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First published online 19 December 2007
doi: 10.1242/dev.008458
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Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, New York, NY 10021, USA.
* Author for correspondence (e-mail: brvnlou{at}rockefeller.edu)
Accepted 26 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: BMP, Gata5, TGIF2, Endoderm, Pancreas, Xenopus, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Over the
past few years extensive investigation has contributed to the elucidation of
the molecular mechanisms underlying initial endoderm formation
(Dickinson et al., 2006;
Ober et al., 2003;
Sinner et al., 2006;
Tam et al., 2003;
Taverner et al., 2005).
However, we still know relatively little about subsequent endodermal
patterning and how different domains of the endoderm progressively become
specialized to generate distinct organ primordia. In this study, we have
focused on the mechanisms underlying the establishment of the pancreatic
domain within the endoderm.
Increasing evidence suggests that regionalization of the endoderm in
vertebrates occurs at relative early developmental stages, beginning with a
broad patterning within the endoderm as it first emerges during gastrulation
(Lewis and Tam, 2006;
Wells and Melton, 2000;
Zorn et al., 1999). In the
mouse embryo, the definitive endoderm might acquire positional identity
already as it exits the primitive streak, such that cells recruited earlier
will form the foregut and those recruited later will contribute to the
posterior gut (Lawson and Pedersen,
1987; Lewis and Tam,
2006). In frog and zebrafish, gene expression and fate mapping
analysis has unveiled the existence of a significant patterning along both the
anteroposterior and dorsoventral axes within the endoderm long before
organogenesis (Chalmers and Slack,
2000; Costa et al.,
2003; Warga and
Nusslein-Volhard, 1999). For instance, in Xenopus embryos
dorsal endoderm explants, once dissected from early gastrula stage and
cultured alone, express pancreatic markers, while ventral explants do not
(Kelly and Melton, 2000). This
suggests that as early as gastrula stage the endoderm is broadly regionalized,
and prospective pancreatic endoderm forms in the quarter of the embryo around
the dorsal blastopore lip. Fate map analysis of the Xenopus endoderm
at neurula stage has shown that the pancreatic primordia emerges from two
distinct regions of endoderm (dorsal and most anterior/ventral endoderm)
(Chalmers and Slack, 2000).
This is in line with the pancreatic specification map of the gastrula embryo,
as both regions originate from dorsal endoderm and, through gastrulation
movements, acquire different positions at neurula stage
(Keller, 1975).
The molecular basis of this early endodermal patterning is clearly less
well understood than those of the ectoderm and mesoderm, but several
observations suggest that the molecular signals regulating early events of
regionalization are shared among the three germ layers
(Harland and Gerhart, 1997;
Henry et al., 1996;
Sasai et al., 1996;
Zorn et al., 1999). For
instance, TGFβ signaling influences anterior specification and patterning
in both mesoderm and endoderm (Henry et
al., 1996; Zorn et al.,
1999). Furthermore, extracellular BMP antagonists, released from
the dorsal mesoderm, specify dorsal fates within the ectoderm (neural tissue)
and the mesoderm (notochord and somites)
(Harland and Gerhart, 1997),
and may also promote endoderm of dorsal character
(Chen et al., 2004;
Sasai et al., 1996). However,
the direct role of BMP signaling in dorsal/ventral patterning of the endoderm
has not been established. More importantly, how this early patterning of the
endoderm leads to the establishment of specific organ domains, such as the
pancreas, has yet to be molecularly characterized.
Indeed, at present, there is a significant gap in our knowledge of
endodermal players acting in the window of time between the early stages of
endoderm formation and the expression of the pancreatic marker Pdx1
(also known as Xlhbox8 in Xenopus)
(Gamer and Wright, 1995;
Jonsson et al., 1994;
Kelly and Melton, 2000;
Offield et al., 1996), which
is induced just before organogenesis. A molecular understanding of this period
of time will explain how the endoderm is progressively patterned to generate
the presumptive pancreatic tissue.
Among the different transcription factors implicated in early embryonic
endoderm formation, the Gata-type zinc-finger transcriptional activators play
a prominent role (Holtzinger and Evans,
2005; Reiter et al.,
2001; Tam et al.,
2003; Weber et al.,
2000; Zhao et al.,
2005). Expression of Gata4, Gata5 and Gata6 has
been described in endodermal cells of the embryo from early gastrula stage
onwards, becoming restricted to anterior endodermal derivatives, such as the
prospective liver and pancreas, as development proceeds in all vertebrates
species so far analyzed (Tam et al.,
2003; Weber et al.,
2000). Although a functional redundancy during endoderm
specification seems to exist among different Gata factors
(Afouda et al., 2005;
Holtzinger and Evans, 2005;
Zhao et al., 2005), loss of
gata5 in the faust zebrafish mutant is sufficient to cause
significant loss of early endoderm and, specifically, of anterior endodermal
derivatives, such as the liver and the pancreas
(Reiter et al., 2001). In
addition, gain-of-function experiments in Xenopus demonstrate that
Gata5 is a potent inducer of endodermal fate and, specifically, of
regionally restricted endodermal markers, such as Pdx1
(Afouda et al., 2005;
Weber et al., 2000), at
tailbud stage. The cascade of molecular events downstream of Gata5 in
the endoderm leading to Pdx1 induction is yet to be defined.
To begin to dissect this cascade we used a microarray approach to identify
genes that operate between Gata5 and Pdx1 in
Xenopus embryos. Overall, we identified 141 genes with expression
that changes in response to Gata5. In this report, we present a
subset of Gata5 putative targets that are expressed in anterior
endodermal derivatives, and provide an in-depth analysis of one of these
targets, the TGFβ-induced factor 2 [also known as TG-interacting factor 2
(TGIF2)] (Imoto et al., 2000;
Melhuish et al., 2001). We
show that TGIF2 defines the pancreatic region by modulating the
TGFβ pathway in the endoderm, highlighting the prominent role of BMP
inhibition in the regionalization of the pancreatic domain within the
endoderm. Importantly, our study uncovers a crucial intermediate step in
pancreas formation, in which TGIF2 acts as a molecular connector
between dorsoventral patterning of the endoderm and specification of
pancreatic fate, linking Gata5 to Pdx1 induction.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For the morpholino antisense oligonucleotides used in this
study, see Fig. S3A in the supplementary material. The sequences of the
morpholino antisense oligonucleotides against Xenopus chordin have
been published (Oelgeschlager et al.,
2003). The oligonucleotides were purchased from GeneTools LLC.
Microarray and plasmid construction
DNA microarrays were prepared as described
(Altmann et al., 2001;
Munoz-Sanjuan et al., 2002).
Sample preparation and hybridization was performed as described previously
(Munoz-Sanjuan et al., 2002)
and below. The hybridization and scan of slides were performed at the Genomics
Core Facility of the Rockefeller University. Array images files were gridded
and analyzed using GenePix Pro image software and the data uploaded into
GeneTraffic software. Sequences of the clones identified in the array were
assembled using Sequencher 4.2.2 and blasted against public databases. The
coding region of Xenopus Gata5 (kind gift of Todd Evans, AECOM, NY)
was subcloned into pCS2++ by PCR. The plasmids Gata5-GR and Gata6-GR were
generously provided by Roger Patient (University of Nottingham, UK). The
majority of the clones regulated in the array contained full-length genes,
including the clone 8B1/xTGIF2. The flag-tagged xTGIF2 was
generated by PCR and ligated into pCS2++. Full-length mTgif2
(BC053438) was purchased from Open Biosystems and used for the rescue
experiments. DN-Alk3 corresponds to truncated Xenopus BMPRI
(1-744 nt) (Suzuki et al.,
1995), and CA-Alk3 was a gift from Bill Smith (UC Santa
Barbara, CA). The RNAs for all these constructs were synthesized on
AscI-linearized templates, with SP6 RNA polymerase using the mMessage
mMachine Kit (Ambion).
In situ hybridization analysis
Whole-mount in situ hybridizations were performed according to Harland
(Harland, 1991). In situ
hybridization on cryostat sections was done as in Schaeren-Wiemers and
Gerfin-Moser (Schaeren-Wiemers and
Gerfin-Moser, 1993). In situ probes from array clones in pCS2++
were prepared as follow: linearized with SalI and transcribed using
T3 polymerase. Two different xTGIF2 in situ probes were used: one
full-length (SalI/T3), and a short one produced by PCR including the
sequence outside the homeodomain and part of the 3'UTR (from nt
568-898). Other in situ probes were prepared as follow: for
Pdx1/Xlhbox8 (PCR cloning into pGEMT, NcoI/SP6); Hex-KSII
(BamHI/T7).
RT-PCR analysis
RT-PCR was performed as described
(Spagnoli and Brivanlou,
2006). Ornithine decarboxylase (ODC) was used as loading control.
Twenty-one PCR cycles were performed for ODC and Endodermin primers, and 25
cycles for the other primers. Real-time PCR reactions were carried out using
the SYBR Green Master Mix (Roche) on Light Cycler Roche. Succinate
dehydrogenase (SDHA) was used as reference gene for mouse samples. According
to the Light Cycler Roche instruction manual, the standard number of real-time
PCR cycles (45 cycles) was performed.
IP and western blot analysis
Immunoprecipitation was performed as previously described in Yeo and
Whitman (Yeo and Whitman,
2001) with mouse anti-FLAG M2 beads (Sigma) or anti-Smad1
monoclonal antibody (Santa Cruz Biotechnology) or anti-Smad2 goat polyclonal
antibody (Santa Cruz Biotechnology). Antibodies used in western blots were:
anti-Flag M2 monoclonal antibody 1:20,000 (Sigma); 1:1000 of a Smad1
polyclonal antibody (Upstate); 1:1000 of a Smad2 monoclonal antibody (BD);
1:10,000 of a 
-tubulin monoclonal antibody (Sigma).
Cell culture and shRNA transfection
BTC6 cells were purchased from ATCC and cultured in DMEM containing 15%
FBS. Short hairpin RNAs for mTgif2 knockdown and the non-silencing
shRNA mir control were purchased from Open Biosystems and transfected into
BTC6 cells using the Fluorescent Arrest-In transfection reagent (Open
Biosystems). C2C12 cells purchased from ATCC were maintained in DMEM
supplemented with 15% FBS. Human recombinant BMP4 for stimulation of C2C12
mouse cells was purchased from R&D. Cells were transfected 24 hours after
seeding using the Lipofectamine Plus reagent (Invitrogen). After 48 hours,
lysates were prepared, and the luciferase activity was determined with the
Dual Luciferase Assay System (Promega). Relative luciferase activities were
normalized by the co-expressed Renilla luciferase activities. All
luciferase assays were repeated at least three times and performed in
triplicate each time.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Munoz-Sanjuan et al., 2002)
(Fig. 1). As positive control
for Gata5 activity, an aliquot of the RNA sample used for the
microarray was assayed for induction of known targets of Gata5, such
as the general endodermal marker, Endodermin
(Sasai et al., 1996), and the
pancreatic regional marker, Pdx1, by RT-PCR
(Fig. 1A). In agreement with
previous observations (Weber et al.,
2000), Gata5 ectopic expression specifically induced a
subset of regionally restricted markers, the expression of which is confined
to the anterior endoderm, but not to mid-posterior endoderm, such as
IFABP (Henry et al.,
1996) (Fig.
1A).
This analysis revealed 141 genes that were upregulated or downregulated
following overexpression of Gata5 in ectodermal explants by a factor
of two or more. Table S1 provides a list of names and sequence identities of
the modulated genes (see Table S1 in the supplementary material). These
Gata5 putative targets were submitted to three independent tests for
priority, focusing initially on genes that were upregulated by Gata5
(Fig. 1B). The first criterion
was a validation of the results of the microarray by RT-PCR analysis on
independent mRNA populations extracted from uninjected and
Gata5-injected ectodermal explants at the same developmental stage
(tailbud). RT-PCR analysis confirmed that more than 80% of the array clones
analyzed were valid (see Fig. S1 in the supplementary material). The second
criterion was based on the establishment of the temporal hierarchy of
Gata5 target activation, by using a hormone-inducible version of
Gata5, referred to as Gata5-GR
(Afouda et al., 2005). This
allowed a classification of the time of induction of a subset of these targets
between neurulation and tailbud stage (see Fig. S2 in the supplementary
material; data not shown). Finally, the third criterion was to identify
putative Gata5 target genes that were co-expressed with
Gata5 (Weber et al.,
2000) in endodermal derivatives by a whole-mount in situ
hybridization approach (Fig.
2). Gata5 targets that underwent RT-PCR confirmation,
were temporally expressed in a window of time between Gata5 and
Pdx1 expression and showed an expression pattern similar to that of
Gata5 were chosen for further analysis, with a special emphasis on
genes that did not show any homology with sequences available in public
databases (Fig. 1B and see
Table S1 in the supplementary material).
|
;
Sumanas et al., 2000)
transcript was detected in dorsal/vegetal cells at the early gastrula stage
and in anterior endodermal derivatives, such as the prospective hepatic and
ventral pancreatic regions, at late stages
(Fig. 2A). This staining
clearly overlaps with the domains of expression of Gata5 at similar
developmental stages (Weber et al.,
2000). Hepatoma derived growth factor [(HDGF);
Clone 7C5] (Lepourcelet et al.,
2005) showed a weak expression in endodermal cells during
gastrulation (Fig. 2B), whereas
Xenopus TGIF2 [(xTGIF2); clone 8B1] expression was enriched in dorsal
and ventral/anterior endodermal cells at gastrula and neurula stages,
respectively (Fig. 2F-J). At
tadpole stage, its endodermal expression is confined to the
pancreatic-duodenal region (Fig.
2K). Some of the array clones were not detected in the endoderm at early embryonic stages, but as development proceeded their transcripts became abundant in endodermal derivatives, including the EST BG410109 (clone 7G9) in the liver, the EST BG410148 (clone 8C11) in the hepatic and duodenum region, and the EST BC094159 (clone 7B7) in the pancreas of the coiled gut (Fig. 2C,D,E). The fact that many of these putative Gata5 targets showed an expression in endodermal territories starting at tailbud stage is consistent with the described temporal regulation that they undergo upon Gata5 expression (see Figs S1 and S2 in the supplementary material). Finally, most of the array clones analyzed also showed sites of expression outside of the endoderm and often in the nervous system at various embryonic stages, suggesting that they may play additional roles during embryogenesis.
Interestingly, a number of Gata5 targets among the identified sets
are genes known to influence the TGFβ signaling pathway, such as, for
example, Coco (clone 57H9) (Bell
et al., 2003), TAB3 (clone 57G10)
(Munoz-Sanjuan et al., 2002)
and TGIF2 (Imoto et al.,
2000; Melhuish et al.,
2001). While the TGFβ signaling pathway has been described to
influence endoderm development (Henry et
al., 1996; Zorn et al.,
1999), Coco, TAB3 or TGIF2 have not previously
been associated with endoderm formation or patterning, suggesting novel
mechanisms of control of the pathway in the endoderm. Among these three
factors, TGIF2 is the only one that showed an endodermal expression
from gastrula stage onwards (Fig.
2F) (Bell et al.,
2003; Munoz-Sanjuan et al.,
2002). We therefore decided to undertake an extensive
characterization of the Gata5 target, TGIF2, in the context
of early endodermal patterning.
TGIF2, a target of Gata5, is a modifying endodermal factor that promotes pancreatic fate
TGIF2, which we identified as a novel target of Gata5,
encodes a homeodomain protein that belongs to the TALE (three-amino-acid loop
extension) superfamily of homeodomain proteins
(Imoto et al., 2000;
Melhuish et al., 2001). No
embryological or endoderm-specific function has been assigned to this
protein.
In order to begin our functional analysis of TGIF2 in the context
of endoderm patterning, we tested its ability to change the character of
endodermal cells using vegetal pole (prospective endoderm tissue) explants, as
embryological assay. Fig. 3A
shows that expression of xTGIF2 strongly induced the expression of
Pdx1 in ventral vegetal cells that are normally devoid of pancreatic
markers. Conversely, the expression of the hepatic marker, Hex (also
known as Hhex - Mouse Genome Informatics)
(Zorn et al., 1999;
Zorn and Mason, 2001),
appeared to be specifically downregulated in both ventral and dorsal vegetal
cells injected with xTGIF2 (Fig.
3A). Interestingly, the transcription factor Foxa2, a
marker of anterior endoderm and hepatic bud
(Zorn and Mason, 2001), was
slightly induced by xTGIF2 on the ventral vegetal side
(Fig. 3A). This reflects the
fact that Foxa2 is more widely distributed than Hex in the
anterior endoderm of tadpole embryos, for instance being expressed also in the
pancreas (Zorn and Mason,
2001). Finally, the level of the posterior gut marker,
IFABP, remained unchanged (Fig.
3A). These results clearly indicated that xTGIF2 has a
modifier activity within the endoderm, changing the character of ventral to
dorsal.
|
). The ectodermal explants were cultured until late tailbud
stage (stage 32) and examined for the expression of the pan-endodermal marker,
Endodermin, and regionally-restricted endodermal markers, such as
Hex and Pdx1 by RT-PCR
(Fig. 3B). Only when
xTGIF2 was presented together with VegT did we observe
induction of the pancreatic marker Pdx1, which was accompanied by
downregulation of Hex (Fig.
3B). These results suggest that xTGIF2 is not an inducer
of pancreatic endoderm, but rather a modifier of endodermal fate, confirming
our findings in vegetal pole explants (Fig.
3A). Finally, we did not observe induction of posterior endodermal
markers, such as IFABP, or of mesodermal and neural fates, as judged
by the absence of expression of the mesodermal-specific marker MyoD
and neural-specific marker NCAM
(Fig. 3B). More specifically,
we also investigated a potential effect of xTGIF2 on mesodermal
markers that are associated with lateral plate derivatives and have been
reported previously to be present in vegetal pole explants
(Horb and Slack, 2001).
Fig. 3C shows that induction of
Pdx1 in ventral vegetal cells injected with xTGIF2 was not
accompanied by induction or modulation of gut-surrounding mesoderm markers,
such as FoxF1, FOG, Tbx5 and Nkx2.5.
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TGIF2 is necessary for the establishment of the pancreatic domain within the endoderm
In order to address the in vivo function of xTGIF2 during the
regionalization of the endoderm and, specifically, of the pancreatic region,
we designed antisense morpholino oligonucleotides (referred to as TGIF2-Mo)
targeting both Xenopus laevis TGIF2 pseudoalleles found in the EST
databases (see Fig. S3 in the supplementary material). To inhibit the
translation of xTGIF2 mRNA specifically within the territory where
pancreas is formed, we injected the TGIF2-Mo into the dorsal vegetal
blastomeres of eight-cell stage embryos. Injection of the TGIF2-Mo into this
region of the embryo resulted in a clear drop of the level of Pdx1
expression, as judged by RT-PCR analysis on embryonic explants
(Fig. 4A). This reduction in
pancreatic character of the dorsal vegetal half was accompanied by a slight
increase of the hepatic marker Hex, whereas the expression of
IFABP and gut-surrounding mesoderm markers (FoxF1 and
FOG) was unaffected (Fig.
4A). Real-time RT-PCR analysis on TGIF2-Mo-injected embryonic
explants confirmed our results, showing a tenfold downregulation of
Pdx1 mRNA when TGIF2-depleted dorsal vegetal explants were compared
with uninjected ones (Fig.
7A).
Similar observations were made by analyzing the expression pattern of Pdx1 and Hex in antisense-injected embryos cultured to stage 35 by whole-mount in situ hybridization using specific probes (Fig. 4B,C). Importantly, the reduction of the Pdx1 domain of expression in TGIF2-Mo-injected embryos was extended to both pancreatic buds and the intermediate duodenum region, indicating that xTGIF2 is crucial for both regions (Fig. 4B). In line with our RT-PCR analysis (Fig. 4A), the domain of expression of Hex was expanded in TGIF2-Mo-injected embryos (Fig. 4C). Notably, the loss of Pdx1 expression in the prospective pancreatic region, due to the knockdown of endogenous xTGIF2 activity, could be rescued by the injection of mRNA encoding mouse TGIF2 (mTGIF2), which lacks the sequences targeted by TGIF2-Mo (Fig. 4B and Fig. 7A). Finally, at late tadpole stage, the pancreatic tissue was drastically reduced or absent in the gut of embryos depleted of TGIF2, as judged by the dramatic downregulation of insulin and amylase expression (Fig. 4D). Taken together, these results indicate that TGIF2 activity is required within the endoderm for proper establishment of the pancreatic region.
TGIF2 inhibits BMP/Smad1 pathway and promotes dorsal fates in Xenopus embryos
In response to TGFβ, an activated Smad complex can interact with
transcriptional co-repressors, such as TGIF, TGIF2, c-Ski or SnoN, which
displace co-activators and limit the extent of TGFβ transcriptional
activation (Massague et al.,
2005). Human TGIF2 has been characterized as a transcriptional
co-repressor for TGFβ-activated Smads, being able to interact physically
with activated SMAD3 (Melhuish et al.,
2001).
|
) or
Xnr1 (nodal-related factor-1)
(Hyde and Old, 2000), and
monitored the expression of immediate response genes activated by these
signaling molecules in ectodermal explants
(Fig. 5B; data not shown). In
this assay, xTGIF2 blocked the induction of a subset of
activin/nodal target genes, such as the lateroventral mesodermal
marker, Wnt8 (Smith and Harland,
1991), but did not block the induction of the dorsal marker,
chordin (Sasai et al.,
1996), or pan-mesodermal marker, brachyury
(Smith et al., 1990)
(Fig. 5B; data not shown).
Thus, a promoter-specific repression of TGFβ-induced activity by
TGIF2 seems to be conserved in vertebrates
(Melhuish et al., 2001).
Second, we considered the possibility that xTGIF2 might regulate
the BMP/Smad1 branch of the pathway. As BMP signaling normally promotes
ventral fates, xTGIF2 modulation of this pathway could explain the
double dorsal axis phenotype (Fig.
5A) and the dorsalized character of the endoderm observed upon
overexpression of xTGIF2 into the ventral vegetal pole
(Fig. 3A). In intact uninjected
ectodermal explants (animal caps), BMP signaling is active and induces ventral
epidermal fate, as judged by the induction of its immediate early response
genes, msx1 and msx2, and epidermal keratin (EK)
(Wilson and Hemmati-Brivanlou,
1995). Fig. 5C
shows that injection of xTGIF2 into animal caps strongly
downregulated msx1, msx2 and EK at gastrula stage, whereas
no induction of mesoderm (xbra) was detected
(Fig. 5C). In line with this,
xTGIF2 behaves as a weak neural inducer, being able to induce
sox2 at early gastrula stage, but only a subset of anterior neural
markers, such as xAG (Bell et al.,
2003), at later stages (Fig.
5C; data not shown). Taken together, these results suggest that
xTGIF2 has the ability to inhibit BMP signaling. Next, we
investigated the effect of xTGIF2 on BMP-activated transcriptional
responses using the BMP-responsive xVent-2 promoter-luciferase (Vent2-Luc)
reporter (Hata et al., 2000)
in Xenopus embryos. As shown in
Fig. 5D, overexpression of
xTGIF2 into the animal pole of the embryos robustly abrogated the
activation of Vent2-Luc transcription in response to BMP4. Expression of
xTGIF2 had no effect on this reporter in the absence of BMP4 (data
not shown). Taken together, these data strongly suggest that xTGIF2
inhibits the BMP/Smad1 branch of the pathway and modifies the TGFβ/Smad2
branch of the pathway by selective inhibition of a subset of regional
markers.
To determine whether TGIF2 is able to interact with the mediator of the BMP
signals, Smad1 (Massague et al.,
2005), we performed immunoprecipitation assays.
Fig. 5E shows that Flag-tagged
xTGIF2 injected into the ventral vegetal zone (the region where BMP
signal is active during gastrulation)
(Faure et al., 2000) of the
embryos interacted with endogenous Smad1
(Fig. 5E). In parallel, we
performed the same assay on the dorsal vegetal pole, and found an interaction
between Flag-xTGIF2 and endogenous Smad2
(Faure et al., 2000)
(Fig. 5E). Similar results were
obtained upon immunoprecipitating Smad1 or Smad2 and immunoblotting to detect
the tag on xTGIF2 (see Fig. S4 in the supplementary material). These results
showed that xTGIF2 is able to interact with both intracellular mediators of
the TGFβ signaling pathway.
|
). To address whether the BMP antagonistic activity of
TGIF2 was conserved in mammals, we performed reporter assays for the mouse
TGIF2 using BMP-responsive promoters both in Xenopus embryos and
C2C12 mouse myoblast cells. Following transfection of mTgif2 DNA in
C2C12 cells, we observed a significant repression of the activation of the
BMP-responsive promoter [BMP response element (BRE)]
(Hata et al., 2000) by BMP4
addition (Fig. 6A). Similar
observations were made in Xenopus ectodermal explants injected with
mTgif2 mRNA (data not shown). These results indicate that TGIF2 is an
inhibitor of the BMP pathway in both systems.
In order to investigate whether the mouse homolog of TGIF2 was
also able to induce ectopic expression of Pdx1, we tested
mTgif2 mRNA in the context of the Xenopus system using
vegetal explant assay, as described above
(Fig. 6B). Importantly, we
observed that mTgif2 was able to induce Pdx1 expression in
the ventral vegetal explants to the same extent as xTGIF2
(Fig. 6B). Next, we moved to a
mammalian system, such as the mouse pancreatic cell line BTC6
(Poitout et al., 1995), that
expresses differentiated pancreatic markers, such as Pdx1 and
insulin, as well as Tgif2
(Poitout et al., 1995) (data
not shown). By a loss-of-function approach using silencing short hairpin RNAs
targeting mTgif2 (shTGIF2), we showed that mTgif2 is
required for the maintenance of the expression of Pdx1 and insulin
(Fig. 6C). Transfection of
increasing dose of shTGIF2 into BTC6 cells resulted in a reduction of the
respective mRNA levels of Tgif2 itself, Pdx1 and insulin, as
judged by real-time RT-PCR analysis, whereas the shRNA control vector had no
effect (Fig. 6C). These results
suggest that the ability of TGIF2 to control the early pancreatic
regional marker Pdx1 is conserved across species.
BMP signaling controls dorsoventral regionalization in the endoderm
Based on our findings, we reasoned that the specification of the pancreatic
territory within the dorsal endoderm might be dependent on the inhibition of
BMP signaling in vivo. To test this hypothesis, we challenged the BMP
signaling activity in the context of endoderm regionalization by two
independent approaches. First, we knocked down the level of chordin, a robust
BMP antagonist released by the organizer
(Harland and Gerhart, 1997;
Oelgeschlager et al., 2003;
Sasai et al., 1996), using the
same assay as for TGIF2-Mo in Xenopus embryos
(Fig. 4;
Fig. 7A). As shown in
Fig. 7A, by real-time RT-PCR
analysis, we detected a reduction of the level of expression of Pdx1
in dorsal/vegetal cells injected with chordin antisense morpholinos (Chd-Mo)
(Oelgeschlager et al., 2003),
whereas the expression of the pan-endodermal marker Endodermin
remained unchanged. Second, we inhibited endogenous BMP signaling in the
ventral vegetal region of the embryo by using a dominant-negative BMP4
receptor I (DN-Alk3), which robustly blocks BMP signaling
(Mishina et al., 1995;
Suzuki et al., 1995).
Fig. 7B shows that
Pdx1 expression was induced in ventral vegetal pole explants injected
with DN-Alk3 mRNA as well as with xTGIF2. A similar
induction was observed also for additional anterior endodermal and pancreatic
markers, such as Foxa2 and Ptf1a
(Kawaguchi et al., 2002)
(Fig. 7B). Consistently, the
injection of constitutive active BMP4 receptor I (CA-Alk3) into the
dorsal vegetal half of the embryo led to a significant downregulation of
Pdx1 expression level, while the expression of gut-surrounding
mesoderm markers was unaffected (Fig.
7C; data not shown).
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By performing both gain- and loss-of-function experiments, we have shown
that TGIF2 behaves as a modifier, imparting a dorsal character to the
endoderm, and is required for the induction of the pancreatic regional marker,
Pdx1, in Xenopus endoderm as well as in mammalian cell
culture. Mouse Tgif2 mimics its Xenopus counterpart in
inducing Pdx1, pointing to an evolutionarily conserved role of
TGIF2 within the context of endodermal patterning. This observation
reflects the finding that TGIF2 transcripts show a similar expression
profile in mouse and frog embryos (Jin et
al., 2005) (our unpublished results).
From a mechanistic point of view, it has been previously reported that
TGIF2 exerts a promoter-specific repression on
TGFβ/Smad2-induced activity (Melhuish
et al., 2001). Our present study complements this finding, by
showing that TGIF2 strongly antagonizes BMP signaling in both
Xenopus and mammalian cells. The balance between Smad co-activators
and co-repressors has been proposed to refine the TGFβ-mediated response
(Massague et al., 2005). Our
study suggests that the main role of TGIF2 in vivo is to bias this balance
more toward BMP/Smad1 inhibition.
From an embryological point of view, we show that overexpression of
TGIF2 into the ventral vegetal blastomeres, where the Smad1 signal is
normally active from gastrulation onward
(Faure et al., 2000), leads to
dorsalization of the endoderm, inducing ectopic expression of Pdx1.
Interestingly, concomitant to Pdx1 induction, we observed a strong
downregulation of another transcription factor, Hex. While
Pdx1 demarcates the future pancreatic territory, Hex is an
endodermal marker that demarcates the future hepatic territory. Interestingly,
in line with our observations Hex has been described as a
BMP-responsive gene in vertebrates (Zhang
et al., 2002) and in amphioxus
(Yu et al., 2007).
BMP signaling has also been shown to play a crucial role in specifying gut
regions in mouse embryos (Bachiller et al.,
2003; Rossi et al.,
2001). For example, BMPs released from the septum transversum
mesenchyme are needed to induce ventral endoderm to adopt hepatic fate and
exclude pancreatic fate (Rossi et al.,
2001). In explants of mouse foregut endoderm cultured in the
presence of the BMP antagonist noggin, Pdx1 is activated, whereas
albumin (a liver marker) is not (Rossi et
al., 2001). Similarly, Tgif2 seems to counteract the
expression of Hex in favor of Pdx1, suggesting a potential
fine regulatory role of Tgif2 over the choice between pancreatic fate
versus hepatic fate. This is consistent with the fact that both the ventral
pancreatic bud and the liver originate from the same anterior/ventral
endodermal cells (Chalmers and Slack,
2000; Deutsch et al.,
2001). In line with these observations in the mouse embryo, a
recent study has shown that exposure of mouse embryonic stem cells to BMP4
also induces differentiation along the hepatic lineage
(Gouon-Evans et al.,
2006).
Finally, we show that modulation of the BMP pathway itself through
TGIF2-independent means modifies the dorsoventral character of the endoderm.
As such, pancreatic fate is inhibited by enhanced BMP signaling upon depletion
of the BMP antagonist chordin and is induced by cell-autonomous BMP
inhibition through expression of the dominant-negative receptor,
DN-Alk3. This latter observation is similar to the endogenous effects
of TGIF2 that we observed. Taken together, these findings propose a
more general mechanism by which patterning of the dorsal endoderm towards a
pancreatic fate relies on the inhibition of the endogenous BMP signaling
(Fig. 7D). This evidence
supports the proposal (Harland and
Gerhart, 1997; Henry et al.,
1996; Sasai et al.,
1996; Zorn et al.,
1999) that the endoderm might be patterned by the same signals
already implicated in mesoderm and ectoderm patterning.
|
). However, work in neural induction in Xenopus has
shown that the prospective neural tissue itself produces inhibitors of BMP
signaling to sustain a prolonged BMP inhibition
(Bell et al., 2003;
Munoz-Sanjuan et al., 2002).
With regard to the endoderm, very few examples of BMP inhibitors have been
detected in this germ layer. For instance, the expression domain of
chordin in Xenopus has been shown to expand from the
organizer into deep dorsal endodermal cell and the so-called bottle cells at
gastrulation, but its expression in the endoderm dramatically decreases at
neurula stage (Sasai et al.,
1996; Zorn et al.,
1999). Similarly, the secreted BMP inhibitor Cerberus shows a
transient expression in the anterior endoderm that does not persist during
later development (Piccolo et al.,
1999). To this scheme we can add a novel intracellular BMP/Smad1
inhibitor, TGIF2, within the endoderm. It is likely that a BMP-BMP
antagonist gradient within the endoderm is initiated by an organizer signal,
such as chordin, with which TGIF2 might synergize.
Subsequently, from neurula stage onward TGIF2 would maintain and
reinforce BMP inhibition intracellularly in order to define the pancreatic
region (Fig. 7D).
In the chick and zebrafish, BMP signaling seems instead to promote
pancreatic identity (Kumar et al.,
2003; Tiso et al.,
2002). For instance, in zebrafish swirl mutant, deficient
in Bmp2b, the expression of the pancreatic marker NeuroD is reduced, whereas
chordino (a BMP inhibitor) mutant embryos show an enlargement of the
pancreas, as detected only by Islet1 expression
(Tiso et al., 2002). This
study does not conclusively address the role of BMPs in patterning the
pancreatic endoderm at early stages, being based on a very limited number of
late-stage pancreatic markers. However, these opposite effects of BMP reported
in zebrafish and chick might be ascribed to differences in the origin and
positioning of the pancreatic precursors within the endoderm among species.
Alternatively, BMPs might have different effects at different stages of
pancreatic development, depending on the competence of the endoderm to respond
to such signals. An answer to this might come from a temporally and spatially
controlled inactivation of BMP signals during development.
Intriguingly, other members of the TALE family of homeodomain proteins,
including Meis and Pbx, have been characterized as co-factors of Pdx1
(Moens and Selleri, 2006).
These interactions can increase the binding specificity and transcriptional
effectiveness of homeodomain proteins. For instance, a Pdx1 complex containing
Pbx1 and Meis2 has been described in pancreatic exocrine cells and seems to
contribute to the switch between endocrine and exocrine fate
(Swift et al., 1998). Similar
mechanisms might also account for the effect of TGIF2 in the context
of pancreatic tissue and, for instance, in the BTC6 pancreatic line, analyzed
here. In support of this, we have found evolutionarily conserved TGIF-binding
sites in the mouse Pdx1 promoter (our unpublished results). Thus,
similarly to other pancreatic factors
(Jensen, 2004), TGIF2
may be involved in the earliest stages of pancreatic induction as well as
later in the maintenance of Pdx1 expression in pancreatic cells.
Although all three GATA factors, Gata4, Gata5 and Gata6,
clearly play a role in the development of the endoderm, recent findings have
indicated a predominant role for Gata6 in the genetic network
orchestrating endodermal programming
(Afouda et al., 2005). In our
analysis, we have found that all the putative Gata5 targets that we
analyzed here, including TGIF2, are also induced by Gata6 at
the same extent and same developmental time, suggesting a functional
redundancy among the different members of the subfamily (our unpublished
data).
In conclusion, our global molecular analysis has provided new insight into the early mechanisms of endodermal regionalization. Notably, we propose that a graded distribution of BMP activity controls the segregation of endodermal territories, where low BMP levels would define the early pancreatic region within the dorsal endoderm. TGIF2, one of the effectors of Gata5, is the endodermal factor that can lower BMP signaling at the appropriate time and location during pancreatic formation, establishing a molecular link between dorsal/ventral patterning of the endoderm and pancreatic induction.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/451/DC1
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ACKNOWLEDGMENTS |
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