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First published online August 25, 2006
doi: 10.1242/10.1242/dev.02516
1 Centre Development in Stem Cell Biology, Institute for Stem Cell Research,
School of Biological Sciences, University of Edinburgh, King's Buildings, West
Mains Road, Edinburgh EH9 3JQ, UK.
2 Discovery Biology, Pfizer Global Research and Development, Ramsgate Road,
Sandwich CT13 9NJ, UK.
Author for correspondence (e-mail:
josh.brickman{at}ed.ac.uk)
Accepted 3 July 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Endoderm, Mesendoderm, Hex, Hhex, PRH, Anterior, Wnt signalling, Nodal signalling, Organiser, Embryonic stem cells, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Marikawa et al., 1997). With
the onset of zygotic transcription, the vegetally localised maternal
transcription factor VegT induces the expression of the Nodal-related genes
Xnr1, Xnr2, Xnr4, Xnr5 and Xnr6
(Clements et al., 1999;
Takahashi et al., 2000;
Xanthos et al., 2001;
Yasuo and Lemaire, 1999),
which synergise with ß-catenin activity to establish the Nieuwkoop
centre. Canonical Wnt/ß-catenin targets such as Siamois
(Carnac et al., 1996;
Lemaire and Kodjabachian,
1996) and Xnr3
(McKendry et al., 1997) are
expressed in the Nieuwkoop centre region and are important for induction of
the Spemann organiser, also referred to as mesendoderm (reviewed by
De Robertis et al., 2000;
Harland and Gerhart, 1997).
Mesendoderm induction is also mediated by Nodal-related molecules, which act
in a dose-dependent manner. The highest levels of Nodal signalling induce the
most anterior mesendoderm derivative (anterior endoderm) and consequently the
dose of Nodal-related activity correlates with anteroposterior positional
information (Chen and Schier,
2001; Dyson and Gurdon,
1998; Green and Smith,
1990; Vincent et al.,
2003).
Following mesendoderm induction, the anterior endoderm expresses
antagonists of both the canonical Wnt and the Nodal-related pathways, such as
Lefty (Meno et al., 1999),
Cerberus (Piccolo et al.,
1999), Dickkopf (Glinka et
al., 1998) and Frzb (Leyns et
al., 1997; Wang et al.,
1997), which insulate the anterior part of the embryo from the
posteriorising influence of continued mesoderm induction
(Niehrs, 1999;
Piccolo et al., 1999).
Accordingly, the anterior endoderm itself may prevent the expansion of trunk
into the head field.
A large component of the spatiotemporal patterns of gene expression
generated during embryogenesis are mediated by homeodomain transcription
factors. However, how these factors integrate their intrinsic activities with
specific signalling pathways is largely unknown. Hex, a homeodomain protein
required for the specification of anterior mesendoderm
(Brickman et al., 2000;
Martinez Barbera et al.,
2000), has been shown to suppress posterior mesodermal gene
expression in the anterior endoderm via the repression of promoters associated
with mesendodermal genes such as Goosecoid
(Brickman et al., 2000). In
addition, overexpression of Hex leads to the expansion of anterior endoderm
expressing Wnt and Nodal antagonists such as Cerberus
(Brickman et al., 2000;
Jones et al., 1999;
Zorn et al., 1999). Thus, Hex
plays a pivotal role in anterior specification.
There are two possible explanations for the ability of Hex to induce anterior endoderm. It either suppresses more posterior identities in the mesendoderm or it promotes the inductive events upstream of anterior endoderm formation. To examine these possibilities, we asked whether Hex influenced signalling by the pathways responsible for mesendoderm induction. Here we show that Hex promotes anterior endoderm induction by amplifying early Wnt signalling. To elucidate the mechanism involved in this process, we looked for Hex target genes and identified the Groucho-related co-repressor/Wnt antagonist Tle4 as a Hex target in both mouse and Xenopus. We also identified four other Hex targets including Nodal. Gain and loss-of-function experiments in Xenopus suggest that anterior specification by Hex is dependent on Tle4 and a subset of Xenopus Nodals. Together, our work supports a conserved model in which Hex promotes anterior endoderm through both amplification of transduced Wnt signals followed by suppression of Nodal-mediated mesoderm induction.
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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).
mRNAs for injection were synthesised from linearised plasmids according to
Smith (Smith, 1993).
ß-catenin, mouse Hex, Hex-
VP2 and nuclear
GFP were linearised with NotI; Xnr1 was linearised
with SmaI; Siamois was linearised with SacII;
Xtle4 (IC 7011138) was linearised with HpaI. All were
transcribed with SP6 polymerase. Activin A (kindly provided by J. C. Smith)
was added to explant culture based on units defined by Cooke et al.
(Cooke et al., 1987). A
morpholino oligonucleotide (GeneTools) was used against the Hex 5'UTR:
CCTTAGCTGTACGTCATGGTCGTGG. The control morpholino was the standard provided by
GeneTools. To inhibit protein synthesis, whole embryos were incubated with 10 µg/ml cycloheximide (CHX) from stage 8 to 10.5 and analysed by in situ hybridisation. To monitor protein synthesis inhibition, embryos were cultured in media containing [35S]methionine with and without CHX, and incorporation of radiolabelled methionine into Xenopus tissue was measured.
RNA isolation and real-time RT-PCR
Total RNA was isolated using Absolutely RNA RT-PCR (Stratagene). RNA (0.5
µg) was used for cDNA synthesis according to Brickman et al.
(Brickman et al., 2000).
Real-time RT-PCR was performed using a LightCycler (Roche). For
Xenopus, cDNA from uninjected DMZ tissue from stage 10.5 embryos was
serially diluted and used as template for a standard curve assayed in each
run. For ES cells, standard curves were generated using varying amounts of
plasmid containing the appropriate gene. Samples were normalised to
Ornithine decarboxylase (Odc) for Xenopus embryos
and to ß-actin for ES cells. PCR primers and conditions are
listed in Table 1. Melting
curve analysis was performed on all PCR runs to ensure that quantification was
based on the accumulation of the specific PCR product.
|
), except that
BMpurple (Roche) was used as a substrate. Xnr3, Goosecoid, Chordin,
Cerberus and Derrière were linearised with
EcoRI; Siamois was linearised with StyI; nuclear
GFP was linearised with BamHI; Xnr1, Xnr2 and
Xtle4 (IC 7011138) were linearised with SmaI; Xnr5
and Xnr6 were linearised with NotI; Xlim1 and
BF1 were linearised with XhoI; and Xbra was
linearised with EcoRV. All were transcribed with T7 polymerase,
except for BF1, which was transcribed with SP6. Double in situ
hybridisation was performed based on a protocol developed in N. Papalopulu's
laboratory (Hardcastle and Papalopulu,
2000).
Luciferase reporter assays
HEK 293T cells (2x105) were plated on a 24-well plate and
transfected a day later with 10 ng of reporter plasmids (TOPflash or FOPflash
from Upstate Biotechnology) and 90-100 ng of test plasmids according to
Brickman et al. (Brickman et al.,
2001).
Embryonic stem cell culture and cell lines
ES cells were cultured as described by Li et al.
(Li et al., 1995). All cell
lines were derived from CGR8s (Nichols et
al., 1990). Details for targeting vector construction are
available on request. Plasmids are described elsewhere
(Brickman et al., 2000;
Soriano, 1999;
Srinivas et al., 2001).
Vectors were electroporated into ES cells
(Schmidt et al., 1997),
G418-resistant colonies expanded and genotyped by Southern blot
(Soriano, 1999). Two
independent clones of Rosa26 HexERT2-VP2 ES cells and one clone of
Rosa26 Hex ES cells were electroporated with pCAGCreIP (gift of A. Smith) and
plated at clonal density. Individual clones were picked, expanded and
genotyped by PCR (5'-CAAACTCTTCGCGGTCTTTC, with either
3'-GCGTAATCAGGAACGTCATA to detect non-recombined locus or
3'-CCAGAGGCCACTTGTGTAGC for recombined), and analysed by Southern and
western blot.
Microarray analysis
ES cells (2x106) were plated on 10 cm2
gelatinised dishes and cultured for 8-12 hours. Cells were then cultured with
or without 600 nM 4-hydroxytamoxifen (4-OHT) for 7.5 hours. RNA was prepared
using RNeasy and QIAshredder (Qiagen). RNA quality was tested with an Agilent
Bioanalyzer and a minimum of three samples applied to Affymetrix MGU74Av2
GeneChip Oligonucleotide Arrays as described in Frye et al.
(Frye et al., 2003). Data were
normalised using the RMA algorithm in Genespring 7.2. Expression measurements
for each gene were represented as average fold-change between each treatment
condition group. The groups used for comparison were Rosa-Hex compared with
Cre-recombined Rosa-Hex (RRosa-Hex); non-recombined cell lines
Rosa-HexERT2-VP2 (clone1) and Rosa-HexERT2-VP2 (clone2) with
and without 4-OHT; and the recombined versions of these lines
(RRosa-HexERT2-VP2 clones 1 and 2) with and without 4-OHT
(Fig. 4A,B). Fold filtering was
performed to select for profiles that differed in both
RRosa-HexERT2-VP2 clones upon 4-OHT treatment and did not change in
the non-recombined controls. The list of genes obtained was further filtered
to remove candidates with low absolute expression levels. During the analysis,
some individual samples expressed lower levels of the pluripotency marker
Oct4. The candidate list was therefore re-examined excluding these samples for
an added level of refinement. All microarray data are available from GEO
(series accession number GSE5141).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability of Hex to anteriorise ß-catenin activity can be interpreted in two ways; Hex suppresses the induction of trunk by ß-catenin or Hex anteriorises ß-catenin signal. To distinguish between these possibilities, we examined whether Hex could anteriorise ß-catenin activity in ventral marginal zone (VMZ) explants. Although modest doses of ß-catenin (500 pg) induced dorsal morphology, it never induced key anterior features such as cement glands or eyes (Fig. 1B, see Table S2 in the supplementary material). However, when this dose of ß-catenin was combined with Hex, VMZ explants were anteriorised and appeared indistinguishable from uninjected dorsal marginal zone (DMZ) explants. Interestingly, a similar phenotype was observed in response to higher doses of ß-catenin (1000 pg), suggesting that Hex increased the ability of low levels of ß-catenin to transduce the canonical Wnt signal. Together, these results support a role for Hex in anteriorising a ß-catenin signal.
Hex amplifies Wnt target activation
To better understand the level at which Hex regulated Wnt signalling, we
looked at Wnt-responsive gene expression. We quantified the expression of the
ß-catenin direct targets Siamois and Xnr3 in VMZ
explants by real-time RT-PCR (Fig.
1C,D). We included in the analysis Hex-VP2, a VP16 fusion
protein that turns Hex into a transcriptional activator
(Brickman et al., 2000). Hex
and Hex-VP2 showed very little or no effect in the absence of
ß-catenin. However, the levels of Siamois and Xnr3
induced by ß-catenin were significantly increased by co-injection of Hex
(Fig. 1C) and suppressed by
co-injection of Hex-VP2 (Fig.
1D). Similar results were obtained by in situ hybridisation where
the upregulation of Xnr3 expression was particularly pronounced in the animal
hemisphere (Fig. 1E, insets and
see Table S3B in the supplementary material).
To reveal the consequence of activating ß-catenin and Hex locally, we
injected Hex, Hex-VP2 and/or ß-catenin mRNA into specific
blastomeres at the four-cell stage, alongside nuclear GFP (nucGFP) used as a
lineage tracer and examined by double in situ hybridisation (light blue,
Fig. 1E). When embryos were
injected on the dorsal side, where ß-catenin is normally active, Hex
strongly increased endogenous Xnr3 expression, whereas
Hex-VP2 inhibited it within the injected cells
(Fig. 1E, see Table S3A in the
supplementary material). Moreover, co-injection of Hex and ß-catenin into
a single-ventral blastomere led to a pronounced enhancement of the ability of
ß-catenin to induce ectopic Xnr3
(Fig. 1E, see Table S4A in the
supplementary material).
To determine whether endogenous Hex was required for
ß-catenin/Nieuwkoop centre activity, Hex was knocked-down by injection of
an antisense morpholino oligonucleotide (MO). We used a previously described
Hex MO known to induce an anterior phenotype in Xenopus laevis
(Foley and Mercola, 2005;
Smithers and Jones, 2002).
Siamois and Xnr3 expression was strongly reduced by Hex
depletion, and this depletion also led to a reduction in the ability of
ß-catenin to induce ectopic expression of both Siamois and
Xnr3 (Fig. 1F, see
Table S7 in the supplementary material). The phenotypes obtained were rescued
by mouse Hex mRNA (Fig. 1F),
which differs with Xenopus Hex in DNA sequence around the ATG
initiation codon, but is indistinguishable in terms of activity
(Brickman et al., 2000;
Smithers and Jones, 2002).
Hex, Wnts and mesendoderm induction
Although the immediate early response to ß-catenin signalling is the
induction of direct targets such as Siamois and Xnr3, these
regulators have complex activities and induce a range of different cell types,
including both the anterior endoderm expressing Cerberus and
Hex itself, but also more posterior axial mesoderm expressing genes
such as Goosecoid. Interestingly, anterior gene products such as
Hex and Cerberus then block the induction of more posterior
cell types, creating a complex network of antagonistic interactions
(Brickman et al., 2000;
Piccolo et al., 1999). Based
on the anteriorised phenotypes observed in
Fig. 1, we hypothesised that
amplification of Wnt signalling by Hex should preferentially induce anterior
endoderm, suggesting a role for Hex in specifying regional identities within
the mesendoderm. To test whether Hex-amplified Wnt signalling preferentially
induced anterior endoderm and determine how this early activity of Hex relates
to its later known role in the suppression of posterior mesendodermal
identity, we looked at the expression of a number of mesendodermal markers in
response to a Hex-amplified ß-catenin signal.
Fig. 2 shows the response of
the anterior endodermal marker Cerberus, alongside the axial
mesendodermal markers Goosecoid and Chordin to a
Hex-amplified Wnt signalling.
|
;
Brickman et al., 2000;
Zorn et al., 1999), we were
not surprised to find that ß-catenin and Hex synergised to induce ectopic
Cerberus expression (Fig.
2A,B, see Table S4 in the supplementary material). Consistently,
morpholino-mediated depletion of Hex resulted in both a reduction in
endogenous Cerberus and in the ability of ß-catenin to induce
ectopic Cerberus (Fig.
2F). However, in contrast to the results obtained with
Cerberus, co-expression of Hex alongside ß-catenin led to a
context-dependent enhancement of Wnt-stimulated ectopic Goosecoid and
Chordin. Thus, in an explant culture that gives an indication of the
net effect in a population of cells, the ability of Hex to stimulate Wnt
signalling appeared to compete with the ability of Hex to inhibit axial
mesoderm (Fig. 2C,D). Modest
levels of Hex (200 pg) inhibited ß-catenin-mediated stimulation of these
genes, whereas at higher doses of Hex (500 pg), ß-catenin-stimulated gene
expression recovered somewhat more pronouncedly in VMZ explants
(Fig. 2D). However, when this
effect is examined at a cellular level, by in situ hybridisation on an
injected clone of cells, Hex-stimulated levels of Wnt signalling appeared to
predominate (Fig. 2E, see
Tables S5 and S6 in the supplementary material). This observation was
particularly striking with respect to Goosecoid, a defined Hex
target, where Hex and ß-catenin synergise to induce Goosecoid,
but the effect is predominately cell non-autonomous
(Fig. 2E, rightmost panels). As
with Goosecoid, Chordin is also synergistically induced by
ß-catenin and Hex, but in this case the induction is primarily
cell-autonomous (Fig. 2E,
rightmost panels), implying that the inhibition of Chordin expression
observed in explant culture (Fig.
2C,D) was probably an indirect effect arising from the ability of
Hex to suppress axial mesoderm.
These observations are consistent with the view that Hex is required for
the maximal level of early ß-catenin signalling to induce anterior
endoderm and then in the mesendoderm to specify/maintain anterior endoderm by
suppressing axial mesoderm. Remarkably, we found that the ability of
ß-catenin to induce all three markers was impaired in Hex-depleted
embryos (Fig. 2F, see Table S7
in the supplementary material). Moreover, in Hex-depleted embryos where
ß-catenin was not overexpressed, Cerberus and Chordin
expression was lost, whereas that of Goosecoid was expanded
(Fig. 2F, see Table S7 in the
supplementary material). These phenotypes were rescued with mouse Hex RNA and
presumably reflect the ability of Hex to both regulate early Wnt signalling
and suppress axial mesoderm. The similar behaviour of both Chordin
and Cerberus may reflect an absolute dependence on Wnt signalling for
the expression of both markers at blastula stages
(Agius et al., 2000). However,
blastula stage expression of Goosecoid and the majority of
mesendoderm, is induced by a combination of both Wnt and Nodal-related
signalling and thus may be less dependent on the ability of Hex to amplify
early Wnt signalling and more sensitive to Hex-mediated suppression of axial
mesoderm. Consistent with this interpretation, we found that other
mesendodermal markers that are dependent on both Nodal-related and Wnt
signalling for their initial induction such as Xlim1 and
Brachyury (Xbra) were increased in Hex-depleted embryos (see
Fig. S1A and Table S7 in the supplementary material). Moreover, the ability of
Hex to synergise with ß-catenin to preferentially induce anterior
endoderm and suppress more posterior mesendoderm was also evident in their
synergistic ability to suppress Derrière, a gene involved in
posterior axial patterning (see Fig. S1B in the supplementary material).
Amplification of Wnt signalling by Hex is mediated by a nuclear antagonist
As the amplification of Wnt signalling by Hex occurs at the level of
immediate-early targets of the Wnt pathway, we asked if it could be
recapitulated in vitro. We tested the ability of Hex to regulate the
activation of TOPflash, a minimal Wnt reporter in HEK 293T cells
(Fig. 3A). Co-transfection of
Hex with ß-catenin enhanced the ability of ß-catenin to activate
TOPflash transcription (Fig.
3B). As Hex is known to act as a transcriptional repressor and the
activator Hex-VP2 suppressed ß-catenin activity in vivo, we
addressed whether in vitro activation of TOPflash was indirect. Consistent
with an indirect mechanism, co-transfection of Hex-VP2 dramatically
reduced the ability of ß-catenin to induce the TOPflash reporter
(Fig. 3B). In no case did Hex,
Hex-VP2 or ß-catenin significantly affect the levels of FOPflash,
a mutant version of TOPflash used as a negative control.
Normally, induction of the Wnt signal suppresses the ability of GSK3 to
phosphorylate ß-catenin (Yost et al.,
1996). The unphosphorylated form of ß-catenin is then
stabilised and enters the nucleus to interact with the Tcf/Lef DNA-binding
proteins and induces Wnt targets. To determine at which level Hex regulates
this pathway, we asked whether Hex or Hex-VP2 could modulate the
activity of: (1) stabilised ß-catenin, a constitutively activated variant
of ß-catenin that had been rendered insensitive to GSK3 regulation
through mutations of GSK3 phosphorylation sites
(Munemitsu et al., 1996); (2)
LefdnßCTA, a nuclear localised fusion of the activation domain of
ß-catenin with Lef1 that is still dependent on the ability of the
activation domain of ß-catenin to stimulate transcription
(Vleminckx et al., 1999); and
(3) Xtcf3-VP2, a fusion of Xtcf3 to two copies of the VP16 activation
domain that acts independently of any ß-catenin specific activation
function. Activation of TOPflash by all three molecules was potentiated by Hex
and efficiently suppressed by Hex-VP2
(Fig. 3C), strongly suggesting
that Hex modulates the expression of a Wnt nuclear antagonist downstream of
ß-catenin that can be recruited by the Xtcf/Lef complex.
The Groucho co-repressor Tle4 and Nodal are novel Hex targets
To gain insight into the mechanism by which Hex positively acts on Wnt
signalling, we sought to identify Hex transcriptional targets. To facilitate
this process, we used a mouse ES cell-based model, as it enabled us to work
with large numbers of cells that reflect an undifferentiated early embryonic
phenotype that can be genetically manipulated in vitro.
|
VP2, an inducible version of
Hex-VP2 that responds to 4-hydroxytamoxifen (4-OHT)
(Indra et al., 1999) were
generated alongside clonally related control cell lines, by targeting Hex or
HexERT2-VP2 to the Rosa26 locus by homologous recombination
(Soriano, 1999;
Srinivas et al., 2001)
(Fig. 4A). These cell lines
contain a stop cassette flanked by LoxP sites that can be removed by
transfection with Cre-recombinase, allowing recombination-mediated induction
of gene expression. Following excision of the stop cassette, Hex or
HexERT2-VP2 were expressed from the Rosa26 promoter and detected by
western blot (see Fig. S2A in the supplementary material). HexERT2-VP2
activity was confirmed using a Goosecoid reporter gene (see Fig. S2B
in the supplementary material).
|
VP2; (2) regulation by Hex; and
(3) invariant expression in parental lines that do not express a Hex
derivative. Although the third criterion may exclude some Hex targets, it was
an essential noise filter for analysis of heterogeneous ES cell cultures
(Fig. 4C). When all three
criteria were applied, a list of five candidates presented in
Fig. 4D was obtained, and we
conclude they represent the most likely Hex targets from this analysis.
Two of the identified genes directly impact on early embryonic signalling:
the Wnt antagonist/Groucho-related co-repressor Tle4 and the mesoderm
inducer Nodal. Induction of these genes by HexERT2-VP2 in ES
cells in the presence of 4-OHT was confirmed by real-time RT-PCR
(Fig. 4E). Based on the
sequence of previously identified Hex-binding sites
(Brickman et al., 2000;
Crompton et al., 1992), we
searched the mouse genome for putative sites in regions around Nodal
and Tle4. In Tle4, we found several clusters of putative
Hex-binding sites located within highly conserved blocks in introns 6, 12, 14
and 16 (see Fig. S3C in the supplementary material; data not shown). The
majority of these sequences are conserved in Xenopus in addition to
other mammalian genomes. In Nodal, Hex-binding sites were found both
upstream and within intron 1 (see Fig. S3A in the supplementary material).
This binding site pattern appears to be conserved in the Xenopus
Nodal homologues Xnr1 and Xnr2 (see Fig. S3B in the supplementary
material).
The Xenopus homologue of Tle4, Xtle4, has been shown to
be a potent antagonist of Wnt/ß-catenin signalling at the level of Xtcf
in early Xenopus embryos (Roose
et al., 1998). Based on the phenotypes obtained in
Fig. 1, we reasoned that
Hex-mediated repression of Tle4 could provide the molecular basis for
the regulation of Wnt signalling by Hex in Xenopus embryos.
Fig. 5 shows expression of
Xtle4 in response to expression of Hex-VP2 and Hex depletion
by MO. At stage 10.5, Xtle4 is normally expressed throughout the
animal hemisphere and in the superficial regions of the marginal zone
(Fig. 5A,B). Hex-VP2
injection in deep dorsal tissue resulted in ectopic induction of
Xtle4 (Fig. 5B, see
Table S8 in the supplementary material). Moreover, Hex MO injection led to a
significant upregulation of Xtle4 in the marginal zone, including the
superficial dorsal lip mesendoderm, while injection of mouse Hex RNA rescued
this phenotype (Fig. 5A, see
Table S7 in the supplementary material).
|
;
Hyde and Old, 2000;
Osada et al., 2000;
Vincent et al., 2003).
Interestingly, Hex depletion by Hex MO injection resulted in a very pronounced
upregulation of Xnr1 and Xnr2, but not of Xnr5 and Xnr6. Moreover, this
ectopic expression of Xnr1 and 2 was rescued by mouse Hex, which had no effect
on Xnr5 and Xnr6 (Fig. 5A, see
Table S7 in the supplementary material). The autoregulatory activity of Xnr1
and Xnr2 also explains why Hex depletion results in ectopic Xnr expression in
cells that do not normally express Hex. Although we were unable to detect a
role for Hex in the regulation of Xnr5 and Xnr6 by
morpholino depletion, Hex-VP2 mRNA injection induced all the Xnrs
tested (Fig. 5B, see Table S8
in the supplementary material). Differential regulation between these subsets
of Xnrs may be due to Xnr-specific co-factors required for Hex repression.
|
; Sinner et al.,
2004) and these factors can complicate interpretation. However,
taken together with the in silico identification of conserved Hex binding
sites and the fact that these genes are induced after a short period of
treatment of 4-OHT in the array data set, these findings support the notion
that Xtle4, Xnr1 and Xnr2 are direct Hex targets.
Tle4 overexpression antagonises Hex-mediated anterior induction
To determine whether Hex regulation of Xtle4 was relevant to Hex-mediated
amplification of Wnt signalling, we overexpressed Xtle4 and asked how this
impacted on the ability of ß-catenin and Hex to stimulate the Wnt targets
Xnr3 and Cerberus in Xenopus embryos.
Fig. 6A shows that
overexpression of Xtle4 blocked the ability of Hex to potentiate
ß-catenin induction of both Xnr3 and Cerberus (see
Table S3B and Table S4B in the supplementary material).
A similar effect was observed when Xtle4 was transfected in the context of the TOPflash reporter in HEK 293T cells (Fig. 6B). Co-transfection of Xtle4 significantly reduced the stimulated expression levels obtained with ß-catenin and Hex co-transfection. Interestingly, while co-transfection of Xtle4 alongside ß-catenin led to a complete suppression of the reporter, the presence of Hex allowed this activity to recover slightly, although in no case to the level of ß-catenin on its own. Together, these data suggest that Xtle4 has a quantitative effect on Wnt-responsive transcription.
|
; Osada et al.,
2000). Moreover, conserved Hex-binding sites are located in intron
1 alongside the Nodal autoregulatory element (see Fig. S3 in the supplementary
material) (Norris et al.,
2002; Osada et al.,
2000). Thus, we reasoned that Hex should interfere with the relay,
behaving as a Nodal antagonist. We next tested whether Hex misexpression
reduced Xnr1 activity. Thus, while ectopic expression of Xnr1 induced a
partial secondary axis marked by a small region of BF1 expression in
Xenopus embryos, this axis was completely suppressed by co-injection
of Hex (Fig. 7A, see Table S9
in the supplementary material). Similarly, the expression of increasing
amounts of Xnr1 in VMZ explants led to a degree of dorsalisation and
concomitant neural induction as evidenced by patchy BF1 staining,
while co-expression of Hex alongside Xnr1 inhibited these phenotypes over a
range of concentrations tested (Fig.
7B, see Table S10 in the supplementary material).
As the induction of Cerberus is not only dependent on
ß-catenin but also on Nodal signalling, we asked whether Hex stimulates
induction of Cerberus by Xnr1. As previously reported and also shown
here, Xnr1 is a potent inducer of Cerberus expression
(Fig. 7C, see Table S11 in the
supplementary material) (Agius et al.,
2000; Niederlander et al.,
2001). However, when Hex was co-injected with Xnr1, this induction
was dramatically reduced (Fig.
7C). This result is particularly surprising, as Hex did induce
Cerberus expression on its own and combinatorially with
ß-catenin. Thus, high levels of Nodal signalling presumably rely on the
ability of the Nodal pathway to stimulate Xnr1 and Xnr2 transcription and Hex
might interfere with this process. In support of this notion, we observed a
general increase in the activity of ectopic Xnr1 in Hex-depleted embryos
(Fig. 7D, see Table S7 in the
supplementary material).
The ability of Hex to suppress Nodal-related activity was also tested with Activin, a related TGFß. Hex inhibited the ability of Activin protein to induce convergent extension phenotypes in animal cap explants (Fig. 7E, see Table S12 in the supplementary material). A quantitative reduction in the ability of Activin to induce Goosecoid, Chordin, Xbra and the endodermal marker Mixer was also observed in these explants (Fig. 7F).
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Perea-Gomez et al., 2002;
Piccolo et al., 1999). The
model in Fig. 8 illustrates the
juxtaposition of these two activities. As Tle4 and Nodal are targets of Hex in
both mouse and frog, these regulatory interactions may be conserved.
|
). Although Xtle4 is expressed in the animal
hemisphere and superficial regions of the marginal zone at gastrula stage
(Molenaar et al., 2000), Hex
is expressed in the deep and early involuting endoderm. These complementary
expression domains support the idea that Hex represses Xtle4 to
establish an embryonic region that is responsive to Wnt signalling.
Evidence from a number of organisms suggest that high levels of Nodal
signalling are required for the induction of endoderm, particularly anterior
endoderm (Aoki et al., 2002;
Vincent et al., 2003). However
although Nodal signalling is seen as required early for anterior endoderm and
later for the continued process of more posterior mesoderm induction,
Wnt/ß-catenin signalling is generally thought of as a posteriorising
signal. Yet, the role of nuclear ß-catenin in establishing the Nieuwkoop
centre makes it one of the earliest determinants of dorsoanterior identity in
both Xenopus and zebrafish (Kelly
et al., 2000; Xanthos et al.,
2002) and our data support an early, dose-dependent role for Wnt
signalling in anterior endoderm specification.
A role for early Wnt signalling in anterior specification is also
consistent with recent revisions to the Xenopus fate map that
demonstrate an anterior fate for the progeny of cells experiencing high levels
of Wnt signalling (Lane and Sheets,
2000). Moreover, in Xenopus, both ß-catenin
(Sinner et al., 2004) and
Siamois (Zorn et al.,
1999) have been implicated in the induction of anterior endoderm
and early Cerberus induction has been shown to be dependent on
ß-catenin (Agius et al.,
2000; Xanthos et al.,
2002). In the mouse, the anterior endoderm is divided into two
populations, the anterior definitive endoderm (ADE), formed during
gastrulation and the anterior visceral endoderm (AVE), an extra-embryonic
tissue formed at peri-implantation stages
(Beddington and Robertson,
1999). Hex is initially expressed throughout primitive endoderm at
the hatched-blastocyst stage and then becomes restricted to the distal region
of the visceral endoderm lineage. These Hex-positive visceral endoderm cells
migrate anteriorly to become the AVE a day prior to gastrulation. Hex is later
expressed in the ADE (Thomas et al.,
1998). Genetic evidence links the induction of these endodermal
tissues to Wnt signalling in both the ADE
(Hsieh et al., 2003;
Kelly et al., 2004) and AVE
(Huelsken et al., 2000). In
zebrafish, Hex is initially expressed asymmetrically as part of a Wnt-mediated
signalling centre in the extra-embryonic yolk syncytial layer
(Ho et al., 1999). This region
is related to the Xenopus Nieuwkoop centre as it induces the earliest
mesendoderm (Fekany et al.,
1999; Kelly et al.,
2000; Yamanaka et al.,
1998) and Hex has also been implicated in this activity
(Ho et al., 1999). Our data
create a molecular framework for these observations and imply that early
expression of Hex may represent a conserved component of the Nieuwkoop
centre.
The anterior endoderm will continue to express Hex, whereas the axial
mesoderm will not. The continued expression of Hex in the anterior endoderm
represses both Goosecoid and Nodal-related gene expression.
Repression of Nodal transcription by Hex would add to the inhibitory effects
of Nodal antagonists such Cerberus and Lefty. A major component of
Nodal-related activity occurs via an autoregulatory loop
(Adachi et al., 1999;
Osada et al., 2000;
Vincent et al., 2003) and we
have shown that Hex inhibits Nodal signalling by interfering with Nodal
transcription. Moreover, the two Xenopus Nodal-related genes that are
Hex targets are also the two genes that exhibit autoregulation
(Hyde and Old, 2000). Thus,
Hex may inhibit Nodal signalling by blocking Nodal production without
interfering with Nodal-mediated signal transduction. The implications of this
idea are that a Hex-expressing cell could receive the high levels of Nodal
signalling required to induce anterior endoderm, but would then no longer
transmit Nodal signals to its neighbours. By blocking the continued Nodal
autoregulation while still leaving cells competent to respond to the signal,
Hex may be in effect defining the end of the anterior axis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/18/3709/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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