA factors induced significant expression, GATA4
was the most effective (Fig.
1E). We therefore conclude that the long isoform of GATA6 is a
potent inducer of endoderm, like GATA4 and 5, and that the three GATA factors
differ in their contributions to the expression of different endodermal
genes.
GATA6 is a direct activator of Sox17 and HNF1ß
To determine if GATA4, 5 or 6 were activating Sox17, HNF1ß or
HNF3ß directly in these assays, we built fusions to the glucocorticoid
receptor (GR) ligand-binding domain (Tada
et al., 1997
; Watanabe and
Whitman, 1999
). In the presence of the inducing hormone and
protein synthesis inhibitors, which prevent secondary gene inductions, only
direct targets for GATA4, 5 or 6 should be activated in explants previously
injected with GATA4-GR, GATA5-GR or GATA6-GR RNA.
GATA4-GR RNA was injected into the animal pole of one-cell embryos,
cap explants were removed around the mid-blastula transition (MBT) and subsets
were treated with the hormone, dexamethasone (Dex), and/or the protein
synthesis inhibitor, cycloheximide (CHX), using the conditions previously
published (Tada et al., 1997
)
(Fig. 2A-C). These conditions
inhibit protein synthesis by around 95% as measured by incorporation of
radiolabelled methionine (Fig.
2A). We also used Western blot analysis to check that protein
synthesis was being inhibited in our assays, detecting the fusion proteins via
their haemagglutinin (HA) tags (Fig.
2B). Significant inhibition was seen in samples treated with CHX,
with residual protein presumably deriving predominantly from synthesis prior
to the addition of CHX.
Expression of Sox17, HNF1ß and HNF3ß in GATA4-GR injected
caps was monitored by real-time RT-PCR
(Fig. 2C). Super-induction of
specific genes by CHX has been reported previously
(Tadano et al., 1993
;
Yasuo and Lemaire, 1999
) and
we observed it here for Sox17
to a much greater extent than for
HNF1ß or HNF3ß. Taking account of this, the data suggest that
Sox17
may not be a direct target for GATA4 because the level of
induction with CHX+Dex was not significantly different from that with CHX
alone, even though Dex alone gave a robust induction
(Fig. 2C). Similarly, for
HNF1ß and HNF3ß, comparison of CHX+Dex with Dex alone shows that
induction was clearly suppressed by CHX, suggesting that the bulk of their
expression requires the activities of factors induced by GATA4, whose
synthesis has been blocked by CHX (Fig.
2C). The residual Dex-induced expression over CHX alone seen for
HNF1ß however, suggests a small direct contribution to the activity of
this promoter from GATA4. The results for Sox17
and HNF1ß with CHX
were confirmed using another protein synthesis inhibitor, emetine
(Edwards and Mahadevan, 1992
),
which, although a slightly less efficient inhibitor of protein synthesis
(85-90% versus 95%, data not shown), does not cause super-induction
(Fig. 2D). Here both the
suppression of Dex-induced Sox17
expression and the residual HNF1ß
expression were clearly apparent in the presence of emetine. We therefore
conclude that GATA4 induction of Sox17
and HNF3ß is indirect,
while that of HNF1ß is partly direct
(Fig. 2E).
We took the same approach to determine if Sox17, HNF1ß or
HNF3ß are direct targets for GATA5 in this assay
(Fig. 2A-D). Inhibition of
protein synthesis by CHX was similarly efficient and CHX again super-induced
Sox17
(Fig. 2A-C).
However, while the outcomes for HNF1ß and HNF3ß were very similar to
that seen for GATA4, induction of Sox17
by GATA5-GR in the presence of
Dex was increased compared to CHX super-induction alone, unlike for GATA4-GR
(Fig. 2C). The conclusions for
Sox17
and HNF1ß were again confirmed using emetine as the
inhibitor (Fig. 2D). We
therefore conclude that activation of Sox17
and HNF1ß by GATA5 in
this assay is partly direct while that of HNF3ß is indirect
(Fig. 2E).
The approach was repeated for GATA6
(Fig. 2A-D). Inhibition of
protein synthesis by CHX was similarly efficient and CHX again super-induced
Sox17 (Fig. 2A-C).
However, in contrast to the data for GATA4, and more strikingly than for
GATA5, induction of Sox17
by GATA6-GR in the presence of Dex was
clearly visible over and above CHX super-induction
(Fig. 2C). Indeed the magnitude
of the combined inductions appeared additive, suggesting that GATA6 induction
of Sox17
was largely unaffected by protein synthesis inhibition. In a
similar fashion to Sox17
, HNF1ß induction by GATA6 was clearly
undiminished by the blocking of protein synthesis by CHX
(Fig. 2C). In contrast, as seen
for both GATA4 and 5, HNF3ß induction was completely blocked by CHX
(Fig. 2C). Again, with emetine
as the inhibitor, the conclusions for Sox17
and HNF1ß were
confirmed (Fig. 2D). We
therefore conclude that GATA6 is a direct activator of Sox17
and
HNF1ß but acts indirectly on HNF3ß in this assay
(Fig. 2E).
GATA4 induction of Sox17 and HNF1ß depends on GATA6: GATA factor interdependence
It has been shown that GATA factors act in a cascade in the process of
endoderm formation in C. elegans
(Maduro and Rothman, 2002). In
addition, GATA4 and 6 induce each other as well as endoderm in mouse ES cells
(Fujikura et al., 2002
).
Furthermore, Sox17
can induce GATA4-6 in Xenopus embryos
(Clements et al., 2003
). It
therefore seems likely that mutual transactivation may be part of endoderm
induction by GATA4, 5 and 6. We therefore monitored GATA4, 5 and 6 expression
in animal caps injected with equal amounts of GATA4, 5 or 6
RNAs (Fig. 3A). Primers and
probes were designed against the 3' UTRs of Xenopus GATA4, 5
and 6 in order to allow distinction between the endogenous
transcripts and the injected RNAs. The data show that GATA4-6 can induce
GATA4, albeit weakly (3-4 fold), with GATA4 and 6 being slightly better at it
than GATA5 (Fig. 3A). Induction
of GATA5 by itself was more efficient (6-7-fold), by GATA4 less so (2-3 fold)
and by GATA6 very inefficient (Fig.
3A). Induction of GATA6 by GATA4 was the most efficient (9 fold),
with GATA6 slightly worse (7-fold) and GATA5 worse still (3-fold)
(Fig. 3A). Thus, of the
inductions tested, the induction of GATA6 by GATA4 was the strongest, raising
the possibility that the induction of Sox17
by GATA4 may be via
GATA6.
To test this, we adopted an antisense approach, using morpholino
oligonucleotides (MOs) to efficiently inhibit translation of specific mRNAs
(for a review, see Heasman,
2002). We designed MOs against GATA4, 5 and 6 and injected them
into the animal poles of one-cell embryos along with 50 pg of GATA4,
5 or 6 RNAs. Explants were removed at stage 8.5, cultured until
sibling whole embryos reached stage 12 and subjected to Western blot and
real-time RT-PCR. The minimal level of each MO, required to block translation
of its cognate RNA and thereby induction of Sox17
was determined by
titration (data not shown). To determine the specificity of the MOs, we
injected equal amounts of GATA4, 5 or 6 mRNAs into the
animal poles of one-cell embryos along with GATA4, 5 or 6 MOs, and monitored
inhibition of translation. The MOs were demonstrated to be very specific:
GATA4 translation was completely inhibited by its cognate MO but not at all by
its non-cognate MOs, and likewise for GATA5 and 6 translation
(Fig. 3B).
With respect to endodermal gene induction, the GATA4 MO efficiently blocked
induction of Sox17 and HNF1ß by its cognate mRNA as expected
(Fig. 3C). However, whereas the
GATA5 MO had no effect, the GATA6 MO also severely inhibited induction of
these genes by GATA4 mRNA injection
(Fig. 3C), even though
translation of the GATA4 mRNA was not affected
(Fig. 3B). These data suggest
that GATA4 induces Sox17
and HNF1ß by inducing GATA6 in animal cap
assays. In contrast the inductions of Sox17
and HNF1ß by GATA5 and
6 were significantly less affected by the non-cognate MOs, although some
reduction of HNF1ß induction by GATA6 was observed for the GATA4 MO, and
Sox17
and HNF1ß induction by GATA5 was reduced to
50% by
either GATA4 MO or GATA6 MO (Fig.
3C,D). Overall, these data are consistent with the cycloheximide
experiments and identify GATA6 as the intermediate in the induction of
Sox17
and HNF1ß by GATA4, and GATA4 and 6 as intermediates in one
of the induction pathways for these genes by GATA5
(Fig. 3E).
Full induction of Sox17 and HNF1ß by activin requires GATA4, 5 and 6 in the order: GATA6>GATA4>GATA5
It has recently been shown that Sox17 expression is initially
induced by the T-box transcription factor, VegT, but that maintenance of
expression depends on TGFß signalling
(Clements and Woodland, 2003
).
The most likely TGFß molecules responsible in the Xenopus embryo
are the Nodal family, Xnr1, 2, 4, 5 and 6, and derrière, which are
expressed around the same time as GATA4, 5 and 6. In view of the evidence
presented here that GATA6 is a strong candidate for a direct activator of
Sox17
, we asked whether GATA6, like GATA4 and 5
(Ariizumi et al., 2003
;
Weber et al., 2000
), is
induced by activin (a mimic for Nodal signalling), and if the induction of
Sox17
or HNF1ß by activin is via GATA4, 5 or 6.
Two levels of activin RNA were injected into the animal pole of
Xenopus embryos at the one-cell stage, and animal caps were removed
at stage 8.5 and cultured until stage 12. With the lower dose of
activin RNA (300 fg), animal caps showed classical signs of
elongation caused by the induction of dorsal mesoderm (data not shown), and
endoderm was induced as revealed by the upregulation of Sox17
(Fig. 4A). At 2 pg of
activin RNA, the caps became white due to turning themselves inside
out, thereby presenting their endoderm on the outside (data not shown). These
caps contained an order of magnitude more endoderm compared to caps injected
with 300 fg activin, as revealed by Sox17
expression
(Fig. 4B compared to 4A). When
caps were pre-injected with MOs to GATA4, 5 and/or 6, their activin-induced
morphology and level of Sox17
expression were affected to varying
degrees. At the low dose of activin, the GATA6 MO restored animal cap
morphology to the uninjected phenotype and reduced Sox17
expression
nearly to background, suggesting that GATA6 is required for many of the
effects of activin at this dose (Fig.
4A and data not shown). In contrast, the GATA4 and 5 MOs had
substantially less effect on cap morphology and Sox17
expression
(Fig. 4A and data not shown).
In the case of GATA5, this presumably reflects the fact that its expression
was substantially less induced at this level of activin, compared to GATA4 and
6 (Fig. 4C, lower panel). Thus,
GATA6 is the main GATA factor mediating activin signalling at low doses.
At the high dose of activin, both GATA4 and 6 MOs returned the cap
morphology to a more uninjected phenotype, and substantially reduced
Sox17 expression (Fig.
4B and data not shown). The GATA5 MO had a greater effect on cap
morphology and Sox17
expression than at the low dose of activin, but
still significantly less of an effect than for the GATA4 and 6 MOs
(Fig. 4B). Expression of all
three GATA factors was induced at this level of activin, although GATA4 and 6
were still more abundant than GATA5 (Fig.
4C, lower panel). This could partly explain why the GATA4 and 6
MOs had more dramatic effects on Sox17
expression than the GATA5 MO. At
these doses of activin, expression of both GATA4 and 6 revealed cross- and
auto-dependence, while GATA5 expression was relatively unaffected by
perturbation of any of the three GATA factors
(Fig. 4C, lower panel).
The data obtained for HNF1ß were similar to those described for
Sox17, except for greater contributions from both GATA4 and 5,
consistent with their direct contributions to the expression of this gene
(Fig. 4C, upper panel).
Overall, these data identify GATA6 as the main player in induction of the
endodermal genes studied at low doses of activin, with some support from GATA4
and very little from GATA5 (Fig.
4D). At higher doses of activin, although GATA6 is still the most
active, GATA4 and 5 make greater contributions, with GATA4 still more
important than GATA5 (Fig.
4D).
GATA4, 5 and 6 are required for full endodermal gene expression and gut formation in vivo
In order to determine the roles played by GATA4, 5 and 6 in endoderm
induction in vivo, we injected MOs into the vegetal hemispheres of one- or
two-cell embryos. The MOs were injected separately or in combination, and
their effects on endoderm formation were monitored by in situ hybridisation
and real time RT-PCR using endodermal gene probes, and also by observing the
morphology of the gut (Fig. 5).
Injected embryos, which developed more slowly than their uninjected siblings,
were collected at stages 12 (judged by blastopore size) and 34 for in situ
hybridisation analysis, and at stage 43 for assessment of gut morphology.
Visual assessment of Sox17 expression by whole mount in situ
hybridisation revealed a clear reduction in a majority of embryos in the yolk
plug (YP) region by GATA6 MO and when all three GATA MOs were injected
together, with less obvious reductions by GATA4 or 5 MOs
(Fig. 5A,B). Embryos were
scored according to the numbers seen in each of three categories: normal
(`high'), reduced (`medium') and substantially reduced (`low') expression of
Sox17
(Fig. 5B). For
uninjected embryos (n=26), 81% had high expression, 18% had medium
expression and less than 1% had low expression. In contrast, for embryos
injected with all three MOs (n=20), the high-, medium- and
low-expressing embryo proportions were 3, 57 and 40% respectively. For embryos
injected with only one of the GATA4, 5 or 6 MOs (n=25, 22 or 22), the
percentages of high-, medium- and low-expressing embryos fell between these
two extremes, with the relative magnitude of the effects being in the order
GATA6 MO>GATA4 MO>GATA5 MO as seen for inhibition of marker induction by
activin. When stained embryos were cut in half to reveal expression in the
involuted endoderm, a reduction of Sox17
expression was apparent for
all three MOs individually as well as all three together
(Fig. 5A, blue arrows).
As an alternative measure, we carried out real-time RT-PCR on RNA extracted
from whole embryos at the same stages (Fig.
5C, n=5). At the highest level of MO injected (40 ng),
Sox17 expression was reduced to 75% by GATA4 MO alone, to 85% by GATA5
MO alone and to 65% by GATA6 MO alone, reflecting the hierarchical order of
activity of the three GATA factors in our earlier assays. When all three MOs
were injected together, the level of Sox17
expression was reduced to
55%. We therefore conclude that all three GATA factors contribute to
Sox17
expression in vivo, with GATA6 making the greatest contribution
followed by GATA4 as seen in the in vitro animal cap assays.
Whole mount in situ hybridisation was less informative for endodermal
HNF1ß expression, because of the low level of expression around the yolk
plug (YP) and the masking of expression in the involuted endoderm, even in
cleared embryos, by mesodermal expression
(Fig. 5D, black arrows).
Therefore, to assess the effects of GATA MO injection on the expression of
HNF1ß in involuted endoderm, embryos were cut in half
(Fig. 5D, blue arrows). All
three MOs individually and together significantly reduced this expression (6/6
embryos in each case), although increases in staining in the blastocoel floor
for all three MOs and in the disrupted Brachet's cleft region for the GATA6 MO
were also apparent for reasons at present unknown. The conclusions of the in
situ hybridisation analyses for HNF1ß were broadly supported by real-time
RT-PCR and similar results were obtained for HNF3ß
(Fig. 5E). We therefore
conclude that, as seen for Sox17 expression, HNF1ß and HNF3ß
expression in stage 12 embryos depends on GATA factor activity. However, the
stronger effect of the GATA6 MO seen for Sox17
was less evident for
these other two endodermal genes, presumably reflecting their reduced
dependence on GATA6 in the in vitro assays.
The effects of GATA factor depletion on later development of the gut and
its outgrowths were studied at stage 34 by whole mount in situ hybridisation
using HNF1ß as a probe and at stage 43 by morphological analysis
(Fig. 5F,G). At stage 34,
HNF1ß is expressed strongly in the liver and more weakly in the
underlying foregut and hindgut (Fig.
5F) (Demartis et al.,
1994). Expression is also strong in the forming pronephros and
pronephric duct, which derive from mesoderm. GATA MO injection led to severe
reduction of expression in the liver for GATA6 MO and for all three MOs
together (7/7 embryos in each case), and an only slightly less severe
reduction for GATA5 MO (5/7 embryos), whereas GATA4 MO had little effect (2/8
embryos showed a small reduction in expression of HNF1ß)
(Fig. 5F). Specificity for the
effect on the liver was demonstrated by the continued strong expression of
HNF1ß in the pronephros and pronephric duct in all the injected embryos.
The effects on the more weakly expressing gut were harder to assess at stage
34, however by stage 43, the effects became apparent as loss of gut coiling
and in extreme cases a reduction in the amount of tissue. Thus, for the GATA6
MO, 24/29 embryos injected with 20 ng had less gut tissue and no coiling,
while all those injected with 40 ng died
(Fig. 5G). Most of the embryos
injected with all three MOs died, but the single surviving embryo had very
little remaining gut tissue (Fig.
5G). Injection of the GATA5 MO resulted in a complete loss of gut
coiling in 5/21 embryos, coiling defects in 8/21
(Fig. 5G) and apparently normal
guts in 8/21 embryos. GATA4 MO had very little effect with only 1/25 embryos
displaying abnormal gut coiling. We therefore conclude that with respect to
effects on gut formation and coiling, and on the liver gene expression tested,
GATA factor requirements are in the order: GATA6>GATA5>GATA4.
![]() |
Discussion |
---|
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---|
GATA4, 5 and 6 are expressed appropriately for a role in maintenance of endoderm
The data on the timing of GATA4, 5 and 6 expression during early
Xenopus development have been somewhat conflicting
(Gove et al., 1997;
Jiang and Evans, 1996
;
Weber et al., 2000
;
Yasuo and Lemaire, 1999
). In
addition, their roles in the complex genetic regulatory network leading to
endoderm formation are unclear (Clements
et al., 2003
; Loose and
Patient, 2004
; Xanthos et al.,
2001
; Yasuo and Lemaire,
1999
). In order to gain more insight, we re-investigated the
temporal and spatial distribution of these factors relative to Sox17
and in comparison to the data already in the literature. Sox17
expression was first detected at stage 8 in comparison to GATA4 and 6 at stage
9 and zygotic GATA5 at stage 10. This timing of Sox17
expression
correlates with previously published accounts and its proposed initial
activation by the maternal T-box protein, VegT
(Clements et al., 2001
;
Engleka et al., 2001
;
Hudson et al., 1997
;
Xanthos et al., 2001
;
Yasuo and Lemaire, 1999
). The
later expression of GATA4, 5 and 6 implicates these factors in the maintenance
of Sox17
expression rather than its initial induction. The presence of
a low level of maternal GATA5 mRNA agrees with previous reports,
although the timing of zygotic expression does not
(Jiang and Evans, 1996
). This
could possibly reflect the greater accuracy of the real-time RT-PCR
method.
The spatial distribution at stage 10 of GATA4 and 6, but not GATA5,
correlates well with the expression pattern of Sox17
(Engleka et al., 2001;
Hudson et al., 1997
). Thus,
GATA4, GATA6 and Sox17 are co-expressed in the supra- and sub-blastoporal
endoderm at this time, whereas GATA5 is restricted to the sub-blastoporal
endoderm (Weber et al., 2000
).
However, by stage 11, none of the GATA factors are co-expressed with Sox17 in
the supra-blastoporal endoderm. Thus, while GATA4 and 6 could be maintaining
Sox17 expression in the pre-involuted endoderm early, they are not involved
later. All three GATA factors, however, could be involved in maintaining Sox
17 expression throughout the sub-blastoporal endoderm. Clearly other factors
are involved in regulating Sox17 in the pre-involuted endoderm later.
Candidate genes include the homeobox-containing transcription factor,
Mixer, and the paired-like homeobox gene, Pitx2
(Engleka et al., 2001
;
Faucourt et al., 2001
;
Henry and Melton, 1998
).
Consistent with such a role for Pitx2, available data suggest that it induces
endoderm in a GATA-independent manner. Overall we conclude that the expression
profiles of GATA4-6 implicate them in the maintenance of the endodermal
programme in a subset of endodermal cells.
GATA factor redundancy?
The similar temporal and spatial expression profiles of GATA4 and 6 raised
the possibility that they carry out redundant functions in elaborating the
endodermal gene expression programme. Consistent with this notion, both
transcription factors induce expression of the same early endodermal genes in
cap explants, albeit with different efficiencies. However, we show here that
much of GATA4's activity in this regard depends on the expression of GATA6.
Indeed we found no evidence for GATA4 acting directly on Sox17, in
contrast to GATA6. This is a striking observation in view of the majority of
the literature pointing to GATA factors binding to the same DNA sequence.
Presumably the context on the Sox17
promoter is critical. This is one
of the clearest demonstrations yet that apparent redundancy amongst GATA
factors may reflect distinct roles in the pathways concerned. An important
parenthetical point about GATA6 here is that, despite both long and short
isoforms being synthesised in vivo (Brewer
et al., 1999
; Brewer et al.,
2002
; Peterkin et al.,
2003
), only the long isoform was able to induce endoderm markers
(this study) (Weber et al.,
2000
).
Models have been proposed in the literature describing gene networks
involved in the formation of endoderm in Xenopus embryos and mouse
embryonic stem cells (Clements and
Woodland, 2003; Fujikura et
al., 2002
; Loose and Patient,
2004
; Xanthos et al.,
2001
; Yasuo and Lemaire,
1999
). Our data agree with these models, but place GATA6 between
GATA4 and to some extent GATA5, and at least two of their endoderm targets. By
using inducible versions of GATA4, 5 and 6 in the presence of protein
synthesis inhibitors, we were able to distinguish the direct or indirect
actions of these transcription factors on Sox17
, HNF1ß and
HNF3ß. Cycloheximide has been used successfully in the past for this
purpose (Clements et al.,
2003
; Tada et al.,
1997
; Watanabe and Whitman,
1999
). However, for Sox17
, superinduction was observed.
This could have been a consequence of blocking the synthesis of the
hypothetical endoderm inhibitor in the cap explants. If that had been the
case, the superinduction should not have been observed with drug
concentrations too low to block protein synthesis. However, Sox17
was
still induced at such drug concentrations (data not shown), suggesting that
cycloheximide acts through a different pathway in inducing Sox17. Thus our
data add Sox17
to the previously reported GATA4 and
Gsc as genes induced by cycloheximide
(Tadano et al., 1993
;
Yasuo and Lemaire, 1999
). The
induction of GATA4 by cycloheximide does not affect the interpretation of our
data as these endogenous induced GATA4 transcripts would not be translated. An
explanation as to why the superinduction was greater when GATA4-GR was
present, is the activation of the p38 MAP kinase pathway by cycloheximide,
which could lead to phosphorylation of GATA4 (but not GATA5 or 6) thereby
overriding the interaction with HSP90
(Charron et al., 2001
;
Kitta et al., 2003
;
Liang et al., 2001
). This
cannot explain all of the observed superinduction though, because
superinduction was seen, albeit at a lower level, with GATA5 and 6 which do
not contain the p38 MAPK target sequence. Because the greater superinduction
in the presence of GATA4 could have exaggerated the apparent block to
induction by cycloheximide, we repeated the experiment with a different
protein synthesis inhibitor, emetine, which does not superinduce, and
confirmed the indirect nature by which GATA4 induces Sox17
. Thus our
data indicate a direct and central role for GATA6 in the genetic network
orchestrating endodermal programming.
GATA4, 5 and 6 and the maintenance of endoderm marker expression by TGFß
A two-step model for the formation of endoderm has been proposed in which
in the initial phase, in early blastulae, the maternal T-box protein VegT
initiates endoderm formation by directly inducing Sox17 and nodal-related gene
expression, followed by the maintenance of Sox17 in the second phase by the
previously induced nodal-related proteins
(Clements et al., 1999;
Clements and Woodland, 2003
;
Yasuo and Lemaire, 1999
). The
second phase correlates well with the time when GATA4 and 6 expression begins,
raising the obvious question as to how these two different families of
proteins are connected. TGFßs, including the nodal-related proteins,
induce GATA factors along with other endoderm-associated genes (this study)
(Ariizumi et al., 2003
;
Chang and Hemmati-Brivanlou,
2000
; Hyde and Old,
2000
; Kofron et al.,
1999
; Weber et al.,
2000
). A dose-response relationship is seen with all three of the
GATA factors and here we reveal differences between them, with sensitivity to
induction by activin in the order GATA6>GATA4>GATA5. Differences such as
these are likely to contribute to the region-specific expression observed in
the embryo (see Fig. 1)
(Weber et al., 2000
).
Furthermore, differences such as these are likely to mean that different GATA
factors mediate TGFß responses at different concentrations of TGFß.
In support of this suggestion, we find that the effects of low concentrations
of activin on animal caps are inhibited by lost GATA function in the order
GATA6>GATA4>GATA5.
At still lower concentrations of activin, Sox17 is induced without GATA
factor induction (Ariizumi et al.,
2003; Hudson et al.,
1997
; Weber et al.,
2000
). The explanation must be that, at lower concentrations,
TGFßs induce endoderm via GATA independent pathways. These are likely to
involve Smad proteins (Germain et al.,
2000
; Massague,
1998
; Massague and Chen,
2000
), possibly in partnership with Pitx2 or Mixer
(Faucourt et al., 2001
;
Henry and Melton, 1998
). Such
alternative pathways presumably also explain the incomplete inhibition by
GATA6 morpholinos of Sox17
expression at low activin concentrations in
our experiments.
Overall the data obtained here identify GATA4, 5 and 6 as mediators of
TGFß (most likely nodal) signalling in elaboration of the endoderm
programme during the late blastula and gastrula stages in Xenopus
laevis. At low concentrations of nodal signalling, in early blastulae,
Sox17 is induced in a GATA-independent manner. However, as nodal builds
up in the sub-blastoporal and early pre-involuting endoderm, our data suggest
that GATA4 and 6, initially alone and then together with GATA5, directly
maintain expression of endodermal gene expression. The consequences of GATA
factor depletion for formation and coiling of the gut, and for gene expression
in the liver, are greatest for GATA6, while being milder for GATA5, with GATA4
having very little effect. Together with the faust (GATA5) mutation
in zebrafish (Reiter et al.,
1999
; Reiter et al.,
2001
), these data strongly implicate GATA5 and 6 in the
development of definitive endoderm. With mouse null mutant data implicating
GATA4 and 6 in primitive, extra-embryonic endoderm formation
(Molkentin, 2000
;
Rossant et al., 2003
), this
branch of the GATA family clearly plays a central role in the development of
all endoderm.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: MRC Molecular Haematology Unit, Weatherall Institute of
Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington,
Oxford OX3 9DS, UK
![]() |
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