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First published online May 1, 2006
doi: 10.1242/10.1242/dev.02358
1 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation and Department of Pediatrics, University of Cincinnati College of
Medicine, Cincinnati, OH 45299, USA.
2 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL,
UK.
* Authors for correspondence (e-mail: h.r.woodland{at}warwick.ac.uk; aaron.zorn{at}chmcc.org)
Accepted 13 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Endoderm, Development, Xenopus, Nodal, Sox17, Gata, Mixer, Microarray, Gene regulatory network
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Stainier,
2002; Tam et al.,
2003; Xanthos et al.,
2001). The key zygotic factors in this pathway are the
Nodal-related TGFß signaling ligands, the Mix-like family of homeodomain
transcription factors, the Gata4/5/6 zinc-finger transcription factors and the
HMG box transcription factor Sox17.
In Xenopus, endoderm development is initiated by the maternal
T-box transcription factor VegT, which is localized to the presumptive
endoderm tissue (Horb and Thomsen,
1997; Stennard et al.,
1996; Zhang and King,
1996). VegT is required for endoderm formation and the expression
of zygotic factors, including the Nodal-related genes Xnr1, Xnr2,
Xnr4, Xnr5 and Xnr6, and Derriere, Mix1, Mix2, Bix1, Bix2,
Bix3, Bix4, Mixer, Gata4, Gata5, Gata6, Sox17
and
Sox17ß (Xanthos et al.,
2001; Zhang et al.,
1998). VegT directly activates the transcription of many of these
(Casey et al., 1999;
Clements and Woodland, 2003;
Engleka et al., 2001;
Hilton et al., 2003;
Tada et al., 1998), but
maintenance of their expression and subsequent endoderm formation requires
Nodal signaling (Clements et al.,
1999; Kofron et al.,
1999; Yasuo and Lemaire,
1999). In Xenopus, Nodal signaling is necessary and, at
high levels, sufficient to induce endoderm development
(Agius et al., 2000;
Henry et al., 1996;
Osada and Wright, 1999) by
promoting the expression of the Mix-like, Gata and Sox17 transcription
factors, which in turn activate downstream target genes
(Afouda et al., 2005;
Clements et al., 2003;
Hudson et al., 1997;
Xanthos et al., 2001).
Ectopic expression of Mixer, Bix1, Bix2, Bix4, Sox17/ß or
Gata4-6 can all induce endoderm differentiation in naïve animal cap
ectoderm (Casey et al., 1999;
Ecochard et al., 1998;
Henry and Melton, 1998;
Hudson et al., 1997;
Tada et al., 1998;
Weber et al., 2000) and
loss-of-function studies have shown that Mixer, Gata4-6 and
Sox17/ß are essential for proper endoderm development
(Afouda et al., 2005;
Clements et al., 2003;
Henry and Melton, 1998;
Hudson et al., 1997;
Kofron et al., 2004).
Although the precise epistatic relationships between Mixer, Gata4-6 and
Sox17 are unresolved, a linear model is commonly proposed where Nodal proteins
regulate Mixer and Gata, and these function upstream of Sox17, which in turn
activates endoderm target genes (Stainier,
2002; Xanthos et al.,
2001). In support of this model, Mixer and Gata5 can induce
Sox17 expression in animal caps and VegT-depleted embryos, but Sox17
cannot induce expression Mixer or any of the other Mix-like
genes (Henry and Melton, 1998;
Sinner et al., 2004;
Xanthos et al., 2001).
Furthermore, a dominant-negative version of Sox17 (Sox17-EnR) has been shown
to inhibit Mixer function, but, conversely, a dominant-negative Mixer
(Mixer-EnR) cannot inhibit Sox17 function
(Henry and Melton, 1998),
suggesting that Mixer acts primarily via Sox17.
However, other evidence suggests that endoderm specification is more
complex than predicted by the linear model. First, Sox17 expression
precedes Mixer, which is principally expressed in equatorial regions
of the endoderm (Henry and Melton,
1998), which is inconsistent with Mixer acting primarily via
Sox17. Second, studies have suggested that that Sox17/ß and
Gata4-6 can regulate the expression of each other
(Afouda et al., 2005;
Clements et al., 2003;
Sinner et al., 2004).
A limitation of many studies to date is that they have relied on only a few
early markers, usually Hnf1ß
(Demartis et al., 1994) and
Endodermin (Edd) (Sasai
et al., 1996) to assay endoderm specification and it is unclear if
their regulation is indicative of all endoderm genes. In addition, most
studies have relied on ectopic overexpression in animal cap ectoderm
(Afouda et al., 2005;
Clements and Woodland, 2003;
Dickinson et al., 2006;
Sinner et al., 2004;
Taverner et al., 2005), which
may lack important co-factors found in the vegetal tissue and it is unclear
how accurately animal cap assays reflect endogenous endoderm development.
Here, we have used microarray analysis and functional experiments to better
resolve the regulatory network controlling Xenopus endoderm
formation. We defined a robust set of genes with enriched expression in the
gastrula endoderm, containing 
90% of the known endoderm-expressed genes
and several hundred uncharacterized sequences. We determined which of these
genes were regulated by Nodal signaling, Mixer or Sox17, and found that only
10% of endoderm genes can be regulated as described by the current linear
model of endoderm development. The bulk of endoderm gene regulation appears to
be much more complex, with Nodal proteins, Mixer and Sox17 having both shared
and distinct sets of target genes. We find that transcriptional repression by
Mixer plays a greater role than previously appreciated and that extensive
autoregulatory loops exist between Sox17 and Bix1/2/4, between Sox17 and Xnr4,
and between Sox17 and Gata4-6. This data challenges the existing models of
vertebrate endoderm development and provides an important resource for
understanding of the complex gene regulatory network that controls
Xenopus endoderm development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and embryos were staged according to the normal table of
development for Xenopus laevis
(Nieuwkoop and Faber, 1994).
Two-cell stage embryos were vegetally injected with the following doses of
antisense morpholino oligos or synthetic RNA: a combination of antisense
morpholino oligos to Sox171 + Sox172 + Sox17ß (20 ng each)
(Clements et al., 2003); Mixer
antisense morpholino oligo (40 ng) (Kofron
et al., 2004); Cerberus-short RNA (1 ng)
(Piccolo et al., 1999);
Mixer RNA (50-500 pg) (Henry and
Melton, 1998); Sox17ß RNA (10-100 pg;
Xenopus tropicalis Sox17ß that is immune to the
laevis Sox17ß morpholino oligo)
(D'Souza et al., 2003); and
Gata4, Gata5 or Gata6 RNA (50-100 pg)
(Afouda et al., 2005). Rescue
experiments were performed with Gata4, Gata5 and Gata6 with
similar results; therefore only the Gata6 data are shown.
Microarray analysis and data processing
Table 1 lists the different
conditions and the number of biological replicates used in the array study.
For each biological replicate, 20 sibling embryos from a single mating or
50 micro-dissected explants from sibling embryos were used. Total RNA was
extracted using Trizol (Invitrogen) and purified on RNAeasy columns (Qiagen).
Ten micrograms of total RNA was used for cDNA syntheses and to make labeled
RNA probe which was hybridized to Affymetrix Xenopus Genechips by the
CHRF microarray core facility, using the standard Affymetrix protocol.
GeneSpring 7.1 software (Silicon Genetics) was used for data normalization,
clustering and filtering. Raw CEL file data from all the samples was
pre-normalized using RMA (Robust Multichip Average). The average log intensity
of the biological replicates was then normalized to the average log intensity
of stage 11 whole embryo. NCBI Unigene cluster nomenclature was used to
describe uncharacterized sequences. All of the raw microarray data are
available from GEO (series record number GSE4448).
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). Details
of the primer sequences used for the 60 genes analyzed in this study are
available on request. For each new primer pair a melt curve analysis was
performed and the PCR product was examined on a gel to ensure that a single
fragment of the predicted molecular weight was amplified. The data for each
sample was normalized to the expression level of the ubiquitously expressed
gene ornithine decarboxylase (ODC).
DNA constructs and In situ hybridization
Plasmids for validation were generously provided by Professor Naoto Ueno
from the NIBB Xenopus EST project (Japan) or clones from the NIH
Xenopus sequencing project were purchased from ATCC. Synthesis of
antisense RNA probes and in situ hybridization to bisected gastrula embryos
were performed as described (Sive et al.,
2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
because the known targets of Mixer, Gata4-6 and Sox17 are expressed.
We compared the transcriptional profile of stage 11 whole embryos (We),
micro-dissected vegetal (Veg) and equatorial regions (Eq), and animal caps
(An) (Table 1). Vegetal regions
isolated from stage 11 gastrulae contained mostly endoderm, and small amounts
of mesoderm. Equatorial regions isolated from stage 11 gastrulae contained
mostly mesoderm but also superficial endoderm. The animal cap tissue isolated
from stage 9 embryos and cultured until stage 11, contained ectoderm. For each
biological replicate, total RNA was prepared from 20 whole embryos or
50 explants from sibling embryos. The RNA was subjected to microarray
analysis using the Affymetrix Xenopus Genechip and the resulting data
were analyzed with GeneSpring software, where the average log intensity of the
biological replicates was normalized to the average expression levels in stage
11 whole embryos.
To identify genes with enriched expression in the endoderm, we examined the
behavior of the known endoderm genes Sox17/ß,
Mixer, Bix1-4, Gata4-6, Hnf1ß, FoxA1 and Edd.
From their characteristics, we empirically determined the following parameters
for selecting endoderm-enriched transcripts from the 15,000 sequences on
the microarray. After filtering the data to eliminate genes that were not
expressed in the gastrula, we selected transcripts with expression in the
vegetal region greater or equal to the expression in the equatorial region.
This eliminated many mesoderm-specific genes (e.g. Xbra), but
retained most genes known to be expressed at the mesoderm-endoderm boundary
(e.g. Eomesodermin). We then selected genes with threefold or greater
expression in the vegetal region than in the animal cap, resulting in a list
of 503 sequences that represented 483 genes based on their NCBI Unigene
designations (see Table S1 in the supplementary material). This list of 483
genes contained 35 of 40 published genes known to have endoderm-enriched
expression (see Table S2 in the supplementary material), providing a strong
validation of our approach. Of the five known genes that were not recovered by
our selection (Siamois, Hex, Xnr4, Xnr6 and FoxA1) four had
low expression levels, just above background, which may explain why they did
not behave as predicted. For further analysis, we selected 276 sequences
(representing 264 genes) that had statistically significant differences in
expression between vegetal and animal cap regions over all biological
replicates using Student's t-test (P< 0.05) (Table S1).
Fig. 1A summarizes the
transcriptional profile of those 276 sequences and Table S3 in the
supplementary material presents the top 60 endoderm-enriched genes.
The predicted molecular function encoded by these 264 endoderm-enriched
transcripts, based on NCBI Unigene annotations, Gene Ontology and blast
analyses is indicated in Fig.
1B. Over 40% of the genes are uncharacterized, while 25%
encode predicted regulatory proteins (38 transcription factors, 15 secreted
ligands/antagonists, nine receptors and eight signal transduction molecules),
a number of which have not previously been implicated in endoderm
development.
Validation of endoderm-enriched transcripts in the Xenopus gastrula
We validated the expression of 25% of the genes not previously known
to have endoderm-enriched expression by RT-PCR and in situ hybridization,
reasoning that this was a representative sample size. By real time RT-PCR
analysis, 51 of 54 (94%) previously uncharacterized genes were expressed at
least three times higher in the vegetal region than in the animal cap
(Fig. 2A; see Table S4 in the
supplementary material). The selection of the 54 genes to validate was largely
random, with an emphasis on those genes for which there were full-length cDNAs
available in the clone repositories. In situ hybridization to bisected
gastrula embryos confirmed that 24 of 35 genes exhibited obvious endoderm
enriched expression in the gastrula embryo
(Fig. 2B). The remaining 11
transcripts that did not exhibit endoderm restricted expression by in situ,
did have enriched endoderm expression by RT-PCR but were either undetectable
by in situ or had expression throughout the embryo with slightly higher levels
in the vegetal region.
The in situ analysis shows that we identified genes with varying endoderm
expression patterns. Xl.11602, Xl.13921, Xl.15375, Xl.2554, Xl.3534
and Xl.46324 were expressed throughout the endoderm in a pattern
similar to Sox17 (Hudson et al.,
1997), while others such as CXCR4, Xl.13033, Xl.215, Xl.13381,
Xl.8924, Xl.18924, FoxA4 and Xl.5418 had varying expression in
the deep endoderm and were enriched at the mesendoderm boundary similar to
Mixer (Henry and Melton,
1998). Xl.15758 (epsin2) and Xl.7782
were expressed in the deep endoderm, but not in the superficial layer of the
blastopore, reminiscent of Gata5
(Weber et al., 2000).
Wnt11-R, Xl.16040 and Xl.8924, which were expressed in the
anterior endoderm reminiscent of Hex or Cerberus, whereas
Pinhead has ventrolateral expression. Finally one unknown gene
Xl.14891 has an expression pattern that suggests it is expressed in
germ plasm. A temporal expression profile of the endoderm-enriched sequences
based on microarray analysis of egg, gastrula and stage 18 is available in
Fig. S1 in the supplementary material.
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Regulation of endoderm gene expression by Nodal proteins, Mixer and Sox17
We next determined which of the 301 endoderm-enriched sequences were
regulated by either Nodal proteins, Sox17 or Mixer. We focused on these three
regulators because specific loss-of-function approaches have been validated
for each of them (Agius et al.,
2000; Clements et al.,
2003; Kofron et al.,
2004). Furthermore this allowed us to test the linear model
predicting that Nodal genes>Mixer>Sox17>endoderm-target genes. If
this model is correct, inhibiting any one of the components should prevent
zygotic endodermal gene expression.
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); (2)
embryos depleted of Mixer (Mixer-) by injection of antisense Mixer
morpholino oligos (40 ng) (Kofron et al.,
2004); and (3) embryos depleted of Sox171/2/ß
(Sox17-) by injection of three antisense Sox17 morpholino oligos (20
ng each) (Clements et al.,
2003). Each of these loss-of-function paradigms has been show to
result in specific defects in endoderm development. Fig. 3 shows the expression profile of the 301 endoderm-enriched transcripts in Nodal-, Mixer- and Sox17- embryos, and a complete list of the average normalized expression levels for each transcript in the different experimental conditions are presented in Table S1 (see supplementary material). The expression profiles in Fig. 3A immediately show that many genes are sensitive to some, but not all, of the experimental conditions. A hierarchal clustering of genes and experimental conditions (Fig. 3B) shows that, as expected, the expression profile of Nodal- embryos is more similar to animal cap tissue than to control embryos or vegetal tissue, indicating that both endoderm and mesoderm development was inhibited by Cerberus-short RNA injection. Sox17-depeleted embryos had an expression profile more similar to equatorial tissue and Nodal- embryos than to control embryos or vegetal tissue, suggesting that mostly endoderm development was compromised, rather than that of mesoderm. Surprisingly, the profile of Mixer- embryos was more similar to isolated vegetal tissue than to any other sample. As we will describe in more detail later, this was due to the fact that many mesendoderm genes are upregulated in Mixer- embryos.
As an initial validation, we focused on known Nodal, Mixer and Sox17
targets (Agius et al., 2000;
Clements et al., 2003;
Clements et al., 1999;
Henry and Melton, 1998;
Hudson et al., 1997;
Kofron et al., 2004;
Rosa, 1989;
Sinner et al., 2004;
Xanthos et al., 2001),
comparing their expression on the array to that determined by real time RT-PCR
(Fig. 4). Although the array
tended to under-represent the fold changes observed by RT-PCR, we found that
14/14 known Nodal targets (Xnr1, Xnr2, Xnr4, Mix1-2, Bix1-4, Mixer,
Gata4-6, Sox17/ß, Edd, HNF1ß), 8/10 of
the known Mixer targets (Xnr5, Gata5, Bix1, Bix4, Cerberus,
Sox17, Eomesodermin, Edd, FGF3, eFGF) and 5/7 of the
known Sox17 targets (Xnr4, Gata4-6, Foxa1, Edd, HNF1ß) behaved
as expected from published results, providing a strong validation of the array
data (Fig. 4; data not
shown).
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74%
(n=53; Table S1 in the supplementary material) of the time the array
data correctly predicted the behavior of a gene in all three experimental
conditions. In 21% of the cases, the array prediction was partially
validated in that the trend in expression change was correct, but the
threshold of more than a 1.4-fold change was not met in one or more
conditions. Only 5% of the time did the array predict a change that was
contradictory to the RT-PCR validation, indicating that the array data were a
very good predictor of a the regulation of a gene. Based on the criterion of more than a 1.4-fold change, we found that 223 of the 301 endoderm enriched genes were regulated by either Nodal signaling, Mixer or Sox17 (Fig. 3C), with 112 Nodal-regulated, 168 Mixer-regulated and 100 Sox17-regulated genes, respectively. Of the 78 genes that were not regulated by Nodal proteins, Mixer or Sox17, 67 had high maternal expression, including germ plasm genes Dazl and Deadsouth (see Table S1 in the supplementary material). Surprisingly, a Venn analysis indicated that only 36/223 transcripts were similarly regulated by Nodal, Mixer and Sox17, including HNF1ß and Edd, two of the early endoderm markers used to establish the current linear model of endoderm development. This suggests that the transcriptional network controlling endoderm development is more complex than predicted by the current model.
Epistatic relationships between Nodal proteins, Mix-like, Gata4-6 and Sox17/ß
The array analysis revealed a number of previously unappreciated
relationships between Xnrs, Mixer, Bix1-4, Gata4-6 and
Sox17/ß, and their downstream targets. For
example, we found that expression of Xnr4, Mix2, Bix1, Bix2, Bix4 and
Gata genes were all downregulated in Sox17- embryos
(Fig. 4), which was unexpected
as they were previously thought to act upstream of Sox17. To test these
observations, we performed a series of loss-of-function and rescue
experiments, comparing the ability of Sox17ß, Mixer or Gata6, to rescue
gene expression in Nodal- embryos with their ability to rescue gene
expression in Sox17- embryos (Fig.
5A). According to the linear model, all should rescue Nodal
inhibition, but only injection of XtSox17ß RNA (Xenopus
tropicalis Sox17ß mRNA lacking the sequence targeted by the Sox17
morpholino) should rescue gene expression in embryos where endogenous Sox17
protein has been depleted.
Sox17 is involved in multiple autoregulatory loops
Our data, in conjunction with published reports, suggests three major
feedback loops: one between Sox17 and Gata4-6; a second unexpected
autoregulatory loop between Sox17 and Bix1/Bix2/Bix4; and a third between
Sox17 and the Nodal ligand Xnr4.
Sox17 and Gata4-6
In animal caps, Gata4-6 and Sox17/ß are known to induce each
others expression, and Gata4-6 are required for full
Sox17/ß expression levels in the gastrula
(Afouda et al., 2005;
Clements et al., 2003;
Sinner et al., 2004). Here, we
show that Gata4-6 are downregulated in both Nodal- and
Sox17- embryos (Fig.
4) (Clements et al.,
2003) and that injection of XtSox17ß can partially
rescue Gata5-6 expression in both Nodal- and Sox17-
embryos. Similarly, we find that Gata6 can rescue
Sox17/ß expression in Nodal- embryos
(Fig. 5A). Together, these data
demonstrate that Sox17 and Gata4-6 autoregulate the expression of one another
downstream of Nodal signaling.
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;
Ecochard et al., 1998;
Tada et al., 1998), the
reverse is not true: Sox17 cannot induce Bix1-4 transcription in
animal cap assays (Sinner et al.,
2004). This implies that Sox17 requires additional cofactors to
promote Bix gene expression and that these factors are absent from the animal
hemisphere. Candidate co-factors include activated Smad2, or possibly Gata4-6
[as Gata6 can partially rescue Bix1 and Bix4 expression in
Sox17- embryos (Fig.
5A)]. Interestingly Sox17-dependent regulation was specific to
only a subset of the Mix-like gene family and was not observed with
Mix1, Bix3 or Mixer. Together with published reports, these
data suggest that Sox17 and Bix1, Bix2, Bix4 regulate each others expression
and that Gatas may also participate in this cross regulatory loop.
Sox17 and Xnr4
The third autoregulatory loop we identified was between Sox17 and Xnr4. We
found that Xnr4 expression, which was thought to act at the top of
the zygotic gene hierarchy regulating endoderm development, was dependent on
Sox17 (but not Mixer or Gatas). Injection of XtSox17ß RNA
rescued the Xnr4 expression levels in Nodal- and
Sox17- embryos (Fig.
5A), suggesting that Sox17 may act in part by maintaining Nodal
signaling, one of the most upstream components of the endoderm specification
pathway. It is intriguing that only Xnr4 and not any other
Xnrs are Sox17 dependent, suggesting that Xnr4 may have some unique
function.
Mixer does not function primarily via Sox17
Although Mixer rescued Sox17 expression in Nodal- embryos
(Fig. 5A), we consistently
found that Sox17 was only moderately downregulated in Mixer-
embryos (Fig. 4)
(Kofron et al., 2004).
Furthermore, of the 268 genes regulated by either Sox17 or Mixer, only 67
genes were regulated by both Mixer and Sox17
(Fig. 3). Thus, the modest
reduction in Sox17 levels observed in Mixer- embryos cannot
account for the Mixer loss-of-function phenotype, indicating that Mixer does
not function primarily via Sox17 as commonly cited.
Endoderm target genes are not all coordinately regulated
Contrary to the current model, we found that the early endoderm markers
Edd, Hnf1ß, Foxa1 and Foxa2 were not all
regulated in same way. As expected, the reduction of Edd, Foxa1 and
Hnf1ß expression in Nodal- embryos was rescued by
injection of Sox17, Mixer or Gata6 RNA
(Fig. 5A; data not shown).
However, in Sox17- embryos, only Sox17 or Mixer, but not Gata4-6,
rescued Edd expression (Fig.
5A; data not shown). These data, along with the fact that
Edd is downregulated in Mixer- embryos
(Fig. 4)
(Kofron et al., 2004),
indicates that Sox17 and Mixer independently contribute to Edd
expression, and that Gatas regulate Edd via Sox17
(Fig. 5B). Finally,
Foxa2, which can be induced in animal cap experiments by ectopic
Sox17 (Sinner et al., 2004),
does not require Sox17 for expression and only Mixer (but not Sox17 or Gata6)
rescued Foxa2 expression in Nodal- embryos. This suggests
that although all three factors, Sox17, Mixer and Gata, participate in
Foxa1 regulation, Mixer is the primary regulator of Foxa2
expression (Fig. 5B).
These results challenge the existing model of endoderm development and establish a new number of epistatic relationships between the known endoderm regulators. Our data suggests that Sox17 is not the most downstream component of the endoderm specification pathway, as commonly cited, but rather participates in auto regulatory loops with Bix1, Bix2, Bix4, Gata4-6 and Xnr4, all of which were previously considered to be upstream of Sox17. In addition, we find that Mixer does not function primarily via Sox17, as predicted by the linear model, and that different endoderm target genes have varying modes of regulation.
The regulatory network controlling endoderm transcription is complex
Having examined the regulation of the known endodermal genes, we next
wanted to determine how endoderm transcription is regulated at a global level.
We therefore examined the array data to identify patterns of Nodal-,
Mixer- and Sox17-dependent gene expression in all 301
endoderm-enriched transcripts. Based on the criterion of a greater than
1.4-fold change in expression levels relative to controls, we grouped genes
into one of three different categories for each of the three experimental
condition. Genes with reduced expression in Nodal-, Mixer-
or Sox17- embryos were classified as positively regulated (+) by
Nodal proteins, Mixer or Sox17, respectively. Genes with increased expression
in Nodal-, Mixer- or Sox17- embryos were considered
negatively regulated (-) and normally repressed by Nodal proteins, Mixer or
Sox17, respectively. Finally, genes exhibiting less than a 1.4-fold change in
expression levels in either Nodal-, Mixer- or
Sox17- embryos relative to controls were considered to be `not
obviously regulated' (0) by Nodal proteins, Mixer or Sox17, respectively.
Based on these criteria, we classified each of the 301 endoderm-enriched genes
as positively, negatively or not regulated by Nodal signaling, Mixer and Sox17
(Fig. 6;
Table 2; Table S1 in the
supplementary material). Overall, Nodal proteins, Mixer or Sox17 regulated 223
of the 301 sequences; of the 78 genes that were not regulated, 67 had
significant maternal expression (Fig.
3C, Fig. 6O).
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10%) were regulated in this manner
(Fig. 6A;
Table 2) and that endoderm gene
expression can be classified into 19 different modes of regulation
(Fig. 6;
Table 2). Table S1 in the
supplementary material provides a full list of how each gene was classified
and the average normalized expression data for each condition. To validate
some of these novel modes of regulation, we performed loss-of-function and
rescue experiments, comparing the ability of RNA encoding, Sox17, Mixer or
Gata6, to rescue gene expression in Nodal- embryos
(Fig. 7).
Nodal proteins, Mixer and Sox17 have both shared and distinct downstream targets
First, we confirmed that the genes Xl.5999 and Xl.8924
(Fig. 6A) were positively
regulated by Nodal signaling, Mixer and Sox17
(Fig. 7A; +N +M +S), similar to
Hnf1ß and Edd. Co-injection of Mixer RNA
produced the best rescue of Xl.5999 and Xl.8924 expression
in Nodal- embryos, while the rescue by Gata and Sox17 was very
modest. These results are consistent with the hypothesis that Mixer induces
Sox17 and Gata expression
(Fig. 5), and then all three of
these contribute to Xl.5999 and Xl.8924 regulation.
We classified 21 transcripts positively regulated by Nodal proteins and
Mixer, but not by Sox17 (Fig.
7B; +N +M 0S); 14 transcripts positively regulated by Nodal and
Sox17 but not Mixer (Fig. 7C;
+N 0M +S); and 22 transcripts positively regulated by Nodal proteins, but not
regulated by either Mixer or Sox17 alone
(Fig. 7D; +N 0M 0S). The
regulation of these different groups of genes is more consistent with a model
where Nodal proteins, Mixer and Sox17 each have distinct sets of target genes.
For example, only co-injection of Mixer RNA rescued Darmin
and Xl.15089 expression in Nodal- embryos, confirming they
are positively regulated by Nodal proteins and Mixer, but not by Sox17 or
Gata6 (Fig. 7B; +N +M 0S). The
rescue experiments also validated genes that were positively regulated by
Nodal proteins and Sox17, but not by Mixer, such as Foxa4 and
Xl.13381 (Fig. 7C; +N
0M +S). In the case of Xl.13381, injection of XtSox17ß
RNA alone could not rescue its expression in Nodal- embryos,
suggesting that other nodal dependent factors are also required for
Xl.13381 transcription. The genes Xenf
(Nakatani et al., 2000),
Xl.2554 and Mig30
(Hayata et al., 2002) are
examples of the 22 endoderm genes that require Nodal signaling, but are not
significantly regulated by Sox17, Mixer or Gata6
(Fig. 6D,
Fig. 7D; +N 0M 0S). We
hypothesize that these may require the combined action of Sox17, Mixer or
Gata6, might be direct Smad2 targets, or might be regulated by some unknown
Nodal-dependent factor.
|
). For
example the 14 genes classified as +N, -M 0S
(Fig. 6G) included the
mesendoderm gene Eomesodermin and the novel gene Xl.2967,
which is also enriched in the equatorial region (see Table S3 in the
supplementary material). In another example, rescue experiments confirm that
Xl.2967 requires Nodal signaling, probably via Gata proteins and that
Mixer represses Xl.2967 expression
(Fig. 7E).
Nodal-independent regulation?
Finally, the array data indicate there are a number of different
categories, comprising 100 genes that were regulated by Sox17 and/or
Mixer, but their expression was not significantly altered by blocking Nodal
signaling (Fig. 6H-N). This
suggests that either a significant proportion of endoderm formation is
independent of Nodal signaling, and/or that Nodal proteins regulate the
expression of both activators and repressors, and the loss of both results in
little overall changes in gene expression.
For example, Xl.4709 is one of the 18 genes with expression unchanged in Nodal- embryos, upregulated in Mixer- embryo and downregulated in Sox17- embryos (Fig. 6M; 0N-M+S). Injection of Mixer RNA or the Sox17-MO repressed Xl.4709 levels, while injection of the Mixer-MO resulted in over expression of Xl.4709 (Fig. 7F). In a second example, Xl.1489 was upregulated by depletion of Mixer or injection of Gata6 RNA (Fig. 7G). We hypothesize that in Nodal- embryos both activators (such as Sox17) and repressors (perhaps Mixer) would be missing, resulting in little change in gene expression. However, in the absence of repression by Mixer, activation by Sox17 predominates; while in the absence of Sox17, repression by Mixer predominates and the gene is downregulated.
Sox17 negatively regulates Wnt/ß-catenin pathways components
Of the 100 Sox17-regulated sequences we observed, 17 were upregulated in
Sox17- embryos and thus normally repressed by Sox17 activity
(Fig. 6E,H,I,K). Interestingly,
at least two of these are components or targets of the Wnt/ß-catenin
pathway: Wnt11 and Xnr3
(McKendry et al., 1997;
Tao et al., 2005). This is
consistent with reports that Sox17 can antagonize ß-catenin/TCF
transcriptional activity in vitro (Zorn et
al., 1999a) and suggests that in the embryo Sox17 may also
restrict Wnt-responsive transcription. In the case of Xnr3, our
rescue experiments indicate that it is also repressed by Mixer, but positively
regulated by Nodal signaling, perhaps via Gata proteins
(Fig. 7H).
In summary, we find that the linear model of endoderm formation does not accurately describe the bulk of endoderm gene expression, which is much more complex than previously appreciated. Importantly, this work provides a complete documentation of how each of the 301 endoderm-enriched transcripts were regulated by Nodal signaling, Mixer and Sox17 (see Table S1 in the supplementary material), providing a comprehensive resource for examining the gene regulator network controlling Xenopus endoderm formation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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300 genes with endoderm-enriched expression, including over a
hundred genes uncharacterized in any species. As our strategy identified most
of the genes known to control endoderm formation, it is likely that many of
these unknown genes may also have important regulatory functions. Using this robust gene list, we interrogated the existing models of endoderm development determining how global endoderm gene expression was regulated Nodal proteins, Mixer and Sox17. In addition to identifying many novel Nodal, Mixer and Sox17 targets, these experiments indicate that the transcriptional hierarchy controlling endoderm gene expression is much more complicated than previously appreciated, with only 10% of the endoderm transcriptome being regulated as predicted by the linear model commonly cited in the literature. Our analysis classified endoderm gene expression into 19 different categories of regulation with Nodal proteins, Mixer and Sox17 having both shared and distinct sets of target genes.
|
;
Casey et al., 1999;
Clements et al., 1999;
Sinner et al., 2004;
Tada et al., 1998). These
results clearly demonstrate that Sox17 is not the most downstream component of
the pathway regulating endoderm formation, as commonly described. We also
found that Mixer does not function primarily via Sox17 as commonly cited.
Although Mixer probably participates in maintaining Sox17 expression,
much of Mixer function is independent of Sox17, and Mixer (but not Sox17) has
a major role in negatively regulating the expression of over a hundred
mesendoderm genes.
Two other recent studies have also used microarrays to identify the genes
involved in Xenopus endoderm development: one by Taverner et al.
(Taverner et al., 2005)
looking at VegT targets; and another by Dickinson et al.
(Dickinson et al., 2006)
attempting to identify Mixer and Sox17ß target genes. An important
distinction between those studies and this one is that they both used
overexpression in animal caps to identify downstream targets. By contrast, we
defined the endogenous endoderm transcriptome and used loss-of-function
approaches to examine gene regulation. Although clearly useful, overexpression
animal cap studies have limitations. For example, we found only 30 of the 71
Mixer and Sox17 target genes identified by Dickinson et al. in our primary
list of 500 endoderm-enriched transcripts. When we examine the expression
profile of the other 41 genes that were not in our list, only four were
endoderm enriched in our array data and the rest were either enriched in the
egg or ectoderm tissue. This indicates that animal cap assays often do not
recapitulate endogenous endoderm development, possibly because animal cap
cells do not contain all the endogenous co-factors that normally interact with
the endoderm transcription factors.
The study we performed here also has limitations. We have not tested cases
where two or more factors are required redundantly to regulate gene
expression. Furthermore, we focused on genes with endoderm-enriched
expression, but clearly there will be genes that are not only expressed in the
endoderm that have crucial functions in endoderm development. In addition,
gene regulatory networks are known to evolve during developmental time
(Bolouri and Davidson, 2003;
Loose and Patient, 2004) and
so far we have only focused on stage 11. It is likely that Nodal proteins,
Mixer and Sox17 may have different functions at different times and in
different regions of the embryo (Clements
and Woodland, 2003; Yasuo and
Lemaire, 1999).
Even with these limitations, we believe that our global analysis adds
substantially to the emerging gene regulatory network describing
Xenopus mesendoderm formation
(Loose and Patient, 2004). In
addition to identifying much of the endoderm transcriptome, this work provides
an essential reference point from which future functional and epistatic
experiments can be devised. In the future, it will be important to identify
which regulatory events described here are directly controlled at the level of
transcription factors binding to promoter elements as opposed to secondary
events, which is an essential step establishing a robust gene regulatory
network.
Based on the data from this study, along with previously published reports, we propose that a `core' auto-regulatory network exists between the Nodal proteins, Mix-like, Gata4-6 and Sox17 factors, with the expression of any one component promoting the expression of the other components. This feed-forward system allows for the rapid establishment of an endoderm transcription profile in vegetal cells in the hours between activation of zygotic transcription at the early blastula to the gastrula stage, when endodermal fate is specified. Coupled with the repressive activity of Mixer and Mix1, such a system could also help establish both the endoderm and its boundary with the mesoderm.
We hypothesize that different species could initiate this conserved `core'
zygotic pathway by different means producing a similar outcome. In
Xenopus, the core pathway is activated by maternal VegT, while in
mouse and zebrafish the pathway may be activated at the level of Nodal
proteins by some unknown mechanism (Tam et
al., 2003). Indeed, a comparison of our data with a transcription
profile the mouse gastrula endoderm (Gu et
al., 2004) identified a number of common genes, suggesting that
the global regulation of endoderm gene expression may be conserved.
We believe this is the first global analysis of the conserved molecular pathway controlling vertebrate endoderm formation during gastrulation. Our data challenge many aspects of existing models of vertebrate endoderm development and provide an important resource for further studies of the complex gene regulatory network controlling Xenopus endoderm development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/1955/DC1
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