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First published online 16 May 2007
doi: 10.1242/dev.001230
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Cincinnati Children's Research Foundation and Department of Pediatrics, College of Medicine, University of Cincinnati, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
Author for correspondence (e-mail:
Aaron.zorn{at}chmcc.org)
Accepted 30 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Endoderm, Patterning, Liver, Pancreas, Foregut, Wnt, ß-catenin, Wnt-antagonists, Gsk3ß, Xenopus, hhex, foxa2, vent2
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Zaret, 2002;
Zhao and Duncan, 2005), the
earlier events linking endoderm formation at gastrulation to organ induction
are less clear. A detailed understanding of these early developmental
decisions is likely to be important for designing efficient strategies to
generate therapeutically useful tissue from stem cells.
Hepatic specification in the ventral foregut endoderm requires signals from
the surrounding mesenchyme (Fukuda-Taira,
1981; Gualdi et al.,
1996; Le Douarin,
1975). In the mouse and chick, these signals include Fgf2 from the
cardiogenic mesoderm and BMPs from the septum transversum mesenchyme
(Jung et al., 1999;
Rossi et al., 2001;
Zhang et al., 2004), whereas
in zebrafish Wnt2bb in the lateral plate mesoderm is required
(Ober et al., 2006). There is
also evidence that prior to these signals, there are earlier developmental
decisions that are crucial for the hepatic lineage. For example,
transplantation and explant studies in amphibian and chick embryos have shown
that only the foregut endoderm, but not the posterior endoderm, is competent
to become liver in response to signals from the cardiogenic mesoderm
(Fukuda-Taira, 1981;
Le Douarin, 1975;
Okada, 1954b;
Okada, 1960;
Takata, 1960). In addition,
tissue recombination experiments have shown that an unknown signal from the
trunk mesoderm represses hepatic potential in the posterior endoderm
(Gualdi et al., 1996;
Le Douarin, 1975;
Takata, 1960). Together, these
findings suggest that establishment of the foregut domain prior to hepatic
induction is an essential prerequisite for liver development.
The regional expression of marker genes along the anterior-posterior (A-P)
axis of the gut tube demonstrates that distinct foregut, midgut and hindgut
domains exist prior to organ specification
(Costa et al., 2003;
Gamer and Wright, 1995;
Grapin-Botton, 2005;
Newman et al., 1997). However,
studies on the mechanisms controlling this have been somewhat contradictory.
Some experiments in amphibians suggested that regional identity in the
endoderm is determined by gastrulation
(Hamburger, 1996;
Holtfreter, 1938), whereas
other studies indicated that continual interactions with the mesoderm are
required (Okada, 1954a;
Okada, 1954b;
Okada, 1960;
Takata, 1960). For example,
anterior vegetal tissue isolated from Xenopus blastulae will
autonomously express foregut genes owing to intrinsic Nodal and Wnt signaling
in the explants (Gamer and Wright,
1995; Henry et al.,
1996; Zorn et al.,
1999). However Horb and Slack subsequently detected cardiac and
lateral mesoderm markers in such vegetal explants
(Horb and Slack, 2001),
suggesting that prolonged mesodermal interactions might be required to
maintain regional identify, although the molecular details of this remain
poorly understood.
In this study we postulated that differential zygotic Wnt signaling might
regulate endoderm patterning similar to the `activation-transformation' model
of A-P patterning in the neural tube. In this model, neural tissue is
initially anterior in character and is then progressively posteriorized by
Wnts expressed in the trunk mesoderm, whereas anterior identity is maintained
rostrally where there is a lack of Wnt signaling
(Christian and Moon, 1993;
Erter et al., 2001;
Kiecker and Niehrs, 2001;
Kim et al., 2000;
Lekven et al., 2001;
McGrew et al., 1995;
Nieuwkoop, 1999;
Takada et al., 1994). In the
canonical pathway, Wnt ligands interact with a Frizzled-LRP5/6 receptor
complex causing the inactivation of a Gsk3ß-containing intracellular
complex that would otherwise promote ß-catenin degradation. This results
in the accumulation of nuclear ß-catenin, which interacts with Tcf/Lef
transcription factors to activate target gene transcription
(Clevers, 2006). Secreted
Wnt-antagonists block signaling by binding to Wnt ligands in the extracellular
space (sFRPs), or by binding to the LRP co-receptor (Dkk1)
(Kawano and Kypta, 2003;
Mao et al., 2001).
In Xenopus, a maternal Wnt pathway results in high levels of
nuclear ß-catenin in the dorsal-anterior endoderm of the blastula, which
specifies the dorsal-anterior axis of all three germ layers
(Heasman, 2006). However,
during gastrula and early somite stages, nuclear ß-catenin levels are
rapidly reduced in the anterior endoderm relative to the posterior endoderm
(Schohl and Fagotto, 2002). We
postulated that this region of low ß-catenin activity is important to
maintain foregut identity and that Wnt ligands (Wnt8, Wnt8b, Wnt3, Wnt3a and
Wnt1) expressed in the trunk mesoderm
(Christian et al., 1991;
Kemp et al., 2005;
Moon, 1993) signal to the
posterior endoderm to inhibit foregut/hepatic fates and promote intestinal
development. By contrast, Wnt-antagonists (Dkk1, Frzb1, Crescent and Sfrp5)
secreted by the anterior endoderm (Kemp et
al., 2005; Leyns et al.,
1997; Pilcher and Krieg,
2002; Wang et al.,
1997) would protect it from the posteriorizing Wnt ligands, keep
nuclear ß-catenin levels low, and maintain foregut identity. Although
such spatial interactions between zygotic Wnt ligands and their antagonists
were known to regulate mesoderm and neural tube patterning, their roles in the
early endoderm had not been established.
Consistent with this hypothesis, we find that forced Wnt/ß-catenin signaling in the anterior endoderm, between gastrula and early somite stages, inhibits foregut development. By contrast, blocking ß-catenin activity in the posterior endoderm is sufficient to initiate ectopic liver and pancreas development. This suggests that the endoderm is indeed patterned by differential Wnt signaling, similar to what has been described in the nervous system. The homeobox gene hhex, one of the earliest foregut markers, is a target of this ß-catenin-mediated patterning and Hhex function is required for both normal and ectopic liver and pancreas development. Analysis of the hhex promoter indicates that ß-catenin/Tcf activity represses hhex transcription in the posterior endoderm indirectly via homeodomain transcriptional repressor Vent2. This is a novel function for Vent factors, which are best known as mediators of BMP signaling in the mesoderm. These results provide a molecular pathway linking endoderm patterning to the initiation of liver and pancreas development, and illustrate that the spatial and temporal activity of Wnt signaling must be tightly controlled during this process.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1994).
Explants were microdissected with tungsten needles and cultured in
0.5xMBS (Zorn et al.,
1999) with antibiotics. For tissue separations, explants were
cultured in 10 µg/ml dispase for 15-20 minutes. Gastrula-stage endoderm
tissue was transplanted into host embryos through an incision in the
blastocoel roof, as previously described
(Slack and Isaacs, 1994).
Embryos with clear pigmentation differences between dorsal-anterior and
ventral-posterior cells were selected for 32-cell stage injections. The
antisense Hex morpholino oligo (HexMO, 80 ng) has been described previously
(Smithers and Jones, 2002).
Synthetic RNA for microinjection was transcribed using the Message Machine Kit
(Ambion) and purified on Microspin-6 columns (BioRad). The following plasmids
were used for mRNA synthesis or directly for microinjection (total amounts of
DNA or RNA injected, and enzymes used to linearize DNA templates and
synthesize RNA are indicated): pCSKA-Xwnt8 (250 pg)
(Christian and Moon, 1993);
pCS2+pt-ß-catenin (250 pg) (Yost et
al., 1996); pCS2+MT Xgsk3ß (500 pg)
(Yost et al., 1996);
pcDNA3-
NXTcf3 (800 pg) (Molenaar et
al., 1996); pRN3 GFP (100 pg; SfiI, T3); pCS2+ß-gal
(100 pg; NotI, Sp6); pCS107 tDkk1 (500 pg; AscI, Sp6);
pCS2+HA-GR-NLEF-ßCTA (800 pg)
(Domingos et al., 2001);
pCS107-Xvent1 (500 pg; AscI, Sp6); pCS107-Xvent2 (500 pg;
AscI, Sp6); pT7TS-HA-Xhex (500 pg; BamHI, T7); pCS2+XfoxA2
(500 pg; NotI, Sp6); pT7TSHA-GR-NTcf3 (800 pg; XbaI,
T7); pT7TSHA-GR-Vent2 (800 pg, XbaI, T7).
Xenopus transgenics and transient luciferase assays
A Xenopus laevis genomic 
-library was screened using
standard procedures to isolate hhex genomic clones and a 
6kb
fragment upstream of the ATG start codon (Accession No. EF059707) was
subcloned into the pGFP3 vector to create the phhex-6kb:GFP plasmid used in
transgenesis. Transgenic Xenopus embryos were generated using sperm
nuclear transplantation as previously described
(Kroll and Amaya, 1996).
Three hhex promoter fragments -6.0, -1.56 and -0.44 kb were PCR amplified and cloned into the pGL2-Basic luciferase vector (Promega). The hhex:luciferase promoter constructs (300 pg) were microinjected along with a pRL-TK renilla (Promega) control vector (25 pg). At gastrula stage, five injected embryos were pooled together and homogenized in 100 µl of 100 mM Tris (pH 7.5) on ice, assayed for luciferase activity and normalized to renilla activity using standard kits (Promega). Each construct was assayed in triplicate.
In situ hybridization and RT-PCR analysis
In situ hybridizations were preformed as previously described
(Costa et al., 2003) using the
following probes: for1 (Seo et
al., 2002), pdx1/xlhbox8
(Wright et al., 1989),
nkx2.1 (Small et al.,
2000), endocut/darmin
(Costa et al., 2003),
hhex and ampb (Zorn and
Mason, 2001), vent2
(Onichtchouk et al., 1998),
ptfa1 (Jarikji et al.,
2007), amylase and elastase
(Horb and Slack, 2002).
Real time RT-PCR analysis was performed on an Opticon PCR machine (MJ
Research) as previously described (Sinner
et al., 2004). The following primers were used (F, forward; R,
reverse): for1 (F, 5'-CTTAATGTGACTGAAGCAGAG-3' and R,
5'-TTTCCATCTGTAGAGCCACAA-3'); c-troponin (F,
5'-AAGTCTCCATGGATCTAC-3' and R,
5'-CAGCTCTAACCATTTCAG-3'); endodermin (F,
5'-AGCAGAAAATGGCAAACACAC-3' and R,
5'-GGTCTTTTAATGGCAACAGGT-3'); foxa2 (F,
5'-CCTATCATGAACTCCTCATAG-3' and R,
5'-GGCCAGAATACATACAGCAGTC-3'); hhex
(Zorn et al., 1999); and
odc (Sinner et al.,
2004).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It is this clearance of zygotic ß-catenin in the
anterior endoderm that we postulate is crucial to maintain foregut fate. To
test this, we microinjected a Wnt8-plasmid into the D1 cells of 32-cell stage
Xenopus embryos (Fig.
1A), which are fated to contribute to the foregut
(Dale and Slack, 1987;
Moody, 1987). The injected
Wnt8 plasmid is not transcribed until the gastrula stage
(Christian and Moon, 1993) and
therefore only activates zygotic ß-catenin signaling. At stages 35-37,
when organ buds are first visible (Chalmers
and Slack, 1998; Nieuwkoop and
Faber, 1994), the embryos were assayed by in situ hybridization
with the liver marker for1 (Seo
et al., 2002), as well as with markers for pancreas/duodenum
(pdx1/xlhbox8) (Wright et al.,
1989), lung/thyroid (nkx2.1)
(Small et al., 2000) and
intestine (endocut/darmin) (Costa
et al., 2003). In almost all of the treated embryos, foregut gene
expression was inhibited and an examination of the gut tube at stage 42
revealed an absence of foregut organ buds in the Wnt8-injected embryos
(Fig. 1B).
To determine if increased repression of Wnt signaling was sufficient to
expand the foregut, we injected RNA encoding the canonical Wnt-antagonist Dkk1
(Glinka et al., 1998) into
cells that contribute to the posterior-lateral endoderm
(Dale and Slack, 1987;
Moody, 1987). Dkk1
overexpression is known to inhibit zygotic but not maternal Wnt signaling
(Glinka et al., 1998). The
resulting embryos exhibited an expansion of for1 and pdx1
expression at the expense of the intestine marker, and at stage 42 the liver
and pancreas buds were conspicuously enlarged
(Fig. 1B).
|
) into the
D1 blastomeres at the 32-cell stage. The stabilized ß-catenin should
persist in the gastrula anterior endoderm long after the time when endogenous
ß-catenin is downregulated, and this resulted in the repression of
foregut markers as well as ectopic intestinal gene expression in the foregut
domain (Fig. 1B and see Table
S1 in the supplementary material). Unlike Wnt8 plasmid injections, there was
little or no effect on head development in ß-catenin-injected embryos,
consistent with this acting in the endoderm and not affecting mesoderm or
neural tissue. Co-injection of ß-galactosidase (ß-gal) RNA as a
lineage-tracer confirmed that the injections targeted the anterior endoderm
(data not shown).
We next asked if reducing canonical Wnt signaling in the posterior endoderm
was sufficient to induce foregut fate. To cell-autonomously repress
ß-catenin signaling we injected RNA encoding Gsk3ß into the D4
posterior endoderm cells at the 32-cell stage. Gsk3ß overexpression
promotes ß-catenin degradation (Yost
et al., 1996) and as maternal ß-catenin is not active in the
ventral-posterior cells, Gsk3ß is expected to repress zygotic
ß-catenin signaling. We observed ectopic for1 and pdx1
expression in the posterior endoderm of 50-60% of the Gsk3ß-injected
embryos, along with inhibition of the intestinal marker endocut
(Fig. 1B and see Table S1 in
the supplementary material). Similar results were obtained with a
dominant-negative NTcf3 construct
(Molenaar et al., 1996) that
represses ß-catenin/Tcf target genes (see Table S1 in the supplementary
material). At stage 42-46, we observed ectopic organ buds in the intestines of
Gsk3ß- and NTcf3-injected embryos. Some of the ectopic buds
expressed early liver and pancreas markers (hhex, pdx1 and
ptf1a) as well as hepatic (for1, ambp and
transferrin) and exocrine pancreas (elastase and
amylase) differentiation markers
(Fig. 1C and see Table S1 in
the supplementary material). However, we did not observe ectopic
insulin expression suggesting that endocrine pancreas development
requires additional signals.
To confirm that the injected Gsk3ß acted autonomously in the endoderm, we co-injected Gsk3ß with ß-gal RNA as a lineage label. In none of the cases in which ß-gal staining was restricted to the mesoderm (n=17) was ectopic for1 expression observed. By contrast, ectopic for1 expression was observed in 19 cases in which the ß-gal staining was in the endoderm and not the mesoderm (Fig. 1D), indicating that Gsk3ß acts in the endoderm.
ß-catenin activity is repressed in the foregut during gastrula and early somite stages
Since our injections did not disrupt head or axial mesoderm structures,
this argues that we did not interfere with the known role of Wnt signaling in
early axial patterning, which occurs during blastula and early gastrula
stages. To rigorously test this, we repeated our experiments with
hormone-inducible constructs that could be temporally controlled.
To confirm that ß-catenin activity inhibits foregut fate after
gastrulation, we used a GR-LEFN-ßCTA construct, which has the
hormone-binding domain of the glucocorticoid receptor fused to the Lef1
DNA-binding domain and the ß-catenin transactivation domain. In the
presence of dexamethasone (Dex), this fusion protein constitutively activates
ß-catenin target genes (Domingos et
al., 2001). By treating GR-LEFN-ßCTA-injected embryos
with Dex at progressively later stages, we determined that ß-catenin
signaling must be excluded from the anterior endoderm between stage 11
(midgastrula) and stage 20 (6-7 somites). Dex treatment during this period
inhibited for1 and pdx1 expression and expanded the
intestinal marker into the foregut territory
(Fig. 2A). Addition of Dex at
stages 25 and 30 had no obvious effect on foregut development when assayed at
stage 35. However, when Dex was added from stage 30 to 42, about half of the
embryos exhibited enlarged liver buds (Fig.
2B). This is consistent with recent reports in zebrafish and mice
suggesting that later in development, ß-catenin promotes hepatoblast
specification and/or proliferation (Ober
et al., 2006; Tan et al.,
2006).
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NTcf3 RNA into D4
blastomeres at the 32-cell stage. GR-NTcf3 consists of the
glucocorticoid receptor ligand-binding domain fused to a dominant-negative
Tcf3, which represses ß-catenin/Tcf target genes in the presence of Dex
(Darken and Wilson, 2001).
Activation of GR-NTcf3 at midgastrula (stage 11) or early neurula
(stage 13) efficiently induced ectopic for1 and pdx1
expression (Fig. 2C), whereas
addition of Dex between stages 15 and 20 had no effect. When GR-NTcf3
was injected into D1 anterior endoderm cells and Dex was added between stages
30 and 42, some embryos exhibited smaller liver buds
(Fig. 2D) consistent with the
late induction of GR-LEFN-ßCTA. These experiments reveal a
ß-catenin function that is distinct from its role in early axial
patterning. We conclude that between the gastrula and early somite stages, repression of ß-catenin activity in the endoderm is necessary and sufficient to initiate liver and pancreas development, probably by regulating the formation of early foregut progenitors. However, during later embryogenesis, ß-catenin has a second role in promoting hepatic development.
The endoderm is a direct target of ß-catenin signaling
Although the lineage-tracing experiments suggested that ß-catenin
signaling acts directly on the endoderm to regulate lineage commitment, it was
important to rigorously test this. We therefore isolated anterior endoderm
from gastrula embryos injected with ß-catenin and GFP RNA or posterior
endoderm injected with Gsk3ß and GFP RNA. This tissue was transplanted
into the blastocoel of uninjected sibling host embryos and the fate of the
cells was determined at stage 42 (Fig.
3A). By the neurula stage, the transplanted cells had incorporated
into the host endoderm near the foregut/hindgut boundary
(Fig. 3B,C). At stage 42,
control transplants injected with GFP only respected their original
anterior-posterior identity. Anterior endoderm was incorporated in the liver
and anterior intestine 80% of the time, whereas control posterior
transplants contributed to the intestine but rarely to the liver. By contrast,
transplanted anterior endoderm overexpressing ß-catenin almost never
contributed to the liver, whereas posterior endoderm tissue injected with
Gsk3ß behaved like anterior endoderm and populated the liver bud 80%
of the time (Fig. 3D-H).
Importantly, GFP-labeled cells were not observed in the heart or other
mesoderm tissue demonstrating that differential ß-catenin activity in the
endoderm directly restricts regional identity.
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|
).
Since cardiac mesoderm induces liver fate in mouse and chick, we asked whether
the mesoderm was also required for normal and Gsk3ß-induced liver
specification in Xenopus. We isolated foregut explants at stage 18
(3-4 somites) and cultured these with or without their associated cardiac
mesoderm (Fig. 4A). RT-PCR
analysis of the explants at stage 35 revealed that, as in mouse and chick, the
mesoderm was required for liver gene expression
(Fig. 4B). By removing the
mesoderm at progressively later times, we found that liver specification
requires an interaction with the mesoderm until stage 28 (20-22 somites) (data
not shown). Similarly, an association with the adjacent mesoderm was also
required for Gsk3ß-injected posterior endoderm to express liver markers
(Fig. 4C). We conclude that
repression of ß-catenin activity in the endoderm initiates a cascade of
reciprocal signaling with the mesoderm leading to hepatic induction.
|
;
Thomas et al., 1998) that is
essential for early liver and ventral pancreas development in mice
(Bort et al., 2004;
Keng et al., 2000;
Martinez Barbera et al.,
2000).
Forcing ß-catenin activity in the anterior endoderm by injection of
either stabilized ß-catenin or GR-LEFN-ßCTA RNA resulted in a
dramatic downregulation of hhex expression at stage 18
(Fig. 5A). Conversely,
repressing ß-catenin activity in the posterior endoderm by microinjecting
Gsk3ß plus ß-gal RNA cell-autonomously induced ectopic hhex
expression in the presumptive hindgut. A similar result was obtained by
injecting either NTcf3 RNA or an antisense ß-catenin morpholino
oligo (Fig. 5A and see Table S1
in the supplementary material), indicating that low ß-catenin activity is
necessary and sufficient to induce early foregut progenitors.
We also examined the expression of foxa2 and gata4, gata5
and gata6 in ß-catenin- and Gsk3ß-injected embryos, as
these transcription factors have been implicated in early mouse liver
development (Lee et al., 2005;
Zaret, 2002;
Zhao et al., 2005). Although
we did not observe obvious changes in gata4-6 expression,
foxa2 foregut expression was downregulated by anterior ß-catenin
injection and modestly increased in the posterior endoderm by Gsk3ß
injection (see Fig. S1 in the supplementary material). Since the effects on
foxa2 expression were subtle, we focused our attention on
hhex in our subsequent experiments.
We tested whether Hhex is required for liver and pancreas development in
Xenopus. Embryos were injected with an antisense morpholino oligo
complementary to the 5' untranslated region of the hhex mRNA
(HexMO), which blocks translation and has been shown to specifically knockdown
Hhex function (Smithers and Jones,
2002). HexMO-injected embryos exhibited very little if any
for1 or pdx1 expression, demonstrating that Hhex is required
for liver and pancreas development (Fig.
5B and see Table S1 in the supplementary material). Furthermore,
injection of HexMO completely blocked the ectopic for1 and
pdx1 expression induced by Gsk3ß
(Fig. 5B and see Table S1 in
the supplementary material), indicating that Hhex function is required
downstream of Gsk3ß. Surprisingly, both the ventral and dorsal pancreas
were absent from HexMO Xenopus embryos (as judged by pdx1
expression), in contrast to the mouse Hhex-/- mutant in
which only the ventral pancreas is compromised
(Bort et al., 2004). The reason
for this difference is not known, but we note that the anterior neural
phenotype in HexMO Xenopus embryos is also more severe than in the
mouse mutant (Smithers and Jones,
2002).
Finally, Hhex overexpressed in the posterior endoderm was not sufficient to induce ectopic for1 or pdx1 expression (data not shown), suggesting that additional factors are required to promote foregut development downstream of repression of Wnt.
Regulation of hhex transcription
To examine how zygotic ß-catenin represses hhex
transcription, we isolated a -6 kb fragment of Xenopus laevis genomic
DNA upstream of the hhex transcription start site and used this to
generate germline hhex:gfp transgenic animals. Confocal analysis of
gastrula- and presomitic-stage hhex:gfp transgenic embryos showed GFP
fluorescence in the anterior endoderm and ventral foregut
(Fig. 6A,B), recapitulating
endogenous hhex expression. To determine if the -6 kb transgene was
regulated by ß-catenin, we injected Gsk3ß into the posterior D4
cells of transgenic embryos, isolated hindgut explants at the gastrula stage
and scored these for GFP at stage 18. As expected, GFP was observed in
50% of control foregut explants (one parent was heterozygous for
hhex:gfp) but was undetected in uninjected hindgut explants
(Fig. 6C,D). By contrast, we
observed ectopic GFP in 4 of 12 Gsk3ß-injected hindgut explants
(Fig. 6E), indicating that the
-6 kb promoter contains elements that confer correct A-P expression and
Gsk3ß responsiveness.
|
Repression of hhex is mediated by Vent homeodomain factors
Because ß-catenin-Tcf complexes normally stimulate transcription, we
postulated that hhex expression must be repressed indirectly, by
activating a transcriptional repressor. Sequence analysis of the hhex
promoter revealed ten putative DNA-binding sites for the Vent class of
homeodomain repressors (Friedle et al.,
1998; Trindade et al.,
1999), with eight of the Vent-sites located between -1.56 and
-0.44 kb. Vent1 (PV.1) and Vent2 (Xbr-1, Vox and Xom) are closely related
transcriptional repressors, best known as mediators of BMP signaling during
dorsal-ventral mesoderm patterning (Ault et
al., 1996; Onichtchouk et al.,
1998; Papalopulu and Kintner,
1996; Schmidt et al.,
1996). Although a function for Vents in the endoderm has not been
described, recent evidence suggests that Wnt signaling promotes vent
transcription in the mesoderm (Friedle and
Knochel, 2002; Ramel and
Lekven, 2004).
To test whether Vents could repress the hhex promoter, we injected the hhex:luciferase reporter constructs into D1 anterior cells, with or without RNA encoding Vent1 or Vent2, and assayed luciferase activity. Co-injection of either Vent repressed the activity of the -6.0 kb and-1.56 kb reporters to levels observed in the posterior endoderm (Fig. 7A; data not shown), whereas the -0.44 kb promoter, lacking eight of the Vent-binding sites, was not efficiently repressed. Thus, the -1.56 to -0.44 kb hhex promoter region exhibits all three activities: (1) it mediates hhex repression in the posterior endoderm; (2) it can be activated by Gsk3ß; and (3) it can be repressed by Vents.
Next, we re-examined the vent1 and vent2 expression
patterns by in situ hybridization of bisected embryos, which exposes the deep
endoderm. In addition to their previously documented mesodermal expression, we
found that vent2 (and to a lesser extent vent1) was highly
expressed in the posterior endoderm during gastrula and early somite stages in
a pattern reciprocal to hhex. Moreover, the vent2-expressing
endoderm was adjacent to wnt8-expressing lateral plate mesoderm
throughout this period of development (Fig.
7B-D and see Fig. S2 in the supplementary material). To test
whether vent2 expression in the posterior endoderm required
Wnt/ß-catenin activity, we injected either Gsk3ß, NTcf3 or
antisense ß-catenin morpholino oligos into the D4 posterior cells, all of
which reduced vent2 mRNA levels
(Fig. 7E and see Table S1 in
the supplementary material). Injection of Vent2 RNA into the D1 anterior
endoderm at the 32-cell stage inhibited hhex and foxa2
expression as well as subsequent liver and pancreas organogenesis
(Fig. 7F; see Fig. S2 and Table
S1 in the supplementary material). In addition, co-injection of Vent2 plus
Gsk3ß RNA in the D4 posterior endoderm blocked the ectopic hhex,
for1 and pdx1 expression induced by Gsk3ß alone
(Fig. 7F and see Table S1 in
the supplementary material).
Although we did not observe any changes in axial mesoderm development in these experiments, it was important to determine whether we were observing a Vent2 function distinct from its known role in gastrula-stage mesoderm patterning. We therefore repeated the experiments with a hormone-inducible GR-Vent2 construct that we could activate after early mesoderm patterning. We found that for1 and pdx1 expression was repressed even when the GR-Vent2 construct was activated by Dex as late as stage 20 (see Fig. S2 in the supplementary material). This indicates that Vent2 has a novel function in the endoderm well beyond the gastrula stage. Together, these data suggest a model in which Wnt/ß-catenin activity promotes vent2 transcription in the posterior endoderm during gastrula and early somite stages, and Vent2 protein then represses hhex transcription and foregut fate in the presumptive intestine (Fig. 7G).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
|
;
Rastegar et al., 1999). The
transcriptional repressor Vent2 would then inhibit foregut fate by repressing
hhex transcription, probably directly via Vent-binding sites in the
hhex promoter. By contrast, the anterior endoderm secretes a number
of Wnt-antagonists (Frzb1, Sfrp5, Crescent and Dkk1) that protect it from the
Wnt ligands, resulting in low ß-catenin levels and the expression of
early foregut genes such as hhex. This appears to be essential for
the development of early foregut progenitors
(Deutsch et al., 2001), as
both the liver and pancreas fates were affected in our experiments.
Our data also suggest that the repression of ß-catenin activity is
sufficient to initiate a cascade of reciprocal signaling that controls some,
but not all, foregut organogenesis. For example, we observed ectopic liver and
exocrine pancreas development, but not endocrine pancreatic fates. In the case
of the liver, it appears that low ß-catenin activity imparts anterior
character to the endoderm and this anteriorized endoderm then induces cardiac
fate in the adjacent mesoderm (Foley and
Mercola, 2005). The cardiogenic mesoderm then signals back to the
foregut endoderm, instructing a subset of the cells to become liver
(Zaret, 2002).
Early targets of Wnt-mediated endoderm patterning
We found that Hhex was required, but not sufficient, for Xenopus
foregut development. Although the direct targets of Hhex in this context are
unknown, recent data in the Xenopus blastula suggest that Hhex acts
in part by repressing the expression of the Groucho-family transcriptional
co-repressor Tle4 (Zamparini et al.,
2006). Repression of Tle4 by Hhex might also be important for
maintaining the foregut precursors in a transcriptionally permissive state.
However, as ectopic Hhex was not sufficient to initiate foregut development,
other factors must also function downstream of the repression of Wnt. One
candidate is the transcription factor Foxa2, which was also negatively
regulated by ß-catenin. Foxa2 is required for foregut development in mice
(Dufort et al., 1998) and
Zaret and colleagues have shown that Foxa binding to liver gene promoters
correlates with hepatic competence (reviewed by
Zaret, 2002). Consistent with
this, liver development fails to initiate in mice in which Foxa1 and
Foxa2 have been deleted from the endoderm
(Lee et al., 2005). Another
possibility is that even in the presence of Hhex or Foxa2, foregut development
requires that intestine-promoting Wnt target genes such as Cdx genes must also
be turned off. The Cdx homeobox genes are expressed in the posterior endoderm
(and mesoderm), they are required for posterior development
(Chawengsaksophak et al., 2004)
and Cdx1 has been shown to be a direct ß-catenin/Tcf target in
mouse and zebrafish (Lickert et al.,
2000; Shimizu et al.,
2005).
|
). In this model, an initial `activation' signal induces
neural tissue of anterior character, followed by a subsequent `transformation'
signal that imparts posterior character to the neural tube in a graded
fashion, with the highest signal generating the most-posterior fates. There is
now good evidence that the posteriorizing signals are a combination of Wnts,
FGFs and retinoic acid (Domingos et al.,
2001; Kudoh et al.,
2002; Sasai and De Robertis,
1997), and recent evidence, including our study, suggests that
these pathways also regulate endoderm patterning. Wells and colleagues found
that high levels of Fgf4 promote intestinal fate and repress foregut
development in mice and avian embryos similar to the Wnt activity we have
described here (Dessimoz et al.,
2006; Wells and Melton,
2000). In zebrafish, BMP signaling has been implicated in
regulating anterior-posterior endoderm patterning
(Tiso et al., 2002) and
retinoic acid appears to specify the position of the pancreatic domain in
Xenopus, zebrafish and mice (Chen
et al., 2004; Martin et al.,
2005; Stafford et al.,
2004). In the future it will be interesting to determine how the
Wnt, FGF, BMP and retinoic acid pathways functionally interact in the
endoderm.
Wnt-mediated endoderm patterning in other vertebrates
Our results are consistent with classical tissue recombination experiments
in chick, which have shown that from the definitive-streak stage, the anterior
endoderm but not the posterior endoderm is competent to become liver
(Fukuda-Taira, 1981;
Le Douarin, 1975). Our
interpretation is that the posterior endoderm had already received a Wnt
signal to repress foregut fate, which is also consistent with explant studies
showing that the posterior axial mesoderm emits signals that inhibit liver
development (Gualdi et al.,
1996; Le Douarin,
1975).
Although it remains to be determined whether ß-catenin patterns the
endoderm in other vertebrates, some genetic studies are consistent with this
model. First, TOPGal transgenic mice, which contain a transcriptional
reporter of ß-catenin/Tcf activity, indicate that the anterior definitive
endoderm experiences little if any Wnt signaling
(Merrill et al., 2004). In
addition, mice lacking both Tcf1 and Tcf4 have hindgut
defects and an expansion of the anterior intestinal tract
(Gregorieff et al., 2004), and
ectopic activation of Wnt signaling in later-stage lung and pancreas
development results in ectopic intestinal tissue
(Heller et al., 2002;
Okubo and Hogan, 2004). The
role of individual Wnt ligands or Wnt-antagonists in the endoderm has been
difficult to determine in mice because of genetic redundancy. Mutations in
Wnt3 or Wnt3A result in posterior truncations
(Liu et al., 1999;
Yamaguchi et al., 1999),
whereas deletion of Dkk1 results in anterior truncations
(Mukhopadhyay et al., 2001);
however, the endoderm in these mutant embryos has not been described in
detail. Sfrp5 mutant mice do not exhibit foregut defects, but the
overlapping expression patterns of Dkk1, Sfrp5, Sfrp1, Sfrp2 and
Frzb (Sfrp3) suggest that they might act redundantly to
repress ß-catenin in the anterior endoderm
(Finley et al., 2003;
Heller et al., 2002;
Kemp et al., 2005;
Leaf et al., 2006).
ß-catenin has multiple roles during hepatic development
The results we describe here, together with previously published reports,
indicate that Wnts regulate multiple events in the hepatic lineage. Our
previous studies have shown that the maternal Wnt pathway stimulates the
initial expression of hhex and Wnt-antagonists in the anterior
mesendoderm of the Xenopus blastula
(Zorn et al., 1999). At this
stage, ß-catenin and hhex appear to cooperate to promote the formation of
the anterior organizer (Zamparini et al.,
2006). Now we show that just hours later, between the gastrula and
early somite stages, zygotic ß-catenin must be repressed to allow
hhex expression and foregut development. Then, by stage 30, the
function of ß-catenin once again changes and it appears to enhance liver
development. This later effect is consistent with recent reports that Wnt2bb
is required for liver specification in zebrafish
(Ober et al., 2006) and that
ß-catenin signaling promotes hepatoblast proliferation in the mouse and
chick liver bud (Hussain et al.,
2004; Tan et al.,
2006). Finally, the inappropriate activation of ß-catenin is
linked to liver cancer (Park et al.,
2001). Thus, one of the keys to understanding foregut organ
development is to determine when a signaling pathway has to be turned on or
off in a lineage and to characterize the resulting genetic program initiated
in each case.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2207/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Texas Children's Liver Center, Baylor College of Medicine,
1102 Bates Street, Houston, TX 77030, USA
|
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