© 2013. Published by The Company of Biologists Ltd | Development (2013) 140, 4903-4913 doi:10.1242/dev.098319
RESEARCH ARTICLE
The Xenopus homologue of Down syndrome critical region
protein 6 drives dorsoanterior gene expression and embryonic
axis formation by antagonising polycomb group proteins
ABSTRACT
Mesoderm and embryonic axis formation in vertebrates is mediated
by maternal and zygotic factors that activate the expression of target
genes. Transcriptional derepression plays an important role in the
regulation of expression in different contexts; however, its
involvement and possible mechanism in mesoderm and embryonic
axis formation are largely unknown. Here we demonstrate that
XDSCR6, a Xenopus homologue of human Down syndrome critical
region protein 6 (DSCR6, or RIPPLY3), regulates mesoderm and
embryonic axis formation through derepression of polycomb group
(PcG) proteins. Xdscr6 maternal mRNA is enriched in the endoderm
of the early gastrula and potently triggers the formation of dorsal
mesoderm and neural tissues in ectoderm explants; it also dorsalises
ventral mesoderm during gastrulation and induces a secondary
embryonic axis. A WRPW motif, which is present in all DSCR6
homologues, is necessary and sufficient for the dorsal mesodermand axis-inducing activity. Knockdown of Xdscr6 inhibits dorsal
mesoderm gene expression and results in head deficiency. We
further show that XDSCR6 physically interacts with PcG proteins
through the WRPW motif, preventing the formation of PcG bodies
and antagonising their repressor activity in embryonic axis formation.
By chromatin immunoprecipitation, we demonstrate that XDSCR6
releases PcG proteins from chromatin and allows dorsal mesoderm
gene transcription. Our studies suggest that XDSCR6 might function
to sequester PcG proteins and identify a novel derepression
mechanism implicated in embryonic induction and axis formation.
KEY WORDS: Transcriptional derepression, Embryonic axis
formation, Mesoderm induction, Down syndrome, Polycomb group
proteins, Xenopus
INTRODUCTION
During amphibian early development, mesoderm is formed through
inductive signals emanating from endoderm cells of the vegetal
hemisphere. The molecular basis of this inductive event has been
extensively studied and now partly elucidated. Several conserved
signalling pathways, including nodal, BMP, Wnt and FGF, are
involved in mesoderm induction and patterning (Heasman, 2006;
Kimelman, 2006). They act as morphogens to exert long-range
effects on responding cells by activating the expression of various
target genes (Smith, 2009). Subsequently, the dorsoventral
1
Laboratory of Developmental Biology, CNRS UMR 7622, University Pierre et
Marie Curie, 9 quai Saint-Bernard, 75005 Paris, France. 2Department of Marine
Biology, Ocean University of China, Qingdao 266003, China.
*Authors for correspondence ([email protected];
[email protected])
Received 26 April 2013; Accepted 18 September 2013
patterning of mesoderm is largely mediated by factors expressed in
the Spemann’s organizer, which is a source of secreted antagonists
for zygotic BMP and Wnt signals (De Robertis et al., 2000; De
Robertis and Kuroda, 2004; Niehrs, 2004).
The polycomb group (PcG) proteins are conserved chromatininteracting factors that are essential for development. They are
transcriptional repressors required for maintaining the correct spatial
and temporal expression of key developmental genes (Kerppola,
2009; Sawarkar and Paro, 2010). The PcG proteins form multiprotein
repressive complexes (polycomb repressive complexes, PRCs), which
repress transcription by a mechanism that involves the modification
of chromatin (Bracken and Helin, 2009). It has been shown that PcG
proteins are concentrated in nuclear foci called PcG bodies, which
contribute to epigenetic silencing of Hox genes in Drosophila
(Bantignies et al., 2011; Pirrotta and Li, 2012). Removal of PcG
proteins in different tissues of vertebrates leads to the transcriptional
activation of genes that are normally repressed (Pietersen et al., 2008;
Ezhkova et al., 2011; Juan et al., 2011). In the Xenopus embryo,
misexpression of PcG proteins alters the expression of anteroposterior
neural genes and produces anterior deficiency (Yoshitake et al., 1999;
Barnett et al., 2001). Thus, the PcG system allows context-dependent
gene expression and an appreciation of its control of the balance
between repression and derepression is crucial in understanding
developmental gene regulation; however, the mechanism of
derepression by PcG proteins is hitherto unclear.
Down syndrome is one of the most frequent human birth defects,
occurring in 1 out of 600 to 1000 births. Phenotypic and molecular
analyses of patients have identified a region of chromosome 21,
called the Down syndrome critical region (DSCR), which is
responsible for many of the characteristic features of Down
syndrome when present in three copies (Delabar et al., 1993).
However, the function of most of the genes within this region in key
early developmental events remains largely unexplored.
Here we report the characterisation of the Xenopus DSCR6
homologue (XDSCR6), which is member of the RIPPLY family
proteins (DSCR6 is also known as RIPPLY3). We found that Xdscr6
is a maternal mRNA that is enriched in the endoderm of the early
gastrula and functions as a novel and distinct mesoderm and axis
inducer. It potently triggers the formation of dorsal mesoderm and
neural tissues in ectoderm explants. XDSCR6 also possesses
dorsalising activity and can induce a secondary axis. Knockdown of
Xdscr6 suggests that it is required for anterior development. We also
show that a WRPW motif, which is present in all RIPPLY family
proteins, is necessary and sufficient for these activities. Furthermore,
we find that XDSCR6 physically and functionally interacts with
PcG proteins through the WRPW motif, preventing their
accumulation in PcG bodies, releasing them from chromatin, and
counteracting their repressive activity. Our results thus identify a
novel derepression mechanism and demonstrate that XDSCR6 is an
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Hong-Yan Li1,2, Raphaëlle Grifone1, Audrey Saquet1, Clémence Carron1,* and De-Li Shi1,*
endogenous inducer for dorsal mesoderm and the embryonic axis.
These findings should also help in understanding the biological
function and activity of the DSCR6 gene in early development and
in the pathogenesis of Down syndrome.
RESULTS
XDSCR6 triggers the formation of dorsal mesoderm and
neural tissues
XDSCR6 is a member of the RIPPLY family of proteins known to
be involved in somite segmentation (Wardle and Papaioannou,
2008); however, whether it plays an early role in development is not
clear. RT-PCR analyses indicated that Xdscr6 is a maternal mRNA
that is expressed at a constant level from fertilisation until midgastrula stage (supplementary material Fig. S1A). In the early
gastrula, Xdscr6 transcripts are enriched in the endoderm
(supplementary material Fig. S1B,C), and its expression could be
induced in the ectoderm by nodal signalling, but not by Wnt
signalling (supplementary material Fig. S1D). This suggests that
Xdscr6 might play an early role during development. We thus
performed functional analyses by injecting Xdscr6 mRNA (0.2 ng)
into the animal pole region and culturing ectoderm explants to
different stages. At the early neurula stage equivalent, uninjected
explants remained rounded (Fig. 1A), whereas Xdscr6-injected
explants exhibited extensive elongation, which is characteristic of
dorsal mesoderm formation (Fig. 1B). At the late neurula stage
equivalent, in situ hybridisation analyses showed that Xdscr6injected explants expressed the muscle-specific myosin light chain
(MLC) gene, the cement gland marker XCG1 and the neuroectoderm
gene Sox3 (Fig. 1C-H). This shows that overexpression of XDSCR6
triggered the formation of dorsal mesoderm and neural tissues.
Development (2013) doi:10.1242/dev.098319
We then injected different amounts (0.1, 0.4 and 1 ng) of Xdscr6
mRNA into the animal pole region and cultured ectoderm explants
to the early gastrula and the late neurula stage equivalent to further
analyse its inducing activity. At the early gastrula stage, Xdscr6 at
both low and high doses potently induced the expression of
dorsoventral mesoderm genes, but not the endoderm gene Sox17α
or the Wnt/β-catenin target gene Siamois (Fig. 1I). However, Sox17α
expression was induced in ectoderm explants treated with activin (1
ng). Injection of Xdscr6 also induced the expression of Xnr1, Xnr2
and Xnr6, but not Xnr4 or Xnr5 (supplementary material Fig. S2).
Consistent with the induction of dorsal mesoderm genes at the early
gastrula stage, ectoderm explants injected with high doses of Xdscr6
mRNA (0.4 or 1 ng) and cultured to the late neurula stage equivalent
expressed somitic mesoderm genes including muscle actin and
MyoD, as well as various anteroposterior neuroectoderm genes (Fig.
1I). Real-time RT-PCR analyses on early gastrula ectoderm explants
injected with low (0.2 ng) and high (1 ng) amounts of Xdscr6
mRNA indicated a dose-dependent induction of mesoderm, but not
endoderm, gene expression. In comparison, ectoderm explants
treated with low (0.2 ng/ml) and high (1 ng/ml) doses of activin
exhibited a dose-dependent induction for both mesoderm and
endoderm genes (Fig. 1J). These analyses suggest that XDSCR6
induces only mesoderm gene expression at the early gastrula stage.
Since mesoderm induction and patterning depend on both
maternal and zygotic factors (Kofron et al., 1999), we tested the
activity of zygotic Xdscr6 in mesoderm and neural induction by
injecting Xdscr6 DNA, which allows the transcription of Xdscr6
mRNA under the control of the CMV promoter to occur only after
the mid-blastula transition (Rupp and Weintraub, 1991). As shown
in Fig. 1I, injection of Xdscr6 DNA (0.4 ng) also induced, although
Fig. 1. XDSCR6 triggers the formation of dorsal mesoderm and neural tissues in Xenopus ectoderm explants. (A,B) Morphology of control (A) and
Xdscr6-injected (B) explants at the early neurula stage equivalent. (C-H) In situ hybridisation analyses of the expression of MLC, XCG1 and Sox3 genes in
control (C,E,G) and Xdscr6-injected (D,F,H) ectoderm explants at the late neurula stage equivalent. (I) RT-PCR analyses of gene expression in ectoderm
explants cultured to stage 10.5 and 20 equivalent, following injection of the indicated amounts of Xdscr6 mRNA, Xdscr6 DNA or after activin treatment. ODC
was used as a loading control. Ms., muscle specific. (J) Real-time RT-PCR analyses of the expression level of the indicated genes in early gastrula ectoderm
explants injected with low and high amounts of Xdscr6 mRNA (top row) or treated with low and high doses of activin (bottom row). Similar results were obtained
using ODC or EF1α as reference.
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weakly, the expression of dorsal mesoderm genes in ectoderm
explants at the early gastrula stage and of neural genes at the late
neurula stage. This result indicates that XDSCR6 is also able to
induce dorsal mesoderm gene expression when expressed after
zygotic transcription.
XDSCR6 induces a secondary embryonic axis
The activity of XDSCR6 in mesoderm and neural induction led us
to examine whether it induces the formation of an embryonic axis.
We first injected Xdscr6 mRNA (0.4 ng) in the ventro-vegetal region
of the 4-cell stage embryo (Fig. 2A); this dose was sufficient to
induce the expression of most dorsal mesoderm genes (see Fig. 1I).
When injected embryos developed to the early gastrula stage, the
formation of a precocious ventral blastopore was apparent (Fig.
2B,C). At the late neurula stage, an ectopic axis became evident
(Fig. 2D,E). At the late tail-bud stage, Xdscr6-injected embryos
formed a secondary axis with essentially trunk and posterior regions
(Fig. 2F,G), although anterior structure including cement gland and
eyes was observed in 9% of the injected embryos (supplementary
material Table S3). Injection of a high dose of Xdscr6 mRNA (1 ng)
did not further change the percentage or character of the secondary
axis (not shown). Ventro-animal injection of Xdscr6 mRNA also
induced a secondary axis, although less frequently. In addition,
ventro-vegetal injection of Xdscr6 DNA (0.4 ng) induced a
secondary axis with high frequency (Fig. 2H; supplementary
material Table S3). We then co-injected lacZ mRNA (0.5 ng) into
one ventral blastomere at the 4-cell stage to analyse how Xdscr6
induces a secondary axis from three independent experiments. When
lacZ mRNA was injected alone, β-galactosidase-labelled cells were
localised to the posterior and ventral region of the embryo at the late
tail-bud stage (Fig. 2I; 100%, n=48). However, in embryos coinjected with Xdscr6 and lacZ mRNAs, β-galactosidase-labelled
cells were mostly localised to the anterior region of the secondary
axis (Fig. 2J; 97%, n=64) and formed a mass of muscle (Fig. 2K),
indicating that Xdscr6 had transformed ventral and posterior cells
into dorsal cells. In situ hybridisation analyses indicated that the
secondary axis expressed the muscle marker MLC (Fig. 2L; 100%,
Development (2013) doi:10.1242/dev.098319
n=47), the forebrain marker Otx2 (Fig. 2M; 67%, n=49) and the
spinal cord marker HoxB9 (Fig. 2N; 100%, n=58) but not the
notochord marker keratin 8 (Fig. 2O; 98%, n=55). Taken together,
these results show that XDSCR6 can induce a secondary axis with
somites and neural tissues.
Although human RIPPLY proteins show relatively low overall
identity (35-40%) with XDSCR6, they similarly induced mesoderm
gene expression and a secondary axis (supplementary material Fig.
S3). Furthermore, an amphioxus DSCR6 homologue (Li et al.,
2006b) could also induce a secondary axis (data not shown). These
observations suggest that RIPPLY family proteins from different
species exhibit similar activity when overexpressed in the Xenopus
embryo.
XDSCR6 possesses dorsalising and neuralising activity
We further characterised the activity of XDSCR6 in mesoderm
patterning. Ventral injection of Xdscr6 mRNA (0.4 ng) at the 4-cell
stage induced ectopic expression of Otx2 and chordin (Fig. 3A-D)
and inhibited the expression of Wnt8 and Xvent1 (Fig. 3E-H) at the
early gastrula stage, indicating that it could dorsalise ventral
mesoderm. To directly test its dorsalising activity, Xdscr6 mRNA or
DNA (0.4 ng) was injected into the two ventral blastomeres at the
4-cell stage and dissected ventral marginal zone (VMZ) was
cultured to the late neurula stage equivalent in order to examine
phenotypic and molecular changes (Fig. 3I). Dorsal marginal zone
(DMZ) explants from uninjected embryos undergo elongation (Fig.
3J), which mimics gastrulation movements. However, VMZ
explants from uninjected embryos remained rounded (Fig. 3K). By
contrast, VMZ explants injected with Xdscr6 mRNA undergo
extensive elongation as DMZ explants (Fig. 3L). VMZ explants
injected with Xdscr6 DNA also elongate, albeit to a lesser extent
(Fig. 3M). RT-PCR analyses indicated that injection of Xdscr6
mRNA or DNA induced ventral mesoderm to express dorsal
mesoderm genes including muscle actin, Myf5 and MyoD, as well
as the anterior neuroectoderm gene Otx2 and cement gland marker
XCG1 (Fig. 3N). Furthermore, radial injection of Xdscr6 mRNA in
the equatorial region at the 8-cell stage produced embryos with trunk
Fig. 2. XDSCR6 induces a secondary embryonic
axis. (A) Xdscr6 mRNA or DNA was injected into one
ventral blastomere (arrow) at the 4-cell stage.
(B,C) Vegetal view of an uninjected early gastrula
embryo (B) and a Xdscr6 mRNA-injected early
gastrula embryo showing the precocious formation of
the ventral blastopore (C, arrow). (D,E) Dorsal view,
with anterior region at the top, of an uninjected late
neurula embryo (D) and a Xdscr6 mRNA-injected late
neurula embryo with a secondary neural tube (E). (FH) Phenotypes of control (F), Xdscr6 mRNA-injected
(G) and Xdscr6 DNA-injected (H) embryos at stage 35.
(I,J) β-galactosidase staining of stage 32 embryos
injected with lacZ mRNA alone (I) or co-injected with
Xdscr6 mRNA (J). (K) Transverse section
corresponding to J showing β-galactosidase staining in
XDSCR6-induced muscles. (L-O) In situ hybridisation
analyses of the expression of the indicated genes in
Xdscr6 mRNA-injected embryos at stage 32. I and II
designate the primary axis and the secondary axis,
respectively.
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Development (2013) doi:10.1242/dev.098319
late neurula stage. However, expression of the cement gland marker
XCG1, which correlates with neural induction, was detected (Fig. 3O;
see also Fig. 1I). This suggests that XDSCR6 could trigger the
formation of neural tissue in the absence of muscle, although this
activity might be achieved by the early expression of chordin.
Xdscr6 knockdown affects anterior development
Since XDSCR6 possesses the activity to induce dorsal mesoderm
and a secondary embryonic axis, we then examined whether it is
required for these processes. We dorsally injected antisense MOs
against Xdscr6 into 4-cell stage embryos and analysed the
expression of mesoderm and neuroectoderm genes. We assayed
three MOs (MO1, MO2 and MO3) targeting the 5′ untranslated
region through the first 25 bases of coding sequence. Since MO1
was more efficient than MO2 and MO3 (data not shown), we used
MO1, hereafter designated Xdscr6MO, in the following
experiments. At the early gastrula stage, in situ hybridisation
analyses revealed that injection of Xdscr6MO (40 ng)
downregulated the expression of chordin, noggin and goosecoid
(Fig. 4A-F), indicating that Xdscr6 knockdown affects dorsal
mesoderm development. At the end of neurulation, Xdscr6
morphants exhibited reduced expression of Otx2, XCG1 and Sox3
(Fig. 4G-L). As development proceeds, injected embryos showed
anterior deficiency characterised by the absence of head structure
(Fig. 4M,N). These effects could be rescued by co-injection of mycXdscr6 mRNA (0.1 ng), which is not targeted by Xdscr6MO (Fig.
4O,P). Western blot analyses of early gastrula embryos previously
co-injected at the 4-cell stage with Xdscr6MO, Xdscr6-Flag mRNA
(0.2 ng) and myc-Xdscr6 mRNA (0.1 ng), showed that Xdscr6MO
strongly inhibited protein synthesis from Xdscr6-Flag mRNA,
which possesses the MO targeting sequence, whereas it had no
effect on protein synthesis from myc-Xdscr6 mRNA (Fig. 4Q),
indicating that the targeting effect of Xdscr6MO should be specific.
We conclude that XDSCR6 is required for dorsal mesoderm
formation and anterior development.
Fig. 3. Dorsalisation of ventral mesoderm by XDSCR6. (A-H) In situ
hybridisation analyses of Otx2, chordin, Wnt8 and Xvent1 expression in
uninjected (A,C,E,G) and Xdscr6-injected (B,D,F,H) embryos at the early
gastrula stage. Ectopic Otx2 and chordin expression sites are indicated by
arrowheads. (I) Scheme of the dorsalisation experiment. Embryos were
ventrally injected at the 4-cell stage (double arrows) and DMZ (D) or VMZ (V)
explants were dissected at the early gastrula stage and cultured to late
neurula stage equivalent. (J-M) Morphology of the uninjected and injected
DMZ or VMZ explants. (N) RT-PCR analyses of gene expression in
uninjected and injected DMZ or VMZ explants. (O) In situ hybridisation of
MLC and XCG1 expression in ectoderm explants injected with the mRNAs
indicated at the top and cultured to late neurula stage equivalent. DMZ,
dorsal marginal zone; VMZ, ventral marginal zone.
and posterior deficiency (supplementary material Fig. S4). These
observations clearly indicate that XDSCR6 could dorsalise ventral
mesoderm during gastrulation.
To examine whether XDSCR6 could initiate neural induction in the
absence of dorsal mesoderm, we injected a low amount of Xdscr6
mRNA (0.1 ng) into the animal pole region along with VegT-EnR
mRNA (50 pg) or antivin mRNA (20 pg). Although RT-PCR analyses
on dissected early gastrula ectoderm explants indicated that neither
VegT-EnR nor antivin blocked the mesoderm-inducing activity of
XDSCR6 (supplementary material Fig. S5), muscle was absent at the
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The WRPW motif confers XDSCR6 activity
RIPPLY family proteins have no clearly defined functional
domain, except for a conserved WRPW tetrapeptide in the Nterminal region (supplementary material Fig. S3A). We
overexpressed various Xdscr6 mutants (Fig. 5A) by injecting the
corresponding mRNA (0.4 ng) into one ventro-vegetal blastomere
at the 4-cell stage to investigate the structure-function relationship
of XDSCR6 protein in axis induction. The results unambiguously
showed that, similar to the wild-type protein, the N-terminal half
of XDSCR6 (XDSCR6N-Flag) induced the formation of a
secondary axis, whereas the C-terminal half of XDSCR6
(XDSCR6C-Flag) and a point mutant (XDSCR6m-Flag), which
transforms WRPW into WRSG, had no axis-inducing activity (Fig.
5B). RT-PCR analyses indicated that wild-type XDSCR6 and
XDSCR6N induced Otx2, chordin and Xbra expression in
ectoderm explants, whereas XDSCR6C and XDSCR6m had no
effect (supplementary material Fig. S6). This indicates that the Nterminal half, in particular the WRPW tetrapeptide, is required for
XDSCR6 function.
Since XDSCR6-related proteins were shown to function as a
repressor during somitogenesis through physical interaction with
Groucho-related proteins (Kawamura et al., 2005; Kondow et al.,
2006), we ventrally injected Xdscr6-EnR or Xdscr6-VP16 mRNA
(0.2 ng) to examine whether the activity of XDSCR6 in mesoderm
induction and axis formation is related to a repressor function.
Phenotypic analyses showed that both XDSCR6-EnR and
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Development (2013) doi:10.1242/dev.098319
XDSCR6-VP16 induced the formation of a secondary axis (Fig.
5B). Furthermore, injection of myc-WRPW mRNA (0.5 ng) was also
sufficient to induce the formation of a secondary axis (Fig. 5B). To
exclude the possibility that myc tags might behave as an activation
domain for transcription (Ferreiro et al., 1998), we injected GFPWRPW mRNA (0.5 ng) and found that it similarly induced a
secondary axis (Fig. 5B,C). However, injection of myc-WRSG or
GFP-WRSG mRNA (0.5 ng) had no effect (Fig. 5B,D). Western blot
analyses indicated that wild-type and mutated proteins were
expressed at a similar level (data not shown). In situ hybridisation
analyses showed that XDSCR6-EnR, XDSCR6-VP16 and mycWRPW similarly induced ectopic chordin expression (Fig. 5E).
These analyses highlight the importance of the WRPW motif in
dorsal mesoderm and axis induction and suggest that the axisinducing activity of XDSCR6 is independent of a repressor or
activator function.
XDSCR6 physically interacts with PcG proteins
Although the WRPW motif is generally involved in interaction with
Groucho-related proteins (Fisher et al., 1996), we found that the
mesoderm- and axis-inducing activity of XDSCR6 was not
influenced by co-expression of the Groucho homologue XGrg4
[Xenopus groucho-related gene 4; also known as transducin-like
enhancer of split 4 (Tle4)], or by inhibition of XGrg4 using an MO
or the naturally truncated XGrg5, a dominant inhibitory Groucho
protein (data not shown). Thus, interaction between XDSCR6 and
Groucho-related proteins cannot be the mechanism underlying its
mesoderm- and axis-inducing activity. We therefore performed a
yeast two-hybrid screen in order to identify novel partners of
XDSCR6 and attempt to provide a mechanism of XDSCR6 in
mesoderm induction. By this approach, we identified enhancer of
zeste homologue 2 (Ezh2). GST pull-down was used to confirm the
physical interaction. Xenopus embryos at the 4-cell stage were
injected with synthetic mRNA (0.5 ng) encoding XEzh2-myc, or
XGrg4-myc as a positive control, and embryo extracts were
prepared at the late gastrula stage. The results indicated that both
XEzh2-myc and XGrg4-myc bound to GST-XDSCR6 (Fig. 6A).
Co-immunoprecipitation further confirmed the interaction between
XDSCR6 and XEzh2 (Fig. 6B).
Ezh2 is a component of PcG protein complex PRC2. We tested
whether XDSCR6 also interacts with the core PRC1 component
BMI1, which was not isolated in the yeast two-hybrid screen. Both
GST pull-down and co-immunoprecipitation showed that XDSCR6,
but not XDSCR6m, interacts with BMI1 (Fig. 6C,D). GST pull4907
Development
Fig. 4. XDSCR6 is required for anterior development.
(A,B) Dorso-vegetal view of chordin expression in a CoMO (A)
and a Xdscr6MO-injected (B) embryo at stage 11. (C,D) Dorsovegetal view of noggin expression in a CoMO (C) and a
Xdscr6MO-injected (D) embryo at stage 10.5. (E,F) Dorsovegetal view of goosecoid expression in a CoMO (E) and a
Xdscr6MO-injected (F) embryo at stage 10. (G,H) Anterior view
of Otx2 expression in a CoMo (G) and a Xdscr6MO-injected (H)
embryo at stage 20. (I,J) Ventral view, with anterior region at
the top, of XCG1 expression in a CoMO (I) and a Xdscr6MOinjected (J) embryo at stage 20. (K,L) Dorsal view, with anterior
region at the top, of Sox3 expression in a CoMO (K) and a
Xdscr6MO-injected (L) embryo at stage 18. (M-O) Phenotypes
of a CoMo-injected embryo (M), a Xdscr6 morphant embryo (N)
and a myc-Xdscr6 rescued embryo (O) at stage 38.
(P) Summary of the rescue results. Numbers at the top indicate
total embryos scored from four independent experiments.
(Q) Western blot analysis shows the blockade of protein
synthesis from Xdscr6-Flag mRNA, but not from myc-Xdscr6
mRNA, by Xdscr6MO. Tubulin is a loading control. CoMO,
mismatch control MO.
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XDSCR6 disrupts the formation of PcG bodies and releases
PcG proteins from chromatin
down using in vitro translated proteins further demonstrated a direct
interaction between XDSCR6, XEzh2 and BMI1 (Fig. 6E,F). Taken
together, these results strongly suggest that Ezh2 and BMI1 are
novel partners of XDSCR6 and that the WRPW motif is involved in
the physical interaction.
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XDSCR6 and PcG proteins mutually antagonise in
mesoderm and axis formation
To understand the functional significance of this antagonism
during development, we co-expressed BMI1 with XDSCR6 in
Xenopus embryos. Dorsal injection of Xdscr6 mRNA (0.2 ng)
alone had no significant effect and most embryos (87%) developed
normally (Fig. 9A,B; supplementary material Table S4). By
contrast, dorsal injection of BMI1 mRNA (0.5 ng) alone produced
anterior deficiency in 70% of injected embryos (Fig. 9C;
supplementary material Table S4). In BMI1 and Xdscr6 co-injected
Development
Fig. 5. The WRPW motif is necessary and sufficient to induce a
secondary axis. (A) Structure of the various Xdscr6 constructs.
(B) Summary of secondary axis formation in control (Ctl) and injected
embryos (constructs numbered as in A). XDSCR6 (1), XDSCR6N (2),
XDSCR6-EnR (5), XDSCR6-VP16 (6), myc-WRPW (7) and GFP-WRPW (9)
similarly induce a secondary axis, whereas XDSCR6C (3), XDSCR6m (4),
myc-WRSG (8) and GFP-WRSG (10) have no effect. The results are
expressed as a percentage and numbers at the top indicate total embryos
scored from three independent experiments. (C) A GFP-WRPW-injected
stage 35 embryo with green fluorescence concentrated in the anterior region
of the secondary axis (arrow). (D) A GFP-WRSG-injected embryo lacks a
secondary axis, with green fluorescence distributed in the posterior region.
(E) In situ hybridisation analyses of chordin expression at stage 10.5
following overexpression of XDSCR6-EnR, XDSCR6-VP16 or myc-WRPW
in the ventro-vegetal region at the 4-cell stage. All embryos are in vegetal
view.
PcG proteins accumulate at pericentric heterochromatin as discrete
loci called PcG bodies, which contribute to epigenetic gene silencing
(Hernández-Muñoz et al., 2005; Bantignies et al., 2011). To explore
the functional significance of the biochemical interaction with Ezh2
and BMI1, we examined whether XDSCR6 can influence the
subcellular localisation of endogenous PcG proteins. Nuclear
localisation and fluorescence intensity of endogenous Ezh2 and
BMI1 in HeLa cells transfected with Xdscr6-RFP (250 ng),
Xdscr6m-RFP (250 ng) or myc-WRPW (250 ng) were analysed by
confocal microscopy. In untransfected cells (arrows in Fig. 7), Ezh2
and BMI1 exhibited a punctate nuclear distribution as previously
described in this cell line (Saurin et al., 1998; Voncken et al., 1999).
However, the nuclear fluorescence intensity for both Ezh2 and
BMI1 was significantly reduced in Xdscr6-RFP- or myc-WRPWtransfected cells (arrowheads, Fig. 7A-C,I-K,M-O,U-W). By
contrast, transfection of Xdscr6m-RFP did not affect the nuclear
staining of Ezh2 and BMI1 (arrowheads, Fig. 7E-G,Q-S),
highlighting the importance of the WRPW motif in interaction with
Ezh2 and BMI1. Quantification of nuclear fluorescence intensity
confirmed these results (Fig. 7D,H,L,P,T,X). Because PcG bodies
represent the site of accumulation of PcG complexes and hence the
site of transcriptional repression (Bantignies et al., 2011; Hodgson
and Brock, 2011), this observation suggests that XDSCR6 might
prevent the targeting of PcG proteins to chromatin and thus avert
their transcriptional repression activity.
We then performed ChIP experiments to test this issue directly. We
chose to examine the goosecoid promoter, which was activated by
XDSCR6 in ectoderm explants. One-cell stage embryos were injected
in the animal pole region with Xdscr6 mRNA (0.5 ng) and ectoderm
explants were cultured until stage 11. Chromatin from uninjected and
injected ectoderm explants was immunoprecipitated and analysed by
semi-quantitative and quantitative PCR. The results showed that, in
the absence of XDSCR6, a −390/–167 (relative to transcription start
site at +1) goosecoid promoter region was amplified from chromatin
precipitated by anti-Ezh2 and anti-BMI1 antibodies. However, in
Xdscr6-injected explants, this region was less efficiently amplified
(Fig. 8A-C). As a specificity control, no goosecoid promoter region
was amplified from chromatin immunoprecipitated by an anti-GFP
antibody, either in the absence or presence of XDSCR6 (Fig. 8A,D).
This indicates that XDSCR6 specifically released Ezh2 and BMI1
from the goosecoid promoter, a region that also exhibits an increased
acetylated histone H4 mark, reflecting an opened state of chromatin
in this location (Shogren-Knaak et al., 2006). As a result, the
goosecoid promoter could be amplified from chromatin precipitated
by the anti-acetylated histone H4 antibody (Fig. 8A,E). These
observations strongly suggest that the activation of mesoderm gene
expression by XDSCR6 results from a derepression mechanism,
which is achieved through the dissociation of PcG proteins from
chromatin.
RESEARCH ARTICLE
Development (2013) doi:10.1242/dev.098319
Fig. 6. XDSCR6 physically interacts with PcG proteins. (A) GST pull-down of embryonically expressed XEzh2-myc or XGrg4-myc by GST-XDSCR6.
(B) Co-immunoprecipitation of XEzh2-myc by XDSCR6-Flag. (C) GST pull-down of embryonically expressed XDSCR6-myc, but not the mutant XDSCR6mmyc, by GST-BMI1. (D) Co-immunoprecipitation of XDSCR6-myc by BMI1-Flag. (E) GST pull-down of in vitro translated XDSCR6-myc, but not the mutant
XDSCR6m-myc, by GST-XEzh2 or GST-BMI1. (F) GST pull-down of in vitro translated BMI1-GFP or XEzh2-myc by GST-XDSCR6. IP, immunoprecipitation;
WB, western blot.
DISCUSSION
This study has uncovered a novel derepression mechanism
underlying dorsal mesoderm and embryonic axis formation during
Xenopus early development. We show that XDSCR6, through
interaction with PcG proteins, removes them from chromatin and
triggers the expression of dorsoanterior genes, which then repress
the expression of ventral genes.
XDSCR6 is a novel dorsal mesoderm and axis inducer
The activity of XDSCR6 in mesoderm and secondary axis induction
presents several characteristic features. First, both maternal and
zygotic XDSCR6 potently induces the expression of dorsal
mesoderm genes, including several Xnr members. However, the
activity of XDSCR6 to initiate the formation of mesoderm and
neural tissue is unaffected in the presence of antivin, implying that
it might function independently of nodal signalling. Second,
XDSCR6 does not have endoderm-inducing activity even at high
concentration, whereas other mesoderm-inducing factors, including
activin and XNR proteins, can induce both endoderm and mesoderm
(Green et al., 1992; Osada and Wright, 1999) (this study). Third, in
the absence of muscle, XDSCR6 potently triggers the formation of
both anterior and posterior neural tissues. Finally, XDSCR6expressing cells participate in the formation of the secondary axis,
which lacks a notochord.
The ability of XDSCR6 to induce a secondary axis may be
attributed to its dorsal mesoderm-inducing and/or dorsalising
activity during gastrulation. Although ventral gene expression could
be detected in Xdscr6-injected ectoderm explants, whether this is
direct or a consequence of mesoderm formation remains to be
determined. In situ hybridisation analyses clearly showed that
ventral overexpression of XDSCR6 dorsalises ventral mesoderm
through ectopic induction of dorsoanterior genes and
downregulation of ventral genes including Wnt8 and Xvent1. Thus,
the induction of dorsal genes and the repression of ventral genes
trigger the formation of a secondary axis. Our findings suggest that
XDSCR6 represents a novel mesoderm and embryonic axis inducer
with both distinct and shared activity with respect to previously
identified mesoderm-inducing factors.
A WRPW motif confers the mesoderm- and axis-inducing
activity of XDSCR6
XDSCR6 belongs to the RIPPLY family, which have no conserved
functional domain except for a WRPW motif in the N-terminal half.
This motif was shown to interact with Groucho-related proteins during
somitogenesis (Kawamura et al., 2005; Kondow et al., 2006);
however, we find that neither overexpression nor knockdown of
XGrg4 affects the activity of XDSCR6 in mesoderm induction and
axis formation. This indicates that XDSCR6 and XGrg4 have distinct
activity in mesoderm induction. Most strikingly, we found that the
WRPW motif is strictly required and sufficient for the dorsal
mesoderm- and axis-inducing activity of XDSCR6, whereas the Cterminal half is totally dispensable. By contrast, the C-terminal region
is required for interaction with Tbx24 during somitogenesis
(Kawamura et al., 2008). Furthermore, we find that the axis-inducing
activity of XDSCR6 is independent of an activator or repressor
function. Together, these differences imply that RIPPLY family
proteins regulate mesoderm induction and somitogenesis through
distinct mechanisms.
The observation that a WRPW motif alone could function as an
axis inducer raises the question of whether other proteins with this
motif have similar activity. It is likely that such proteins, to some
4909
Development
embryos, 76% of embryos were morphologically normal (Fig. 9D;
supplementary material Table S4), indicating that XDSCR6
counteracts the activity of BMI1 during anterior development.
Conversely, ventral injection of Xdscr6 mRNA (0.1 ng) induced a
secondary axis in 66% of injected embryos (Fig. 9E,F;
supplementary material Table S5), whereas ventral injection of
BMI1 mRNA (0.5 ng) had no effect (Fig. 9G; supplementary
material Table S5). Co-injection of BMI1 mRNA with Xdscr6
mRNA reduced the prevalence of a secondary axis to 36% (Fig.
9H; supplementary material Table S5), which correlated with a
blockade of ectopic chordin expression induced by XDSCR6
(supplementary material Table S6, Fig. S7). In accordance with
these in vivo observations, in vitro analyses showed that XDSCR6
strongly activated a −1500/+3 goosecoid and a −907/–5 Xnr1
promoter-luciferase reporter gene, whereas these were repressed
by co-expression of BMI1 (Fig. 9I,J). Altogether, these results
suggest that XDSCR6 releases PcG proteins from chromatin (Fig.
9K) and thus contributes to transcriptional derepression.
RESEARCH ARTICLE
Development (2013) doi:10.1242/dev.098319
Fig. 7. XDSCR6 interferes with the nuclear localisation of endogenous Ezh2 and BMI1. Confocal microscopy analyses of Ezh2- and BMI1-associated
PcG bodies in HeLa cells. In cells transfected with Xdscr6-RFP or myc-WRPW (arrowheads in A-C,M-O,I-K,U-W), the intensity of endogenous Ezh2 and BMI1
staining is significantly decreased compared with neighbouring untransfected cells in the same culture dish (arrows). In Xdscr6m-RFP-transfected cells
(arrowheads in E-G,Q-S), the intensity of fluorescence is comparable to that of neighbouring untransfected cells (arrows). Scale bar: 10 μm. (D,H,L,P,T,X)
Quantification of nuclear fluorescence intensity. Red lines represent the median for each condition. Data are presented as mean ± s.e.m. P-values (Student’s
t-test) and number of measures (n) are indicated.
XDSCR6 counteracts the repressor activity of PcG proteins
We also demonstrated that the WRPW motif is indispensable for
physical and functional interactions with protein components
within both PRC1 and PRC2, which are chromatin-associated
multiprotein complexes involved in the maintenance of stable gene
repression, especially during early development and stem cell fate
decision (Schwartz and Pirrotta, 2008; Kerppola, 2009; Sawarkar
and Paro, 2010; Bantignies et al., 2011; Ezhkova et al., 2011). In
Fig. 8. XDSCR6 releases endogenous Ezh2 and BMI1 from the goosecoid promoter. ChIP experiments using uninjected (uninj.) or Xdscr6-injected
Xenopus ectoderm explants. (A) A representative semi-quantitative PCR result. (B-E) Quantitative PCR analyses of the goosecoid promoter using chromatin
immunoprecipitated from uninjected or Xdscr6-injected ectoderm explants by the indicated antibodies. Data represent the mean of triplicate experiments (error
bars indicate s.d.).
4910
Development
extent, exhibit similar function to XDSCR6. For example, Hairyrelated proteins may function as dual transcriptional regulators
(activator and repressor) in embryonic axis formation (Murato et al.,
2006). In the ascidian embryo, Posterior end mark (PEM), which
has a WRPW motif and lacks a DNA-binding domain, plays a role
in anteroposterior patterning (Yoshida et al., 1996). Thus, WRPWcontaining proteins might regulate embryonic patterning with both
shared and distinct specificity.
RESEARCH ARTICLE
(Chen et al., 2009; Juan et al., 2011). BMI1 has been shown to
bind to a specific locus in pancreatic β-cells and decreased
binding leads to transcriptional derepression (Dhawan et al.,
2009). Thus, it is likely that XDSCR6 triggers mesoderm
induction by releasing PcG proteins from chromatin to allow
transcriptional activation of mesoderm genes.
In conclusion, the DSCR on chromosome 21 is associated with
specific features of Down syndrome that are likely to be caused by an
increased gene dosage (Pritchard et al., 2008). Functional analyses of
these genes should help to understand how they regulate key aspects
of embryogenesis and how their duplication leads to the pathogenesis
of Down syndrome. The finding that XDSCR6 antagonises the
repressor activity of PcG proteins in embryonic axis formation
suggests a novel mechanism of derepression during development.
MATERIALS AND METHODS
Embryo manipulations
Xenopus eggs were obtained from females injected with 500 IU of human
chorionic gonadotropin (Sigma) and artificially fertilised. For activin
treatment, ectoderm explants at the blastula stage were incubated in 0.2 to
1 ng/ml activin solution for 1 hour and then cultured to appropriate stages.
Plasmid constructs
The full-length Xdscr6 (AB073615) and human RIPPLY2 (NM_001009994)
sequences were PCR amplified and cloned into the pCS2 vector (Rupp and
Weintraub, 1991). Xdscr6-Flag, Xdscr6N-Flag and Xdscr6C-Flag were cloned
in the pCS2 vector, upstream of the sequence coding for the Flag epitope.
Fig. 9. XDSCR6 and BMI1 are mutually antagonistic during embryonic axis formation. (A-D) XDSCR6 rescues anterior deficiency produced by BMI1
overexpression. Embryos at the 4-cell stage were either left uninjected (A) or dorsally injected with Xdscr6 mRNA (B), BMI1 mRNA (C) or co-injected with BMI1
and Xdscr6 mRNAs (D), and cultured to the stage 32. (E-H) BMI1 inhibits the axis-inducing activity of XDSCR6. Embryos at the 4-cell stage were either left
uninjected (E) or ventrally injected with Xdscr6 mRNA (F), BMI1 mRNA (G) or co-injected with BMI1 and Xdscr6 mRNAs (H), and cultured to the stage 35.
(I,J) BMI1 represses the goosecoid promoter (gsc-luc) and Xnr1 promoter (Xnr1-luc) activity that had been stimulated by overexpression of XDSCR6 in
ectoderm explants. Values were expressed relative to the value obtained from uninjected control explants. Mean ± s.e.m.; P<0.05 (Student’s t-test). RLU,
relative luciferase units. (K) Model of XDSCR6 function in transcriptional derepression. (Top) In the absence of XDSCR6, PcG proteins bind to chromatin and
repress the transcription of mesoderm genes. (Bottom) The interaction between XDSCR6 and PcG proteins removes PRC1 and PRC2 from chromatin and
contributes to transcriptional derepression.
4911
Development
functional assays, XDSCR6 prevents the proper nuclear
localisation of both Ezh2 and BMI1 in PcG bodies, which
contribute to epigenetic silencing. Thus, XDSCR6 may regulate
embryonic induction and patterning through interference with
endogenous PcG protein function and derepression of dorsal
mesoderm genes.
At present, it is unclear how PcG protein-mediated
transcriptional silencing established during development is
reversed to allow gene expression. In Drosophila, it has been
demonstrated that testis-specific TATA box-binding protein
(TBP)-associated factors bind to target promoters, reduce binding
of PcG proteins and promote gene expression (Chen et al., 2005).
XDSCR6 might counteract PcG proteins through both a similar
and a distinct mechanism. Based on the interference of XDSCR6
in the formation of PcG bodies and on the binding of PcG
proteins to chromatin, we propose that a physical interaction
between XDSCR6 and PcG proteins may remove the latter from
target promoters, open the chromatin and thus promote gene
expression. In support of this hypothesis, it has been shown that
several key genes involved in mesoderm induction, including
nodal and brachyury, are targets of PcG proteins. In the absence
of PcG function, these genes are expressed independently of the
inductive signals (Dahle et al., 2010). Furthermore, there are
several lines of evidence showing that conditional knockout of
Ezh2 in different mouse tissues is accompanied by transcriptional
activation of genes that are normally repressed in these cells
Development (2013) doi:10.1242/dev.098319
RESEARCH ARTICLE
Morpholinos (MOs)
The MOs for Xdscr6 (MO1, 5′-AACTCCGCTGTTGATTCGGCTCCAT-3′;
MO2, 5′-CCGCAACTCCGCTGTTGATTCGGCT-3′; MO3, 5′-AGAACAGTTCGGCCGTGTCTCTGAC-3′) and the control MO (CoMO)
incorporating five mismatches (indicated in lowercase) in the MO1 sequence
(5′-AAgTCCcCTGTTcATTCcGCTgCAT-3′) were from Gene Tools, and
suspended in sterile water.
In situ hybridisation and cell lineage tracing
Whole-mount in situ hybridisation was performed according to a standard
protocol (Harland, 1991). Probes were labelled using digoxigenin-11-UTP
and appropriate RNA polymerases. Cell lineage tracing was performed by
injection of lacZ mRNA followed by β-galactosidase staining using X-Gal
as substrate.
RT-PCR and real-time RT-PCR
For RT-PCR, total RNA extraction and reverse transcription were as
described (Li et al., 2010). PCR primers are listed in supplementary material
Table S1 or were described previously (Shi et al., 2002). PCR products
amplified in a reaction mixture containing 1 μCi [α-32P]dCTP were resolved
on a 5% non-denaturing polyacrylamide gel and visualised using a
phosphorimager (Bio-Rad).
For real-time RT-PCR, total RNA from 15 ectoderm explants was extracted
using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to
the manufacturer’s instructions. Quantitative PCR was performed using
SsoAdvanced SYBR Green Supermix (Bio-Rad) according to the
manufacturer’s instructions. PCR primers are listed in supplementary material
Table S2. The data were analysed using both ornithine decarboxylase (ODC)
and EF1α as reference.
Yeast two-hybrid screening
The Xdscr6 coding sequence was cloned in the pGBKT7 vector (Clontech) in
frame with the Gal4 DNA-binding domain (Gal4-Xdscr6). The AH109 yeast
strain was transformed with this construct and expression of the fusion protein
was detected by western blot using anti-Gal4 or anti-myc antibody. The
AH109-Gal4-Xdscr6 strain was mated with an 11-day mouse embryo
Matchmaker cDNA library pre-transformed into yeast strain Y187 (Clontech)
and ~2.4×106 independent clones were screened at high stringency.
GST pull-down and co-immunoprecipitation
GST-Xdscr6 and GST-BMI1 constructs were obtained by cloning their
coding sequences in pGEX4T1, and fusion proteins produced in E. coli cells
were bound to glutathione-agarose (Sigma). Synthetic mRNAs encoding
epitope-tagged proteins were injected into Xenopus embryos and lysates
were incubated with glutathione-agarose-bound GST fusion proteins
overnight at 4°C. Bound proteins were subjected to western blot analyses.
The experiment was also performed with in vitro translated proteins using
the TNT Coupled Reticulocyte Lysate System (Promega). Coimmunoprecipitation was performed as previously described (Carron et al.,
2003).
4912
Cell transfection and confocal immunofluorescence microscopy
Xdscr6-RFP, Xdscr6m-RFP or myc-WRPW plasmids were transfected into
HeLa cells using Fugene 6 reagent (Roche). Transfected cells were cultured
for 24 hours and then fixed in PBS-buffered 4% paraformaldehyde. After
permeabilisation in PBS containing 0.1% Tween 20 and blocking in PBS
containing 5% BSA, immunodetection was performed with rabbit anti-Ezh2
antibody (1:800; Abcam, 1 mg/ml stock, code ab3748), mouse anti-BMI1
antibody (1:100; Abcam, 1 mg/ml stock, code ab14389) or 9E10 antibody
(1:2000; Santa Cruz Biotechnology, 200 µg/ml, code sc-40), followed by
Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody. The
cells were then stained with 4⬘,6-diamidino-2-phenylindole dihydrochloride
(DAPI). Images were acquired with a Leica SP5 confocal microscope and
quantification of nuclear fluorescence intensity was by fluorescent particle
counting in an area of 20 μm2 using ImageJ software (NIH). Box plots were
generated using R software. Statistical significance was assessed by
Student’s t-test in triplicate.
Chromatin immunoprecipitation (ChIP)
ChIP was based on a published method (Havis et al., 2006). One-cell stage
embryos were injected in the animal pole region with Xdscr6 mRNA (0.5
ng) and ectoderm explants were dissected at stage 11. Sonicated chromatin
from 100 ectoderm explants was incubated with 10 μg anti-Ezh2 antibody
(Abcam), 5 μg anti-BMI1 antibody (Abcam), 8 µl anti-acetylated histone
H4 antibody (Millipore, unpurified serum, code 06-866) or 10 μg anti-GFP
antibody (Roche, 400 mg/ml stock, code 11814460001). The goosecoid
promoter regions (−390/–167 and −390/–290) were analysed by semiquantitative PCR or by quantitative PCR in triplicate using the primers listed
in supplementary material Tables S1 and S2.
Luciferase assay
The −1500/+3 goosecoid and −907/–5 Xnr1 promoter-luciferase reporter
plasmid DNAs (Watabe et al., 1995; Cao et al., 2007; Cao et al., 2008) were
injected at 100 pg with synthetic mRNAs in the animal pole region at the 2cell stage, and the luciferase assay was performed using ten early gastrula
ectoderm explants as described (Cao et al., 2012). The experiments were
carried out in triplicate using different batches of embryos and the data were
analysed using Student’s t-test.
Acknowledgements
We thank E. Jones, H. Clevers, A. Ciechanover, Y. Cao, B. Thisse and M. van
Lohuizen for reagents; the imaging facility at IFR83 (University Pierre et Marie
Curie) for help with confocal image acquisition; and C. Antoniewski, R. Gautier, D.
Cladière, E. Mouchel-Viehl, R. Le Bouffant and E. Havis for helpful discussion and
technical advice.
Competing interests
The authors declare no competing financial interests.
Author contributions
H.-Y.L. performed the experiments with assistance from R.G. and A.S.; C.C. and
D.-L.S. designed and performed the experiments, analysed results and wrote the
paper.
Funding
This work was supported by grants from Association Française contre les
Myopathies (AFM), Ligue Nationale Contre le Cancer (LNCC) and Agence
Nationale de la Recherche (ANR) [ANR-09-BLAN-0262-03].
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.098319/-/DC1
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