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First published online 9 April 2008
doi: 10.1242/dev.018853
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1 Embryology Unit, Children's Medical Research Institute, University of Sydney,
Locked Bag 23, Wentworthville, New South Wales, NSW 2145, Australia.
2 Faculty of Medicine, University of Sydney, Locked Bag 23, Wentworthville, New
South Wales, NSW 2145, Australia.
3 Laboratory of Mammalian Genes and Development, National Institute of Child
Health and Human Development, National Institute of Health, Bethesda, MD
20892, USA.
4 The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade,
Parkville, Victoria 3050, Australia.
* Author for correspondence (e-mail: ptam{at}cmri.usyd.edu.au)
Accepted 14 March 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Head development, WNT signalling, DKK1, WNT3, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Lhx1 encodes a LIM class homeodomain protein
(Li et al., 1999) that forms
complexes with LIM-domain binding protein (LDB1) and single-strand DNA binding
protein 1 (SSDP1) (Enkhmandakh et al.,
2006). This complex interacts synergistically with OTX2 to
activate target genes (Nakano et al.,
2000). Interestingly, loss of the functions of Ldb1,
Ssdp1 and Otx2 individually, like that of Lhx1, also
leads to head truncation in the mouse embryo, albeit with different degrees of
severity (Mukhopadhyay et al.,
2003; Nishioka et al.,
2005; Acampora et al.,
1995; Acampora et al.,
1997; Ang et al.,
1996; Matsuo et al.,
1995). The activity of the genes that influence head morphogenesis
is potentially linked to WNT signalling, principally via effects on the
expression of antagonistic factors. Mutant embryos that lack Otx2 or
Foxa2 (which directly trans-activates Otx2) also lose the
expression of a WNT antagonist, Dkk1, in the visceral endoderm
(Kimura et al., 2001;
Zakin et al., 2000;
Kimura-Yoshida et al., 2007).
Similarly, loss of Ssdp1 or Ldb1 function affects the
expression of Dkk1, as well as of other WNT antagonists such as
Sfrp1, Sfrp2 and Frzb/Sfrp3
(Hoang et al., 1998;
Nishioka et al., 2005;
Mukhopadhyay et al.,
2003).
To date, the most compelling evidence for a role of WNT antagonist activity
in head formation is that the loss of Dkk1 results in anterior
truncation (Mukhopadhyay et al.,
2001). Dkk1 encodes a secreted protein that inhibits
canonical WNT signalling by sequestering the LRP6 co-receptor so that it can
no longer function alongside the frizzled receptors to activate the WNT
signalling cascade (Glinka et al.,
1998; Mao et al.,
2001; Zorn, 2001).
Dkk1 is expressed first in a girth of visceral endoderm in the
mid-region of the embryonic day (E) 5.5 embryo. It is then expressed in a
crescent-shaped domain of visceral endoderm in the anterior region of the
E6.0-6.5 embryo (Kimura-Yoshida et al.,
2005) and similarly in the anterior endoderm of E7.0-7.5 embryos
(Lewis et al., 2007;
Pfister et al., 2007).
Dkk1 is later restricted to the prechordal plate and the ventral
foregut endoderm (Lewis et al.,
2007). At every stage of development from E5.5 to E7.5,
Dkk1-expressing cells appear to demarcate the anterior and lateral
border of the expression domain of Fzd8 (which encodes a frizzled WNT
receptor) in the anterior endoderm (Lu et
al., 2004). Taken together, these findings imply a link between
the modulation of WNT signalling by the antagonists and the development of
anterior structures. Consistent with this concept, blocking WNT signalling in
embryonic stem cells by DKK1 promotes the formation of precursors of forebrain
neurones (Watanabe et al.,
2005). By contrast, ectopic expression of Wnt1 in the
anterior tissues, in conjunction with the loss of Six3 or
Hesx1 function, is associated with truncation of anterior structures
or posteriorization of the forebrain tissues
(Lagutin et al., 2003;
Andoniadou et al., 2007).
Despite the demonstration of a crucial requirement for Dkk1
activity for head formation, it is not clearly known when its function is
required during embryogenesis. Some findings suggest that an earlier function
of Dkk1 in the visceral endoderm may be dispensable at least for the
induction of forebrain tissues. These include: (1) that in the
Dkk1-null mutant embryo, the molecular markers for anterior visceral
endoderm are expressed correctly
(Mukhopadhyay et al., 2001);
(2) that chimeric embryos comprising Dkk1-deficient extra-embryonic
tissues, including the visceral endoderm, develop normally
(Mukhopadhyay et al., 2001);
and (3) that, although the enforced expression of Dkk1 in the
Otx2-null mutant can restore the patterning of the visceral endoderm,
it is not sufficient to rescue the truncated head phenotype
(Kimura-Yoshida et al., 2005).
There is, however, indirect evidence for a role of Dkk1 later in the
anterior mesendoderm for head formation, which is revealed by a synergistic
interaction of Dkk1 and Gsc via their negative action on WNT
signalling. Both genes are expressed in the prechordal plate and the foregut
endoderm. Gsc may suppress the transcription of Wnt8a and
acts in concert with Dkk1 to attenuate WNT signalling in the anterior
tissues (Lewis et al.,
2007).
As the function of Dkk1 is to antagonize WNT signalling, the
phenotypic effect of the loss of Dkk1 function may be caused by an
inadequate control of the level of WNT signals. Several genes encoding WNT
ligands are expressed in the mouse embryo during the immediate
postimplantation period (Yamaguchi,
2001; Kemp et al.,
2005). Of these, Wnt3 is one of the first to be
expressed: transiently in the posterior visceral endoderm, then restricted to
the posterior proximal epiblast and later to the nascent primitive streak
(Rivera-Perez and Magnuson,
2005). Wnt2b and Wnt8a are expressed primarily
in the posterior epiblast, Wnt3a in the primitive streak and
Wnt5a in the mesoderm (Yamaguchi,
2001; Kemp et al.,
2005). In contrast to the posterior localization of the ligand
transcripts, genes coding for WNT antagonists, such as Dkk1, Sfrp1
and Sfrp5 (Kemp et al.,
2005; Finley et al.,
2003; Kimura-Yoshida et al.,
2005; Lewis et al.,
2007), are expressed in the anterior visceral endoderm and later
the anterior definitive endoderm. The regionalization of antagonist and ligand
expression in the embryo is consistent with the maintenance of a graded
signalling activity from low in the progenitor tissues of the head to high in
the posterior germ layer and the primitive streak
(Pfister et al., 2007).
However, it is not known which of these WNT activities is modulated by DKK1 to
control head morphogenesis.
In the present study, we have shown by marker and reporter analysis of mutant embryos that loss of Dkk1 leads to ectopic activation of WNT/β-catenin signalling during gastrulation. Our results demonstrate that Dkk1 and Wnt3 activities are regulated in a negative feedback manner in vivo. Analysing the development of compound Dkk1;Wnt3 mutants reveals that Dkk1 and Wnt3 interact genetically, and that balancing the Dkk1 and Wnt3 activity at early gastrulation is essential for head development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and Wnt3+/-
(Liu et al., 1999) mice,
maintained on a 129xC57BL6 background, were intercrossed to generate
compound Dkk1;Wnt3 mutant mice. These mice were then interbred to
produce mutant embryos of different combinations of Dkk1 and
Wnt3 genotype. A total of 323 embryos were collected at E7.75 to
E10.5 for the analysis of compound mutant phenotype. Embryos were also
collected from pregnant mice of the Dkk1+/- intercross for
the analysis of phenotype and marker expression. For detecting the response of
cells to WNT signalling in embryos of different Dkk1 genotypes, two
lines of transgenic mice, TOPGal (DasGupta
and Fuchs, 1999) and BATGal
(Maretto et al., 2003), were
crossed with Dkk1+/- mice to introduce the lacZ
transgene onto a Dkk1+/- background. Mating of the
resultant mice generated wild-type, Dkk1+/- and
Dkk1-/- embryos that express one of the transgenic
reporters, which can be detected by X-Gal staining for β-galactosidase
activity. For transplantation experiments to examine the differentiation of
Dkk1-/- cells, a stock of mice was produced by crossing
Dkk1+/- mice with transgenic mice that co-express two
transgenes (pCAGG-EGFP and Hmgcr-nls-lacZ) widely in
embryonic tissues (Kinder et al.,
2001). Offspring from this mating were selected for the
Dkk1+/-;EGFP;lacZ genotype. Embryos containing
Dkk1-/- cells that harboured the EGFP and
lacZ transgenes were obtained from pregnant female mice generated by
intercrossing Dkk1+/-;EGFP;lacZ mice. Genotyping by PCR of Dkk1 and Wnt3 was performed on tail tissues of newborn mice and the yolk sac of embryos (primer sequences and PCR conditions available upon request). Mice harbouring the lacZ and EGFP transgenes were identified by X-Gal staining and visualizing the fluorescence of ear tissues.
Phenotypic analysis of Dkk1 mutant embryos
Whole-mount in situ hybridization
Embryos were processed for in situ hybridization (ISH) according to the
protocol of Wilkinson and Nieto (Wilkinson
and Nieto, 1993) with the following modifications. Riboprobes were
labelled with digoxigenin-11-UTP (Roche) using the AmpliScribe kit (Epicentre
Technologies) or the MAXIscript T7/T3 Kit (Ambion). SDS was used in place of
CHAPS in both prehybridization and hybridization, no RNA digestion was
performed after hybridization, and formamide was omitted from
post-hybridization washes. The following riboprobes (and the source) were
used: Dkk1, Sfrp1 and Sfrp5 (H. Westphal); Lhx1,
Wnt3 and Chrd (R. Behringer); Six3 (O. Guillermo);
Fgf8 (G. Martin); Hesx1 (S. Dunwoodie); T (B.
Hermann); Tbx6 (D. Chapman); En1 (A. Joyner); Dlx5
(J. Rubenstein); Sox17 (J. Gad); Cer1, Foxa2 and
Sfrp5 (W. Shawlot); Wnt2b and Wnt8a (S. Aizawa);
Mixl1 (L. Robb); Nkx2.1 (S. L. Ang); Pax6 (K.
Backs); Axin2 (J. Martinez-Barbera); and Shh (B.
St-Jacques).
Immunofluorescence
Mid-streak stage (E7.0) wild-type and Dkk1-/- embryos
were processed for whole-mount immunofluorescence according to the protocol of
Ciruna and Rossant (Ciruna and Rossant,
2001) using anti-β-catenin (rabbit) (Abcam) and Alexa
488-conjugated secondary antibody (Invitrogen). Nuclei were counterstained by
propidium iodide. Phallotoxin (Invitrogen) was used to stain the F-actin to
reveal the cell outline. Stained embryos were dissected into anterior and
posterior halves, and mounted separately for confocal fluorescence
microscopy.
Cell transplantation experiment
Pregnant Dkk1;EGFP;lacZ mice were euthanized at E7.0 to harvest
embryos for isolating cells for transplantation. Embryos were genotyped by PCR
on yolk sac tissues (primer sequences available upon request). Tissue
fragments were dissected from the anterior region of the primitive streak
[APS, containing progenitors of mesoderm and endoderm
(Kinder et al., 2001)] of the
mid-streak stage Dkk1-/-;EGFP;lacZ embryos.
Similar APS fragments were obtained from
Dkk1+/+;EGFP;lacZ for the control experiment.
These fragments were dissected into clumps of about 10-15 cells, which were
transplanted into the APS of wild-type mid-streak stage ARC/s host embryos for
assessing the differentiation of Dkk1-/- APS cells. The
recipient embryos were examined by fluorescence microscopy 2 hours after
transplantation to ascertain the location of the EGFP-expressing graft.
Embryos showing incorrect positioning of the graft were excluded from further
analysis.
Recipient embryos were cultured (Sturm
and Tam, 1993) for 24 hours until they reached the six- to
eight-somite stage. The host embryos were examined by fluorescence microscopy
to visualise the distribution of the graft-derived EGFP-expressing cells.
Embryos were then briefly fixed in 4% paraformaldehyde, stained in X-Gal
solution overnight to detect the lacZ-positive graft-derived cells
and then processed for paraffin wax histology. The number and the distribution
of X-Gal stained cells in the host tissues were scored in serial sections of
the specimen.
Electroporation of the endoderm
Mid-streak stage wild-type and Dkk1-/- embryos were
harvested from E7.0 pregnant mice. Cells in the endoderm layer of the embryo
were marked by electroporating a pCMV-EGFP expression vector
(Davidson et al., 2003). The
embryos were then cultured in vitro (Sturm
and Tam, 1993). The sites of labelling were ascertained by the
localization of the EGFP-expressing cells 3 hours after electroporation. After
24 hours of in vitro culture, the distribution of the EGFP-expressing cells
along the anterior-posterior body axis was recorded.
RT-PCR analysis of Dkk1 induction
NIH3T3 cell lines containing pLNCX retroviral vector expressing full-length
cDNA, encoding Wnts or lacZ
(Kispert et al., 1998) were
used to test the induction of Dkk1. The expression of
β-galactosidase, Wnt1, Wnt3a, Wnt4, Wnt5a, Wnt7a and
Wnt11 in respective cell lines was confirmed by RT-PCR using primers
described by Lako et al. (Lako et al.,
2001). Total RNA was prepared from the cultured cells using the
RNeasy Kit (Qiagen) following the manufacturer's instructions. RNA was
reverse-transcribed using Superscript III Reverse Transcriptase using
oligo(dT) as a primer. RT-PCR for Dkk1 and Hprt was
performed on the products of the reverse transcription (primer sequences
available upon request).
Quantitation of Dkk1 and Wnt3 expression level
Quantitative RT-PCR analysis was performed to determine the level of
Dkk1 and Wnt3 expression of E7.0-7.5 embryos of mice of
Wnt3+/+, Wnt3+/- and
Wnt3-/- genotypes (from Wnt3+/-
inter-cross) and of Dkk1+/+;Wnt3+/+,
Dkk1+/+;Wnt3+/-,
Dkk1+/-;Wnt3+/+ and
Dkk1+/-;Wnt3+/- genotypes (from the
Dkk1+/-;Wnt3+/- intercross). Total RNA was
isolated using the RNeasy Micro Kit (50) (Qiagen). cDNA was generated using
the SuperScript III First Strand Synthesis System (Invitrogen) with oligo-dT
primers. Quantitative RT-PCR of Dkk1 transcript was performed using
the Rotorgene 6000 thermal cycler (Corbett Research) with SYBR green I
(Molecular Probes) using Platinum Taq DNA Polymerase (Invitrogen). Primer
sequences and reaction conditions are available upon request. The levels of
Gapdh were used for the normalisation of sample results. Initially,
PCR products were run in a 2% gel to confirm correct band size in order to
validate the results, and routine melting curve analysis was performed later
to verify the presence of a single amplified product.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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In the E7.0-7.75 Dkk1-/- embryos, BATGal reporter is
expressed in a wider domain encompassing more anterior germ layer tissues
(11/14 embryos) or even the whole embryo (3/14 embryos)
(Fig. 1F-H). Stronger and
broader TOPGal expression is found in the posterior proximal region and the
primitive streak of Dkk1-/- embryos (see Fig. S1F-H in the
supplementary material). At both the early-somite and early-organogenesis
stages, BATGal- or TOPGal-free tissues are reduced or absent from the rostral
region of the mutant embryo (Fig.
1I,J; see Fig. S1I,J in the supplementary material). To further
assess the cellular response to WNT signalling in the Dkk1-null
embryo, we examined the overall level of expression and the subcellular
localization of β-catenin in the anterior endoderm of the mid-streak
(E7.0) embryos. In five Dkk1-null embryos, endoderm cells in the
anterior-proximal, the anterior-distal and the posterior regions (see Fig.
S1L,N,P in the supplementary material) are stained more strongly for
β-catenin, which was also localized more frequently in the nucleus of the
endoderm cells than in the equivalent population in the wild-type embryo (see
Fig. S1K,M,O in the supplementary material). The abundance of stabilised form
of β-catenin in the anterior endoderm of the Dkk1-/-
embryo and the elevated expression of WNT activity reporters in the anterior
germ layer tissues are consistent with an elevated response to WNT signalling
in the progenitor tissues of the embryonic head. Concurrent with the expanded
expression domain of BATGal (Fig.
2A: BATGal) and TOPGal (see Fig. S1I,J in the supplementary
material), a WNT-downstream gene, Axin2, is expressed more widely in
the head folds of the early-somite-stage Dkk1-/- embryo
(Fig. 2A, Axin2)
(Jho et al., 2002). By E9.5,
Dkk1-/- mutant embryo lost all craniofacial structures
rostral to the upper hindbrain (Fig.
2A, Shh) (Echelard et
al., 1993).
Loss of Dkk1 activity does not affect the allocation of germ layer precursors but may affect cell spreading in the endoderm
Marker expression was analysed to test whether the truncation of the head
is associated with an inappropriate allocation and/or patterning of progenitor
tissues (Fig. 2B,C). No
significant difference in the expression pattern of markers of the primitive
streak (Mixl1, T) (Robb et al.,
2000; Wilkinson et al.,
1990), the organizer (Foxa2, Chrd)
(Ang and Rossant, 1994;
Klingensmith et al., 1999),
the mesoderm (Lhx1, Tbx6)
(Shawlot and Behringer, 1995;
Chapman et al., 1996) and the
endoderm (Cer1) (Belo et al.,
1997) was found between the wild-type and mutant embryos. There
is, however, a more extended domain of T expression in the head
process mesoderm (Fig. 2B,
T). Sox17 expression is reduced in the definitive endoderm
of the early-bud stage mutant embryo, and the expression of both
Foxa2 and Sox17 is reduced in the anterior foregut endoderm
of the early-somite stage mutant embryo
(Fig. 2C; Sox17,
Foxa2) (Kanai-Azuma et al.,
2002; Ang and Rossant,
1994). The differentiation of the endoderm may therefore be
affected by the loss of Dkk1. It is not known, however, whether the
loss of Dkk1 may have affected the allocation of the germ layer
progenitors to the endoderm and mesoderm or cell fate specification.
To further test the impact of loss of Dkk1 on cell differentiation, cells from the APS of the mid-streak (E7.0) Dkk1-/- embryos were transplanted orthotopically to the APS of the E7.0 wild-type host embryo and the distribution of the descendants of the transplanted cells in the host was examined (see Fig. S2A in the supplementary material). Cells from the wild-type donor embryos contributed extensively to anterior structures, including the foregut and anterior axial mesoderm. Similarly, Dkk1-/- cells were able to colonize the cranial paraxial mesoderm, the anterior axial mesoderm and the foregut endoderm of the host embryo (see Fig. S2A-D in the supplementary material). Loss of Dkk1 therefore does not affect the ability of APS cells to colonize the anterior tissues that are missing in the Dkk1-null mutant. Intriguingly, most graft-derived Dkk1-null cells were found in the cranial mesenchyme and few in the trunk and posterior region of the host embryo (see Fig. S2A in the supplementary material, boxed section). The lineage analysis results show that Dkk1-null cells can contribute to both mesoderm and endoderm of the host embryo. It is likely that the altered expression of cell markers is related to defective differentiation of mutant cells after allocation to the respective lineages.
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).
To test whether loss of Dkk1 affects cell movement in the endoderm,
groups of cells in five different regions of endoderm of the E7.0
Dkk1-/- embryo were electroporated with a pCMV-EGFP
expression vector so that the descendants of these cells can be tracked as the
embryo develops in vitro (e.g. Fig. S3A,B in the supplementary material). In
contrast to the extensive displacement of endoderm cells in the wild-type
embryos, endoderm cells of the Dkk1-/- were more
restricted in their distribution in the anterior-posterior axis of the embryo.
Cells in the anterior endoderm were not wholly displaced to the yolk sac (see
Fig. S3C in the supplementary material) or tended to localize more frequently
to the mid-embryonic sites than those in the wild-type embryo (see Fig. S3D in
the supplementary material). F-actin immunostaining revealed that endoderm
cells in the anterior region of the E7.0 Dkk1-/- embryo
have a smaller apical cell surface area, suggesting that these cells may be
more tightly packed together (see Fig. S3H,I in the supplementary material).
Cells in the distal endoderm failed to extend along the anterior-posterior
axis (see Fig. S3E in the supplementary material) and those in the
posterior-distal site were restricted to the posterior region and did not move
to anterior sites (see Fig. S3F in the supplementary material). Only cells in
the posterior-proximal region of Dkk1-null mutant embryo behaved like
the wild type (see Fig. S3G in the supplementary material). Loss of
Dkk1 function may therefore impede the anterior-posterior spreading
of endoderm cells in the anterior and middle parts of the embryonic gut.
Wnt3 activity influences Dkk1 expression
In the early to mid-streak (E6.5-7.25) embryo, Dkk1-expressing
cells are distributed in a crescent-shaped domain that delimits the anterior
and lateral border of the anterior endoderm
(Fig. 3A,B)
(Kimura-Yoshida et al., 2005;
Pfister et al., 2007). At
similar stages, Wnt3 is expressed in the visceral endoderm, with the
domain extending anteriorly along the mid-girth of the pre-streak embryo
(Fig. 3A)
(Rivera-Perez and Magnuson,
2005). During gastrulation, Wnt3 is expressed in the
posterior epiblast and the primitive streak
(Fig. 3B,C), where two other
WNT genes (Wnt2b and Wnt8a) are also expressed
(Fig. 3D,E)
(Kimura-Yoshida et al., 2005).
Among these genes, Wnt3 displays the broadest expression domain,
which extends anteriorly to the vicinity of the Dkk1-expressing
endoderm cells (Fig. 3A,B). In
embryos that lack Dkk1 activity, the expression domain of
Wnt3 remains unchanged (Fig.
3C'), whereas those of Wnt2b (6/6 embryos) and
Wnt8a (1/3 embryo) expand slightly
(Fig. 3D',E'). In
addition to Dkk1, two WNT antagonists, Sfrp1
(Kemp et al., 2005) and
Sfrp5 (Finley et al.,
2003), are also expressed in the anterior region of the embryo. In
the Dkk1-/- embryo, the expression of Sfrp1 is
reduced but Sfrp5 does not change
(Fig.
3F,F',G,G').
Dkk1 is directly targeted by WNT/β-catenin signalling
(Gonzalez-Sancho et al., 2005;
Niida et al., 2004;
Chamorro et al., 2005) through
the binding of TCF/β-catenin complex to the Lef/Tcf sites in the
Dkk1 promoter (Chamorro et al.,
2005). To test whether Dkk1 expression is influenced by
Wnt3 activity in vivo, we examined Dkk1 expression in the
E7.0 Wnt3-/- embryo. Dkk1 is not expressed in
four out of five null-mutant embryo, and the remaining embryo shows very weak
and restricted expression in the anterior endoderm
(Fig. 3H,
Wnt3-/-). Wnt3-null embryos fail to gastrulate
but they do form the anterior visceral endoderm
(Liu et al., 1999), suggesting
that the lack of Dkk1 expression is not due to the loss of this
tissue. Dkk1 expression domain is more restricted in the
Wnt3+/- embryo (Fig.
3H, Wnt3+/-). Q-RT-PCR analysis of embryos of
different Wnt3 genotypes showed that Dkk1 expression is not
changed when Wnt3 dose is halved but is markedly reduced in the
absence of Wnt3 (Table
1A). In view of the fact that Dkk1 expression depends on
Wnt3 activity, we predict that halving the gene dose of Dkk1
in Wnt3+/-embryo might reduce the expression of
Dkk1. This was found to be the case: Dkk1 expression in the
Dkk1+/-;Wnt3+/- is significantly downregulated
(Table 1B). In situ
hybridization was also performed on embryos of different Dkk1;Wnt3
genotypes. The results show that in the
Dkk1+/-;Wnt3+/+ embryo, the domain of
Dkk1 expression in the anterior endoderm is reduced (seven out of
eight embryos; Fig. 3J,
Dkk1+/-;Wnt3+/+) to a similar extent as in the
Dkk1+/+;Wnt3+/- embryo (five out of
five embryos; Fig. 3J,
Dkk1+/+;Wnt3+/-). The
Dkk1+/-;Wnt3+/- embryos display weaker and more
restricted Dkk1 expression than both
Dkk1+/-;Wnt3+/+and
Dkk1+/+;Wnt3+/- embryos (five out of
five embryos; Fig. 3J,
Dkk1+/-; Wnt3+/-).
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|
Dkk1 and Wnt3 interact genetically in head morphogenesis
To test whether a reduced level of Dkk1 activity may affect head
morphogenesis, we undertook a phenotypic study of the compound
Dkk1+/-;Wnt3+/- mutant embryo. Like
Dkk1-/- embryos, some
Dkk1+/-;Wnt3+/- embryos have smaller
head folds at E8.5 and forebrain at E9.5
(Fig. 4A). Altogether, about
70% of E8.0-9.5 Dkk1+/-;Wnt3+/-
embryos display abnormal head morphology, compared with 5.5% of
Dkk1+/+;Wnt3+/- and 9.5% of
Dkk1+/-;Wnt3+/+ embryos from the same cross
(Table 2), a finding consistent
with the degree of Dkk1 downregulation for these genotypes
(Table 1B). Analysis of BATGal
reporter expression (Fig. 4B:
BATGal) revealed that ectopic signalling activity is perceived by cells in the
anterior region of the E7.75 compound heterozygous embryo, suggesting that the
elevated canonical WNT signalling may precede or be coincidental with the
emergence of anterior defects. The phenotype of
Dkk1+/-;Wnt3+/- embryos is variable
but can be categorized in four ways:
|
Class II, reduced forebrain size (51%);
Class III forebrain truncation with additional patterning defect such as malformed eye and branchial arches (8%); and
Class IV, severe head truncation and/or trunk defects (11%)
(Fig. 4A,
Table 3). In the Class II
mutant embryos, the expression domains of Fgf8, Hesx1 and
Six3, which mark the rostral forebrain tissues, are reduced
(Fig. 4B; Fgf8, Hesx1,
Six3) (Crossley and Martin,
1995; Thomas and Beddington,
1996; Oliver et al.,
1995). In the mutant embryo that developed a relatively intact
forebrain, Nkx2.1 expression was reduced, suggesting that the brain
tissues lack the molecular characteristics of the ventral forebrain
(Fig. 4B; Nkx2.1)
(Camus et al., 2000). By
contrast, expression of Pax6 is similar to that in the wild-type
embryo (Fig. 4B; Pax6)
(Inoue et al., 2000),
suggesting the dorsal forebrain tissues are probably not affected. The most
severely malformed embryos (Class IV) display an open neural tube, reduced
head size and the formation of a solitary tissue mass in place of the paired
branchial arches (see Fig. S4 in the supplementary material). The increase in
the proportion of the Class I embryos from 30% at E9.5 to 88% at E10.5 might
be due to the loss of the severely malformed (Class III-IV) embryos
(Table 3). Alternatively, the
morphological defects of forebrain might have been corrected during the
development of some Class II embryos. The results of the compound heterozygote
study suggest that Dkk1 and Wnt3 interact genetically, and
that the reduction of Dkk1 activity in
Dkk1+/-;Wnt3+/- embryos leads to
abnormal head morphogenesis.
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|
). In the E8.5
Dkk1-/-;Wnt3+/- embryo, TOPGal-free
tissue, which is greatly reduced in the
Dkk1-/-;Wnt3+/+ embryo, is restored in the
rostral part of the head folds (Fig.
5B, TOPGal), suggesting that a WNT-signal free zone is
re-established. In summary, lowering the level of Wnt3 activity has
partially compensated for the loss of Dkk1 function, suggesting that
Dkk1 modulation of Wnt3 activity at gastrulation is crucial
for normal head development. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Lewis et al., 2007), which
demarcates the proximal and lateral border of the Fzd8 expression
domain (Lu et al., 2004). The
alignment of the Dkk1-expressing cells with the border between the
Wnt3 and the Fzd8 domains suggests that Dkk1
activity may be induced by WNT3 signalling in cells that also express the Fzd8
receptor. A potential role for DKK1 is therefore to delineate the boundary of
the prospective head and/or forebrain field. In the skin, WNT and DKK proteins
are shown to act as molecules in a reaction-diffusion mechanism for the
spatial patterning of hair follicles (Sick
et al., 2006). The activity of Dkk1 and Wnt3 in
the anterior endoderm may be involved in a similar mechanism for delimiting
the tissue domain of the head from the body.
Dkk1 expression in vivo is responsive to Wnt3 gene dosage
In the Dkk1-null mutant, the expression domain of Wnt3
and Sfrp5 remains unchanged, whereas that of Wnt2b and
Wnt8a is slightly expanded and that of Sfrp1 is reduced.
Overall, these changes are associated with an elevated level of WNT signalling
perceived by cells in the anterior germ layer tissues. On the contrary,
Dkk1 expression changes with decreasing Wnt3 gene dose: the
expression domain is slightly altered with a halved dose of Wnt3
(Wnt3+/- embryo), significantly reduced to a `hypomorphic'
level in Dkk1+/-;Wnt3+/- embryo and almost
completely lost in the Wnt3-/- embryo. The induction of
Dkk1 by WNT signal, which in turn antagonizes the signalling
activity, constitutes a negative-feedback mechanism for regulating WNT
signalling. That Dkk1 can be induced specifically by Wnt1 in
the NIH3T3 cells (this study) and that this regulatory loop can be blocked by
RNAi against Dkk1 in 293T cells
(Niida et al., 2004) suggests
that DKK1 is the key component in modulating Wnt1 signals in these
cell models. A similar negative-feedback mechanism is found for
Axin2, which is directly activated by β-catenin/Tcf and inhibits
WNT signalling by downregulating β-catenin
(Jho et al., 2002;
Aulehla et al., 2003).
It is possible that the activity of other canonical WNT genes [Wnt2,
Wnt2b, Wnt6 and Wnt8a (Kemp
et al., 2005)] that are expressed at similar developmental stages
to Wnt3 may contribute to outcome of loss of Dkk1. However,
the finding that reducing Wnt3 gene dose alone may substantially
rescue the head truncation phenotype suggests that other WNT genes may have a
relatively minor role in the initial phase of head induction.
|
)
(Fig. 6E), whereas those
lacking Wnt3 fail to gastrulate
(Liu et al., 1999)
(Fig. 6H). By studying the
phenotype of the compound mutant embryo, we showed that the genetic
interaction of Dkk1 and Wnt3 during gastrulation is
essential for head morphogenesis. However, the interaction is more complex
than a simple stoichiometric balance of gene dose. Halving the dose of each
gene in the Dkk1+/-;Wnt3+/- embryo is expected
to produce a reduced but otherwise balanced agonist versus antagonist activity
(Fig. 6B). On the contrary, the
level and the domain of Dkk1 expression in the compound heterozygous
mutant embryo are lower and more restricted, respectively, than those in the
Dkk1+/- embryo. In view of that, Dkk1 and WNT act
in a negative-feedback pathway, the loss of one allele of Wnt3 may
result in a reduced level of agonist activity that may then induce less
Dkk1 activity when only one wild-type Dkk1 allele present
(Fig. 6C). The head phenotype
of Dkk1+/-;Wnt3+/- embryo may be
caused by the unbalanced WNT3 signalling resulting from the `hypomorphic'
Dkk1 activity, which cannot be substituted for by other antagonists.
The phenotype of the compound heterozygous embryo is variable, which may
suggest that the reduced gene dose has resulted in an unstable but generally
excessive signalling activity (Fig.
6F). As well as anterior defects, some
Dkk1+/-;Wnt3+/- embryos also display posterior
defects, indicating that the WNT signal may not have been maintained at the
required level in the posterior tissue of these embryos for the formation of
trunk structures. This suggests that a proper level of interaction between
Dkk1 and Wnt3 is essential for maintaining a balanced
signalling activity for both anterior and posterior morphogenesis. Our results
further show that reducing the Wnt3 dose in
Dkk1-/- embryo can partially rescue the brain and
pharyngeal defect, despite the absence of Dkk1
(Fig. 6G). This finding
suggests that WNT3 signal is specifically targeted by DKK1 in the anterior
germ layers to maintain at a proper level of activity for head
morphogenesis.
DKK1 and cell movement in the endoderm
In the Otx2-null mutant embryo, absence of Dkk1 activity
is accompanied by the accumulation of nuclear and cytoplasmic β-catenin,
and by the lack of cell movement in the visceral endoderm. Defects of cell
movement can, however, be rescued by expressing Dkk1 from the
Otx2 locus or lowering the level of WNT/β-catenin signalling by
reducing the gene dose of Ctnnb1 [which encodes β-catenin
(Kimura-Yoshida et al.,
2005)].
The definitive endoderm is reputed to be an essential source of inductive
signals for patterning the anterior region of the embryo and for the
specification of forebrain tissues. A delay or failure to move the definitive
endoderm to the anterior region of the embryo may underpin the loss of
anterior structures in mutant embryos, owing to the lack of provision of
patterning cues to the overlying tissues (Lewis and Tam, 2007). In the
Lhx1-null mutant, definitive endoderm cannot move anteriorly to
populate the foregut because of the immobility of the pre-existing anterior
visceral endoderm (Shimono and Behringer,
2003; Tam et al.,
2004). In the Mixl1-null mutant, abnormal anterior
development is associated with impaired endoderm cell movement owing to the
lack of propulsive action caused by inefficient tissue accretion in the
posterior endoderm (Tam et al.,
2007). By tracking the movement of the endoderm cells in the
Dkk1-null embryo, we have shown that loss of Dkk1 impedes
the movement of the definitive endoderm. It is interesting that in the
Dkk1-deficient zebrafish embryo, there is an accelerated anterior
movement of mesoderm cells (Caneparo et
al., 2007). In the mouse, descendants of
Dkk1-/- APS cells were absent from the posterior mesoderm
of the wild-type host, which may reflect a preferential anterior localization
of Dkk1-deficient mesoderm cells. These findings of two different
embryo models raise the possibility that DKK1 may have cell-autonomous and/or
a germ-layer (endoderm versus mesoderm)-specific role in navigating cell
movement.
|
). A
switch between the WNT/β-catenin and the WNT/PCP pathway may be achieved
by regulating the level of Dkk1 activity. It may be envisaged that as
DKK1 sequesters LRP6 to block the β-catenin-LEF/TCF pathway, the frizzled
receptors may become available for engagement with the PCP pathway. Presently,
it is not known whether the PCP pathway is activated whenever Dkk1
activity is absent and whether Dkk1/Wnt3/β-catenin activity has
a direct role in endoderm movement. In the zebrafish, a switch may be mediated
by the binding of Dkk1 to Knypek, thereby enhancing PCP activity that
influences gastrulation cell movement
(Caneparo et al., 2007). In the
mouse, the expression of glypican 4 (the Knypek homologue) overlaps with that
of Dkk1 in the anterior region of the embryo
(Ybot-Gonzalez et al., 2005).
This raises the possibility that Dkk1 acting in conjunction with
glypican 4 may regulate cell movement in the endoderm in addition to its other
role in modulating WNT3/β-catenin activity.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1791/DC1
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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