1 Cancer and Developmental Biology Laboratory, Center for Cancer Research,
National Cancer Institute-Frederick, NIH Frederick, MD 21702, USA
2 School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501,
USA
* Author for correspondence (e-mail: tyamaguchi{at}ncifcrf.gov)
Accepted 23 September 2005
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SUMMARY |
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Key words: Mouse, Wnt3a, Left-right determination
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Introduction |
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Members of the transforming growth factor ß (Tgfß) family,
specifically Nodal, Lefty1 and Lefty2, are the first genes
to be asymmetrically expressed along the LR axis
(Hamada et al., 2002).
Nodal is expressed in the periphery of the node, where it functions
as the left-determinant (Brennan et al.,
2002
; Saijoh et al.,
2003
). Nodal transcription is controlled by the Notch
signaling pathway. Activation of Notch receptors by the ligand Delta-like 1
(Dll1), leads to the cleavage and nuclear translocation of the Notch
intracellular domain, where it acts as a transcription factor when bound to
the DNA-binding protein RBP-J (Rbpsuh - Mouse Genome Informatics)
(Schweisguth, 2004
). Loss of
function mutations in components of the Notch pathway lead to loss of LR
asymmetry, and RBP-J-binding sites found within the Nodal
node-specific enhancer are required for Nodal expression in the node
(Krebs et al., 2003
;
Raya et al., 2003
). These data
demonstrate that Nodal is a direct target gene of the Notch signaling
pathway; however, the relationship between Notch activity and
symmetry-breaking events in the node is not clear.
Cilia emanating from the ventral surface of the node play a crucial role in
the breaking of bilateral symmetry
(McGrath and Brueckner, 2003).
Embryos carrying mutations in genes required for cilia formation or motility
display laterality defects (Marszalek et
al., 1999
; Nonaka et al.,
1998
, Supp et al.,
1999
). Motile cilia generate a leftward flow of extra-embryonic
fluid at the node, termed nodal flow, that is necessary for the generation of
LR asymmetry (Nonaka et al.,
1998
; Okada et al.,
1999
). Artificial reversal of nodal flow is sufficient to reorient
the LR axis (Nonaka et al.,
2002
) demonstrating that nodal flow is both necessary and
sufficient for LR axis specification. These experiments led to the development
of the morphogen flow model that proposed that nodal flow, generated by node
cilia, set up a morphogen concentration gradient that directs asymmetric gene
expression at the node (Nonaka et al.,
1998
; Okada et al.,
1999
).
A second population of node cilia, known as mechanosensory cilia, have been
proposed to participate in LR determination, largely owing to the observation
that mutations in the polycystic kidney disease 2 (Pkd2) gene cause
abnormal LR development (Pennekamp et al.,
2002). Pkd2 encodes polycystin 2 (PC2), a
Ca2+-permeable cation channel expressed in node cilia that is
necessary for the generation of asymmetric Ca2+ flux
(McGrath et al., 2003
). These
results led to the development of the two-cilia model for LR initiation in
which a centrally located population of Lrd-containing motile cilia generate
nodal flow, while a second population of PC2-expressing nonmotile
mechanosensory cilia sense nodal flow on the left side of the node and convert
it into an asymmetric Ca2+-dependent signal transduction event
(McGrath and Brueckner, 2003
;
Tabin and Vogan, 2003
).
Activation of the Wnt/ß-catenin pathway by members of the Wnt family
of secreted signaling molecules elevates levels of ß-catenin, a
transcription co-factor with T cell factor/lymphoid enhancer factor (Tcf/Lef),
leading to the activation of target genes (see Wnt homepage,
http://www.stanford.edu/~rnusse/wntwindow.html).
Although it is well-known that Wnts are important molecular components of the
vertebrate organizer (Niehrs,
2004), playing crucial roles in AP patterning
(Yamaguchi, 2001
), little is
known about the potential roles that Wnts may play in LR determination.
Gain-of-function experiments in the chick embryo have implicated the
Wnt/ß-catenin pathway in LR patterning
(Rodriguez-Esteban et al.,
2001
); however, loss-of-function mutations have not demonstrated a
requirement for Wnts in this process. Interestingly, of the 19 known Wnt
genes, Wnt3a is the only one whose expression initiates in the
gastrulating mouse embryo at E7.5 (Takada
et al., 1994
), a stage that correlates with node formation, LR
determination and somitogenesis. We hypothesized that Wnt3a may be an
important component of the trunk organizer, functioning to coordinate LR axis
specification with trunk development.
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Materials and methods |
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Antibodies
The following reagents were obtained commercially: mouse monoclonal
anti-ß-catenin (BD Transduction Laboratories), anti-acetylated tubulin,
clone 6-11B-1 (Sigma), goat polyclonal anti-PC1 (M-20) (Santa Cruz),
Rhodamine-Phalloidin, DAPI, anti-mouse IgG(H+L) goat Alexa-Fluor 488,
anti-goat IgG(H+L) donkey Alexa-Fluor 488 (Molecular Probes), anti-rabbit
IgG(H+L) goat Cy3, and anti-mouse IgG(H+L) goat Cy3 and Cy5 (Amersham). The
YCC anti-PC2 antibody directed against amino acids 687-962 of human PC2 has
been characterized previously (Cai et al.,
1999).
Whole-mount immunofluorescence and confocal microscopy
Mouse embryos were fixed with 2% PFA for 20 minutes at room temperature,
washed with PBS and stored in 0.1% sodium azide/PBS at 4°C until use.
Embryos were permeabilized with 0.1% Triton X-100 and 100 mM glycine in PBS
for 10 minutes at room temperature, blocked with 10% calf serum, 0.1% BSA
(Sigma) and 3% normal goat serum (NGS) in TBST (20 mM Tris-HCl, pH 8.0, 150 mM
NaCl. 0.05% Tween-20), and then incubated with primary or secondary antibodies
diluted in TBST containing 0.1% BSA and 1.5% NGS. All blocking and antibody
incubation steps were performed overnight at 4°C followed by multiple TBST
washes. For imaging (Zeiss LSM510), embryos were placed in Glass Bottom
Culture Dishes (MatTek Corporation) in PBS containing 50% Vectashield mounting
medium (Vector Labs), or node regions were dissected and mounted on a slide
glass with a spacer in SlowFade Light Antifade Kit (Molecular Probes). Four
Wnt3a+/- and four Wnt3a-/- 0- to
2-somite stage embryos were analyzed for expression of the cilia markers PC1,
PC2 and acetylated tubulin. PC-positive cilia were quantitated manually.
Mice
To make the BATlacZ mouse, two oligos (TBS-1, 5'-AAT TCA GAA
TCA TCA AAG GAC CT-3'; and TBS-2, 5'-AAT TAG GTC CTT TGA TGA TTC
TG-3') containing a Tcf/Lef binding site sequence flanked by
EcoRI sites, were annealed and ligated to construct an 8x
multimer, and then subcloned into pBluescript to generate a plasmid designated
8x TBS-pBS. A 130 bp Xenopus Siamois minimal promoter was
amplified by PCR from p01234 (kindly provided by D. Kimelman) using primers
xSiamois-1 (5'-CGT GAA TTC TAT TTA TAT TTT TTT CAT-3') and
xSiamois-2 (5'-AGC GGA TCC CTC TGT CTC CCA AAA TG-3'), and then
subcloned into the EcoRI/BamHI sites of 8x TBS-pBS.
NLS-lacZ from pCS-nß-gal was subcloned into the
BamHI/XbaI sites of 8x TBS-xSiamois-pBS to generate
the BATlacZ transgene. Transgenic mice were generated in the
Transgenic Core Facility by pronuclear injection following standard
procedures. From the four lines that were generated, the one that most
faithfully replicated domains of Wnt signaling was designated as the
BATLacZ line. These mice are similar in design to the BATgal mice of
Maretto et al. (Maretto et al.,
2003). All animal experiments were performed in accordance with
the guidelines established by the NCI-Frederick Animal Care and Use
Committee.
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Results |
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Examination of E8.75-9.5 embryos homozygous for a null allele of
Wnt3a (Takada et al.,
1994) revealed multiple laterality defects. Although wild-type and
Wnt3a+/- hearts invariably looped to the right
(Fig. 1A),
Wnt3a-/- embryos displayed hearts that looped to the right
(45%, n=49) (Fig. 1B),
left (31%) (Fig. 1C) or
remained in the midline (25%, see Fig. S2B in the supplementary material). The
direction of axial rotation or embryonic turning was randomized with 47%
(n=17) of Wnt3a-/- embryos correctly turning
clockwise such that the tail and allantois lay on the right side of the
embryo, and 53% turning in the opposite direction (not shown). The
heart-looping defects were not secondary to earlier defects in cardiogenesis,
as several heart markers were expressed normally in the mutants (see Fig. S2
and Fig. S3G-J in the supplementary material).
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Wnt3a is necessary for asymmetric gene expression
Wnt3a-/- embryos were examined for the expression of
the asymmetrically expressed genes Nodal, Lefty1, Lefty2 and
Pitx2 at stages prior to the morphological manifestation of LR or AP
phenotypes. Nodal transcripts were detected in the ventral node of
Wnt3a-/- mutants at presomitic, headfold (E7.75-8) stages;
however, the spatial domain was smaller
(Fig. 2B,F) compared with
wild-type controls (Fig. 2A,E)
(Lowe et al., 1996;
Collignon et al., 1996
). This
domain became increasingly restricted to the posterior edge of the mutant node
as development proceeded, and was approximately one-third the size of the
wild-type domain (compare Fig.
2H with 2G) by the two- to four-somite stages
(Fig. 2D,H,J,N). Nodal
mRNA was not detected in the Wnt3a-/- left lateral plate
mesoderm (LPM) at these stages (Fig.
2D) when Nodal was normally expressed there in wild-type
embryos (Fig. 2C), but was
bilaterally expressed in the posterior LPM and streak starting at the four- to
five-somite stage (Fig. 2J,N),
and remained bilaterally expressed in the LPM
(Fig. 2L,P) at stages when
Nodal was normally turned off in wild-type embryos (six- to
eight-somite stages; Fig.
2K,O). The anterior limit of the Nodal LPM expression
domain was posteriorized in mutants (arrowhead,
Fig. 2L), never extending
anteriorly into the heart as in earlier staged wild-type embryos (arrowhead,
Fig. 2I).
Lefty1 and Lefty2 are required for proper LR patterning, functioning as
negative regulators of Nodal (Hamada et
al., 2002). Lefty2 is a direct target gene of Nodal, and
is normally expressed in the left LPM between the three- and six-somite stages
(Meno et al., 1998
)
(Fig. 2C,Q,R). Lefty2
expression in Wnt3a-/- embryos mirrored Nodal
expression, with expression in the LPM initially delayed, then restricted to
the posterior streak (Fig. 2D
and not shown), and later bilateral and posteriorized (seven somites,
Fig. 2Q,R). Similarly,
Pitx2, a bicoid-type homeobox gene expressed in the left LPM and
heart (Yoshioka et al., 1998
),
was delayed, and then bilaterally expressed in mutant posterior LPM (Fig. 2S;
data not shown). Lefty1 is asymmetrically expressed in the left
prospective floor plate (PFP) in wild-type embryos
(Meno et al., 1998
)
(Fig. 2C,G), but was never
detected in the mutant PFP, being expressed in only a few individual cells in
the posterior node and anterior streak
(Fig. 2D,H). Loss of
Lefty1 expression is not due to the physical loss of a midline
barrier (Hamada et al., 2002
)
as several markers, including Shh, Foxa2, T, Wnt11, Gdf1 and cryptic
(Cfc1 - Mouse Genome Informatics) were easily detected in the mutant
node, notochord or PFP (see Fig. S3A-L in the supplementary material; data not
shown). Thus, molecular marker analyses demonstrate that a cascade of genes
necessary for the generation of LR asymmetry are abnormally expressed in the
Wnt3a-/- node and LPM, indicating that Wnt3a
functions early in the genetic hierarchy of LR determination.
If Wnt3a is upstream of left determining genes, then ectopic
activation of Wnt signaling should alter their expression. To test this, we
examined the expression of left determining genes in embryos lacking
Axin, a negative regulator of the Wnt/ß-catenin signaling
pathway (Zeng et al., 1997).
Homozygous AxinTg1 embryos continued to express
Wnt3a and Nodal normally in the primitive streak; however,
Nodal expression in the node was slightly expanded
(Fig. 2U,W) and large ectopic
domains of symmetrical Nodal (Fig.
2U) and Lefty1/2 (Fig.
2W) expression were observed. Thus, both gain- and
loss-of-function alleles of genes in the Wnt/ß-catenin signaling pathway
lead to aberrant expression of left determining genes.
Cilia are structurally normal but display reduced polycystin 1 (PC1) expression
To determine whether a relationship between Wnt3a and cilia
structure or function exists, we first determined whether cilia were present
on the Wnt3a-/- node. Scanning electron microscopy (SEM)
analysis of mutant nodes at E7.75 revealed the presence of monocilia in the
ventral node (Fig. 3B), similar
to that observed in wild-type nodes (Fig.
3A). Immunofluorescent labeling of cilia with anti-acetylated
tubulin confirmed this, and further showed that the general morphology of the
node remained normal (compare Fig.
3C with 3D; data not shown). Quantitation of node cilia in
Wnt3a+/- (mean=158+/-13.7, n=4) and
Wnt3a-/- (mean=144+/-30.7, n=4) stage-matched
embryos revealed no significant differences in total cilia number. The
presence of structurally normal cilia indicates that Wnt3a does not lie
upstream of genes required for ciliary structure, such as Kif3a or
Kif3b as these mutants lack cilia
(Marszalek et al., 1999;
Nonaka et al., 1998
;
Takeda et al., 1999
).
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The presence of mechanosensory cilia in Wnt3a mutants was
evaluated by examining nodes for the expression of polycystin 1 (PC1) and PC2.
The Pkd1 gene product, PC1, interacts with PC2 and is thought to
control the gating of PC2 Ca2+ channels
(Delmas et al., 2004). While
cilia co-expressing PC1, PC2 and acetylated tubulin were easily found in the
wild-type node (arrows, Fig.
3J,O), similarly labeled cilia were rarely found in the mutant
(Fig. 3N,P,Q). Interestingly,
PC1 expression was significantly downregulated (P<0.0001, Welch's
t-test) in the Wnt3a-/- node cilia
(Fig. 3K,N), with only
6.8±5.8% (n=4 embryos) of the mutant cilia expressing
detectable levels of PC1, compared with the 46.9±0.9% (n=4) of
cilia that were PC1-positive in Wnt3a+/- embryos
(Fig. 3G,J). PC2 was strongly
expressed in more than 92% of central and peripheral cilia in both wild-type
and Wnt3a-/- nodes
(Fig. 3H,L). The rare cilium
that co-expressed PC1 and PC2 in Wnt3a-/- nodes expressed
PC1 weakly and in a much smaller spatial domain (arrow,
Fig. 3P) than in wild-type
cilia suggesting that mechanotransduction may be perturbed in the absence of
Wnt3a.
Wnt3a signals directly to the node and presomitic mesoderm via ß-catenin
Although Wnt3a is expressed in the dorsal posterior node, gene
expression in the Wnt3a mutants is perturbed in both the dorsal and
ventral node. To determine which tissues respond directly to Wnt signals, we
examined two independent transgenic lines that report sites of presumed
Wnt/ß-catenin activity in vivo. Both the TOPgal and BATlacZ (see
Materials and methods) transgenes were expressed in the node, primitive streak
and posterior mesoderm during LR determination stages
(Fig. 4A-C,E; see Fig. S4A-C in
the supplementary material) (Merrill et
al., 2004). The node was the strongest site of
ß-galactosidase (ß-gal) expression in TOPgal embryos at early somite
stages (Fig. 4A), with
particularly robust expression detected in the ventral node
(Fig. 4B).
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We reasoned that if the transcriptional activator ß-catenin was
asymmetrically expressed in the node, then target genes of the canonical
pathway should also be asymmetrically activated in the node. One such target
gene, Nkd1, a mammalian homolog of the Drosophila segment polarity
gene naked cuticle (Wharton et
al., 2001; Yan et al.,
2001
), was expressed symmetrically in the primitive streak, psm
and node at E7.75, but was asymmetrically distributed in the node by the
two-somite stage. Elevated levels were observed on the left side of the
ventral node, while expression in the psm remained symmetric
(Fig. 5E,F). Analysis of
Nkd1 expression in three- to six-somite stage
Wnt3a-/- embryos (n=4) revealed that transcript
levels and asymmetric expression were reduced in the node
(Fig. 5G,H). Expression in the
mutant psm was also downregulated. Thus, Wnt3a signals directly to the psm and
ventral node to activate expression of the Wnt/ß-catenin target gene
Nkd1.
Wnt3a regulates the Dll1/Notch pathway during LR determination and somitogenesis
As the Dll1/Notch signaling pathway directly controls Nodal
expression in the node (Raya et al.,
2003; Krebs et al.,
2003
), we investigated the possibility that the abnormal
Nodal expression domain in the Wnt3a mutant node may be due
to aberrant Notch signaling. Using Dll1 and Lfng as
reporters of Notch activity (Raya et al.,
2003
), we examined Notch activity in Wnt3a-/-
embryos. At E8, Dll1 was expressed in the streak and in psm
(Fig. 6A) in a pattern similar
to the Wnt reporter (Fig. 4C).
Dll1 was expressed in psm cells immediately adjacent to
Nodal-expressing peripheral ventral node cells
(Fig. 6C). Notably, expression
of Dll1 in Wnt3a-/- psm was posteriorized such
that Dll1-expressing cells only contacted the posterior-most node
(Fig. 6B,D). This domain
correlated well with the abnormally small domain of Nodal expression,
suggesting that Wnt3a regulates Nodal expression indirectly, via Dll1
and the Notch signaling pathway.
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Lunatic fringe (Lfng) expression oscillates in the psm and is
dependent upon Notch signaling (Barrantes
et al., 1999) and Wnt3a
(Aulehla et al., 2003
) at
tailbud stages. At early somitogenesis and LR determination stages when Nodal
is expressed in the left LPM, Lfng is expressed in a dynamic manner
that can manifest in a diffuse patch in the posterior primitive streak and as
two stripes adjacent, and anterior, to the node in the psm
(Fig. 6K). Interestingly,
Lfng is also expressed in the node periphery, overlapping with
Nodal expression in the node (Fig.
6K and not shown). Wnt3a-/- mutants (0-7
somites, n=7) displayed only a single abnormally shaped stripe of
Lfng expression posterior to the node and no expression was detected
in the node periphery (Fig.
6L). More than one set of stripes was never observed, suggesting
that dynamic oscillating Lfng expression did not occur in the absence
of Wnt3a. Together, the Axin2 and Lfng expression
patterns indicate that the segmentation clock does not function properly at
early somitogenesis stages in the absence of Wnt3a. Wnt3a appears to
play dual roles at these stages, signaling to the node and psm to regulate LR
determination and somitogenesis.
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Discussion |
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Wnt signaling and organ laterality
Although posterior organs did not develop in Wnt3a mutants because
of a requirement for Wnt3a for posterior development
(Takada et al., 1994),
laterality phenotypes in anterior viscera such as the heart, lungs and liver
were assessed. The majority of E11.5-12.5 Wnt3a-/- embryos
were heterotaxic, i.e. at least one organ displayed laterality defects. The
laterality phenotypes were not secondary to the posterior truncation phenotype
as aberrant gene expression in the node was observed well before AP phenotypes
emerged. In fact, Dll1 and Nodal are two of the earliest
known genes to be affected by the Wnt3a mutation and display aberrant
expression prior to somitogenesis, arguing that Wnt3a, signaling via
its target gene Dll1, regulates LR determination first, and
somitogenesis and AP elongation second.
The process of cardiac looping determines the relative positions of the
heart chambers and their connections with the aorta and pulmonary artery.
Alterations in the direction of cardiac looping lead to alignment defects that
result in a range of cardiovascular abnormalities such as TGA, PTA, double
outlet right ventricle (DORV) and atrioventricular septal defects (AVSD)
(Maclean and Dunwoodie, 2004).
Several of these anomalies were observed in the
Wnt3avt;Dll1 compound mutants, consistent with
Wnt3a and Dll1 functioning in a common genetic pathway to
regulate cardiac laterality.
The aberrant expression of Nodal in Wnt3a-/-
embryos presents an opportunity to examine the importance of the timing and
asymmetric nature of Nodal and Lefty signaling for organ
laterality. As Nodal is required in the node to activate
Lefty1 expression in the dorsal node and Nodal expression in
the LPM (Brennan et al., 2002),
we suggest that the reduced levels of Nodal in the
Wnt3a-/- node are insufficient to activate Lefty1
or Nodal at the two-somite stage when they are normally activated,
but are sufficient to account for the delayed Nodal expression
observed in the LPM. Lefty1 was never detected in the mutant PFP,
indicating that the midline barrier to Nodal diffusion was absent, and
providing an explanation for the bilateral expression of Nodal in the
>4-somite stage LPM. Interestingly, left pulmonary isomerism was not
observed in Wnt3a mutants, as would be predicted from the
Lefty1-/- phenotype
(Meno et al., 1998
). Instead,
the Wnt3a mutants more closely resembled cryptic mutants, which lack
Nodal expression in the LPM and display randomized situs and right
isomerism (Yan et al., 1999). As Nodal is not expressed bilaterally
in the Wnt3a-/- LPM until the five-somite stage, these
phenotypes suggest that asymmetric Nodal expression in the LPM must
be established between the two- to four-somite stages (a 6-hour window) to
establish proper LR asymmetry in anterior organs. Bilateral Nodal
expression in the LPM after the five-somite stage appears to be insufficient
to induce left isomerisms in any of the organs examined; however, it should be
noted that Nodal expression in the LPM never extended anteriorly into
the heart, as it did in wild-type embryos.
Wnts and polycystins in LR determination
The membrane receptor PC1 colocalizes with PC2 in renal mechanosensory
cilia where it senses mechanical bending of the primary cilium induced by
fluid flow, transducing it into a chemical Ca2+ flux by activating
PC2 (Nauli et al., 2003).
Kidney cells lacking PC1 form cilia but do not display Ca2+ influx
when stimulated by fluid flow (Nauli et
al., 2003
) or activating antibodies
(Delmas et al., 2004
). Our
observation that PC1 is co-expressed with PC2 in node mechanosensory cilia
suggests that a similar regulatory relationship between PC1 and PC2 exists in
node cilia. Embryos lacking Wnt3a display structurally normal node
cilia that robustly express PC2 but display reduced levels of PC1. These
results predict that Ca2+ asymmetry will be perturbed in the
Wnt3a-/- node, despite the presence of PC2, and this will
be addressed in future experiments. As PC1 and PC2 activity appear to be
mutually dependent, and mutations in either Pkd1 or Pkd2
result in identical polycystic kidney disease phenotypes
(Delmas, 2004
), it seems
likely that Pkd1 mutants will also display laterality defects and a
loss of Ca2+ asymmetry. The cardiovascular defects observed in
embryos homozygous for a targeted allele of Pkd1
(Boulter et al., 2001
) are
consistent with a role for Pkd1 in the regulation of cardiac
laterality.
Despite reports in the literature that PKD1 is a direct target
gene of Wnt/ß-catenin signaling
(Rodova et al., 2002), our
results suggest otherwise. Examination of 5 kb of the mouse Pkd1
promoter revealed five consensus Tcf1-binding sites; however, activation of
the Wnt/ß-catenin pathway did not activate Pkd1 promoter
luciferase reporter constructs in transient transfections in vitro (data not
shown). Furthermore, mutational analysis showed that the Tcf sites were not
necessary for basal expression. It is unclear how Wnt3a indirectly regulates
ciliary PC1 expression; however, it is tempting to speculate that the
mechanism involves inversin, another ciliary protein required for proper LR
determination (Watanabe et al.,
2003
) that has recently been shown to bind dishevelled and
regulate Wnt signaling (Simons et al.,
2005
).
Wnt3a signaling and target gene expression in the node
Although much of the LR phenotype observed in Wnt3a mutants can be
directly attributed to the aberrant expression of Dll1 in the psm,
and consequently of Nodal in the node, our data indicate that Wnt3a
also directly regulates gene expression in the ventral node. Two independent
Wnt/ß-catenin reporters, as well as the Wnt/ß-catenin target genes
Nkd1and Axin2, were expressed there. Interestingly,
Nkd1 expression was asymmetric in the ventral node. The significance
of this asymmetric expression, and the mechanisms underlying it, are presently
unclear. Given that Wnt3a is symmetrically expressed in the streak
and node, one possible mechanism, interpreted in the context of the morphogen
flow model, is that the Wnt3a ligand itself becomes asymmetrically distributed
at the ventral node surface by cilia-generated nodal flow. This hypothesis
would require that Wnt3a, which is expressed by the dorsal epiblast, is able
to traverse the ventral node epithelium to reach the apical surface of the
node where the cilia are located. This is unlikely to occur as the transverse
movement of secreted molecules across an epithelial tissue is blocked by the
tight junctions of the polarized epithelium. More importantly, this postulate
is not supported by our data demonstrating that neither of the
Wnt/ß-catenin reporters, nor Axin2, were asymmetrically
expressed in the node. Perhaps a more likely scenario is one in which
Nkd1 is symmetrically activated in the node by Wnt3a, but
Nkd1 mRNA becomes graded because of asymmetric localization or decay.
Although Nkd1 has also been shown to exhibit oscillatory gene
expression in the psm (Ishikawa et al.,
2004), its function remains unclear as animals lacking
Nkd1 do not display embryonic phenotypes
(Li et al., 2005
).
Wnt3a is a major component of the trunk organizer
Embryological studies performed primarily in amphibians, fish and chick
have demonstrated that the Spemann-Mangold organizer is a dynamic structure
that can be subdivided into head, trunk and tail organizers based on their
distinct cell subpopulations and differing inductive capacities
(Niehrs, 2004). In the mouse,
evidence for the distinction of all three organizers remains relatively scant
(Robb and Tam, 2004
); however,
a strong argument can be made for trunk organizer activity residing in the
node: (1) transplantation experiments demonstrate that the node is sufficient
to induce patterned ectopic trunks, but not heads
(Beddington, 1994
;
Tam et al., 1997
); (2)
surgical ablation studies show that the node is necessary for DV and LR
asymmetry, and proper segmentation and AP elongation of the prospective trunk,
but is not required for AP polarity
(Davidson et al., 1999
). The
timing of node formation, which occurs after AP polarity and head structures
have been specified, but before LR determination and trunk development, is
also consistent with the node functioning as a trunk organizer.
Our demonstration that Wnt3a is expressed in the node and is
required for LR determination and segmentation, coupled with previous studies
demonstrating that Wnt3a is required in a dose-dependent manner for
the formation of the entire posterior trunk and tail
(Greco et al., 1996;
Takada et al., 1994
), suggests
that Wnt3a is a major component of the trunk organizer. We present a model for
how Wnt3a could function in this capacity
(Fig. 7). A source of Wnt3a is
established at E7.5 in the primitive streak and node progenitors at the
posterior end of the gastrulating embryo. Wnt3a specifies mesoderm fates in
the streak by directly regulating T transcription
(Galceran et al., 2001
;
Yamaguchi et al., 1999
). Wnt3a
also regulates Dll1 expression in the psm directly, and indirectly
via T (Galceran et al., 2004
;
Hofmann et al., 2004
). Dll1
expression in the psm stimulates Notch activity at the psm/node boundary, to
activate Nodal transcription in the node periphery
(Krebs et al., 2003
;
Raya et al., 2003
). Activation
of Nodal in the lateral aspects of the node establishes an axis of
Nodal expression that is perpendicular to the AP axis, leading to the
orthogonal orientation of the LR axis. Elevated Notch activity also activates
Lfng in the node periphery, which could serve to restrict
Nodal to the node periphery by inhibiting Notch in a
negative-feedback loop (Dale et al.,
2003
).
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Our work implicates ß-catenin as the primary transducer of canonical Wnt signals that coordinately regulate LR specification and segmentation. The use of conditional ß-catenin alleles and Cre drivers that are expressed in the node or psm will help address the specific roles that ß-catenin plays in the node during LR determination, and in the psm in the regulation of oscillating gene expression during somitogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/24/5425/DC1
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ACKNOWLEDGMENTS |
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