The Salk Institute for Biological Studies, Gene Expression Laboratory,
10010 North Torrey Pines Road, La Jolla, CA 92037, USA
*
Equal first authors
Author for correspondence (e-mail
belmonte{at}salk.edu
)
Accepted 25 May 2001
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SUMMARY |
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Key words: ß-Catenin, Left-right, Nodal, PKA, Sonic hedgehog, TGFß, Wnt, Chick
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INTRODUCTION |
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Together with TGFßs, Hh and FGFs, the Wnt family of secreted factors
is involved in many developmental decisions in a variety of organisms (Wodarz
and Nusse, 1998; Peifer and
Polakis, 2000
). Although in
Xenopus Wnt signaling appears to be involved in orienting the LR
embryonic axis (Danos and Yost,
1995
; Nascone and Mercola,
1997
; Yost,
1998
), so far no clear role in
LR development has been described for any specific Wnt member in either chick
or mouse. With the aim of extending earlier observations in Xenopus
and improving our understanding of the role of Wnt factors in LR development,
we have analyzed in detail the expression patterns of several Wnt
genes during early development of the chick embryo. We show that a
Wnt gene that operates through ß-catenin, Wnt-8c, is
expressed on the right side of Hensen's node, although it actually acts as a
left determinant in the chick embryo. We also show that the activity of
ß-catenin is both necessary and sufficient to regulate Nodal
expression. Additionally, protein kinase A (PKA) also acts as a positive
regulator of Nodal, in a pathway independent of Wnt-8c.
Antagonism of Wnt/ß-catenin signaling (by N-cadherin) and of PKA activity
(by the endogenous PKA inhibitor PKI) may also contribute to further restrict
and localize Nodal expression to the left side of the embryonic
node.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridization
Antisense riboprobes for Nodal, Shh, Car, Fgf-8, cSnR and
Pitx2, and in situ hybridization procedures were as described
(Rodríguez-Esteban et al.,
1999). The Wnt-8c
antisense riboprobe was derived from the entire ORF of chick Wnt-8c,
cloned by RT-PCR from HH stages 12-16 chick embryos. Antisense riboprobes for
chick ß-catenin and the gene encoding subunit
of chick
PKI were similarly derived form RT-PCR fragments. The entire ORF of chick
ß-catenin was cloned using primers derived from the published
sequence (GenBank Accession Number, U82964). In the case of PKI, degenerated
primers were designed after comparison of consensus amino acid residues from
human and mouse PKI
subunit sequences. The 230 base pairs PCR fragment
obtained is identical to part of GenBank sequence U19496.
Bead implants
For treatment with blocking anti-Shh antibody
(Pagán-Westphal and Tabin,
1998), Affigel blue beads
(BioRad) were soaked in the antibody for several hours before they were
implanted in the left or right side of HH stages 4-5 chick embryos. Controls
were obtained by implanting beads soaked in PBS, and no phenotypic consequence
or changes in gene expression were observed. As previously described, and
based on its effects on Nodal and patched expression
(Pagán-Westphal and Tabin,
1998
), this antibody is a
powerful blocker of Shh signaling in vivo. Implantation of beads soaked in a
blocking anti-N-cadherin antibody was exactly as described (Garcia-Castro et
al., 2000
). We must point out
that Garcia-Castro et al. did not describe changes in Nodal
expression after implantation of the blocking antibody. This may be due to
slight differences in the experimental conditions used by these authors and by
ourselves. In previous experiments, we have noticed that visualization of
ectopic domains of Nodal expression may require long periods of
development of the in situ detection reaction, which may lead the researcher
to score some experimental embryos as false negatives for ectopic
Nodal expression. Beads soaked in forskolin (an activator of PKA via
its effect on adenylyl cyclase) or H89 (a specific inhibitor of PKA), both at
concentrations of 150 µM (dissolved in 4% DMSO in embryo medium) were
applied into the New cultured blastoderms at HH stage 4. These chemicals
appear to reach large areas of the embryo even when applied locally. Control
embryos were obtained by similar treatment with 4% DMSO in embryo medium, and
we never detected any phenotypic alteration or change in gene expression.
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RESULTS AND DISCUSSION |
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We next explored the pathway by which the Wnt-8c signal might influence LR
development in the chick. ß-catenin has been shown to act as an
intracellular mediator of Wnt signals (including Wnt-8c) in a variety of
systems, by translocating to the nucleus in response to Wnt and interacting
with TCF/LEF factors to generate transcriptionally active complexes that
regulate expression of Wnt targets (reviewed by Wodarz and Nusse,
1998; Peifer and Polakis,
2000
). The chick
ß-catenin gene is transcribed in the whole embryo at the early
stages examined, although it is more strongly expressed throughout the
primitive streak, with no detectable asymmetry in the node
(Fig. 1E). After infection of
the right side of HH stage 4 embryos with a retroviral construct expressing an
activated form of ß-catenin (RCAS-ß-cateninACT;
Capdevila et al., 1998
), which
activates Wnt targets independently of the presence of Wnts, we observed a
high incidence of reversal of heart situs and of hearts located in a middle
position that failed to loop towards either side of the embryo. This is
similar to what was observed with right-sided Wnt-8c implants, and
infections of the left side had no detectable effect (data not shown). This
result, together with the fact that Wnt-8c has been shown to operate through
ß-catenin in other organisms, led us to conclude that ß-catenin is
likely to mediate the effects of Wnt-8c on LR development in the chick embryo.
Moreover, and consistent with a requirement for ß-catenin in normal LR
development, overexpression of Axin (a well-characterized negative regulator
of ß-catenin) (Wodarz and Nusse,
1998
; Peifer and Polakis,
2000
) in the left side of HH
stage 4 embryos (schematized in Fig.
1F), results in heart alterations that reveal disruption of the LR
axis (in 9/26 embryos; Fig. 1I,
compare with control heart in Fig.
1G). Right-sided overexpression of Axin had no detectable effect
on LR development (n=15).
In order to analyze the molecular changes associated with the observed LR
defects, we repeated the experiments harvesting the embryos 5-10 hours after
implantation of Wnt-8c cells. Whole-mount in situ hybridization was
performed using riboprobes to detect expression of the Shh, Fgf-8, Car,
Nodal, cSnR and Pitx2 genes, all known to be involved in LR
determination. As indicated in Fig.
2A-G, ectopic Wnt-8c to the right side of the node
(schematized in Fig. 2A) has no
effect on the left determinants Shh (n=12) and Car
(n=16), and the right determinant Fgf-8 (n=20).
However, it strongly activates Nodal (in 6/20 embryos;
Fig. 2H,I) and the
Nodal target Pitx2 (in 5/13 embryos;
Fig. 2L,M), and represses
cSnR (in 4/12 embryos; Fig.
2J,K), normally strongly expressed on the right side of the embryo
(Isaac et al., 1997). Again,
very similar results were obtained after ectopic expression of
RCAS-ß-cateninACT on the right side (data not shown).
Moreover, antagonism of ß-catenin in the left side of the embryo by
overexpression of Axin (schematized in Fig.
2N) resulted in strong repression of both Nodal (in 8/20
embryos) and Pitx2 (in 7/20 embryos;
Fig. 2O,P, respectively) and
strong activation of cSnR (in 8/22 embryos;
Fig. 2Q). Shh and
Car were both unaffected (data not shown). These results confirm that
Wnt-8c acts as a left determinant in the chick embryo and that ß-catenin
is a very likely transducer of the Wnt-8c signal that induces Nodal.
Functional ß-catenin appears to be necessary for normal Nodal
expression in the left side of the embryo. Moreover, we believe that the
induction of Nodal by ectopic Wnt-8c or activated ß-catenin
observed on the right side of the embryo strongly suggests that this induction
also operates inside and around the node, especially considering that
expression of Wnt-8c is mostly restricted to the node and primitive
streak at the stages at which the experiments were performed.
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Because Shh has been shown to be both necessary and sufficient for
Nodal expression in the chick embryo
(Pagán-Westphal and Tabin,
1998), we decided to further
investigate a possible interaction between the Wnt-8c and Shh pathways
regarding induction of Nodal. Interestingly, while left application
of a blocking anti-Shh antibody (Fig.
3A) has been shown to repress Nodal expression (as we
observed in 9/10 embryos; red arrow in Fig.
3B), a graft of Wnt-8c cells implanted together with the
blocking anti-Shh antibody (Fig.
3C) is able to counteract this repression, thus maintaining
Nodal transcription (in 8/10 embryos;
Fig. 3D). Application of the
blocking anti-Shh antibody alone does not appear to alter the pattern or the
level of expression of Wnt-8c, whereas expression of the Shh target
patched is repressed, further confirming the blocking activity of the
antibody in our experimental setting (data not shown). These results, together
with the expression patterns of Wnt-8c and Shh in the node,
and the fact that Wnt-8c misexpression (or Wnt/ß-catenin
antagonism by Axin) do not affect transcription of Shh or
Car, indicate that specific levels of Wnt-8c may activate
Nodal in a pathway independent of Shh.
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A role for N-cadherin in the control of Nodal
The presence of Wnt-8c on the right side of the node would seem to
argue against its role as a left determinant, and yet our results show that
Wnt-8c is an upstream regulator of Nodal (a key left
determinant itself). As other Wnts are also expressed in the node (i.e.
Wnt-3a, which also signals through ß-catenin (Shimizu et al.,
1997), and Wnt-11), it is
possible that a combination of Wnt activities, rather than a single Wnt,
actually acts as the left determinant identified by our experiments. However,
neither Wnt-3a nor Wnt-11 show a left-specific expression
pattern that could counteract the right bias of Wnt-8c expression in
the node. And even if that is actually the case, why isn't Nodal also
transcribed on the right side of the node in response to Wnt-8c? One way to
resolve this apparent discrepancy would be to assume the presence of a
repressor or repressors of Nodal transcription exclusively on the
right side of the node, which could antagonize the activation of
Nodal by Wnts on the right but not on the left side of the node
(where Wnt-8c and other Wnt proteins, and also ß-catenin, are presumably
present). Accordingly, Wnts would act normally as regulators of Nodal
on the left side of the node (where the putative antagonist or antagonists are
not present).
A good candidate to act negatively on the transcription of Nodal
on the right side of Hensen's node is the adhesion molecule N-cadherin.
Recently, it has been described that expression of N-cadherin in the node of
the chick embryo is restricted to the right side, and that blocking its
activity results in LR alterations (Garcia-Castro et al.,
2000). Moreover, it is known
that high levels of cadherin expression negatively correlate with the
activation of Wnt targets by ß-catenin, because high levels of cadherin
may reduce the pool of cytoplasmic ß-catenin available for activating
nuclear targets of Wnt signals (Fagotto et al.,
1996
). Thus, we reasoned that
high levels of N-cadherin on the right side of the node could potentially
interfere with the activation of Wnt targets (such as Nodal) by
ß-catenin. To test this hypothesis, we blocked N-cadherin activity by
implanting beads soaked in a blocking anti-N-cadherin antibody to the right
side of the node of HH stage 4 embryos
(Fig. 3E), and analyzed
Nodal expression 6-12 hours after implantation. As indicated in
Fig. 3F, blocking N-cadherin
activity on the right side of the node results in induction of Nodal
expression (in 6/15 embryos). This effect is independent of Car
(which is negatively regulated by Fgf-8 on the right side of the
embryo (Rodríguez-Esteban et al.,
1999
; Yokouchi et al.,
1999
; Zhu et al.,
1999
), since Car
expression is normal in the treated embryos (data not shown). These results
indicate that N-cadherin normally represses Nodal, suggesting that
the presence of N-cadherin on the right side of the node may serve as a
mechanism that ensures that the Wnt pathway is unable to activate
Nodal transcription in that region of the node, despite the presence
of Wnt proteins and ß-catenin.
Control of Nodal by PKA
Given that both Shh and Wnt-8c control Nodal transcription, we
reasoned that the analysis of modulators of the Shh pathway could reveal
additional interactions between these two signaling pathways. As PKA has been
shown to antagonize Hedgehog signaling pathways in a variety of organisms, we
decided to explore a possible role for PKA as a repressor of Nodal,
which is a target of Shh during LR determination in the chick. We first
implanted a bead soaked in the PKA activator forskolin to the left side of the
node of HH stage 4 embryos, expecting a repressive effect on Nodal
transcription. Surprisingly, Nodal and its target Pitx2 were
unaffected on the left side (data not shown), but they were strongly activated
on the right side (in 9/20 and 4/20 embryos, respectively), and exactly the
same effect was obtained when implanting the bead on the right side (as shown
in red in Fig. 4A; ectopic
domains of gene expression are indicated by red arrows in
Fig. 4D,E). This suggests that
PKA, contrary to our expectations, acts as an activator of Nodal. Shh,
Car and Wnt-8c were unaffected (data not shown), indicating that
the activation of Nodal was not mediated by an ectopic maintenance of
Car expression on the right side of the embryo, or by an alteration
in the distribution of Wnt-8c expression. Alterations of heart
looping and gene expression are shown in
Fig. 4G (14/32 embryos had
either isomeric or left-looped hearts; compare with normal heart in
Fig. 4F). The fact that we
observe the same effect independently of the location of the bead suggests
that forskolin most probably reaches a large area of the embryo, unlike
secreted proteins or antibodies also applied using beads, both of which appear
to have a more localized effect. When embryos were treated in a similar way
with the specific PKA inhibitor H89 (left-sided implant is schematized in
green in Fig. 4A), expression
of both Nodal (in 7/20 embryos) and Pitx2 (in 4/10 embryos)
was completely repressed (red arrows in
Fig. 4H,I), and alterations of
heart looping occurred in 8/20 embryos
(Fig. 4J), which suggests that
PKA activity is necessary for normal expression of left-specific genes in the
chick embryo. The effect of H89 is also independent of the location of the
bead. Whilst both activation and repression of Hedgehog-dependent targets by
PKA has been described in Drosophila (Ohlmeyer and Kalderon,
1997), it appears that this is
the first case described in vertebrates of activation of Shh targets
by high levels of PKA activity. Complementing the results presented above, we
also found that the endogenous PKA inhibitor PKI has an asymmetric pattern of
expression, being more strongly expressed on the right side of the node
between HH stages 6 and 7 (Fig.
4L). This pattern of expression suggests that PKI could prevent
PKA (which is present in all cells) from activating Nodal
(Fig. 4N) on the right side of
the node. Interestingly, PKI and Nodal also appear to be mutually
exclusive at earlier stages of development
(Fig. 4K,M). Ectopic
application of Wnt-8c to the right side of the embryo failed to alter
PKI expression (data not shown).
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From all these data, we conclude that a PKA-dependent process is required for left-sided Nodal expression in the chick embryo, and that inhibition of PKA activity on the right side of the node by the endogenous inhibitor PKI is a possible mechanism by which Nodal is prevented from being transcribed in that location. This PKA-dependent pathway operates as a mechanism that controls Nodal expression in parallel to both Shh and Wnt-8c signals.
Multiple pathways contribute to localize Nodal
expression
Our results describe a situation where multiple regulatory mechanisms
control and restrict Nodal expression in and around the node. But why
is it important to have several independent ways of controlling Nodal
expression in Hensen's node? Left-specific expression of Nodal in the
node is likely to play a key role during LR determination, as hinted by the
fact that it is the earliest molecular asymmetry known to be conserved among
vertebrates (reviewed by Capdevila et al.,
2000). Moreover, recent data in
mice and zebrafish suggest that the presence of Nodal on the left
side of the node (and also the normal transduction of the Nodal signal) is
absolutely required for the transfer of LR positional information from the
node to the lateral plate mesoderm (LPM), including left-specific expression
of lefty-1 (in the presumptive floor plate), and of lefty-2
and Nodal (and hence Pitx2) in the LPM (reviewed by Schier
and Shen, 2000
). The exact
reason why left-sided expression of Nodal in the node is required for
the transfer of LR positional information to the LPM is still unknown. In any
event, and complementing the mouse and zebrafish data mentioned above, our
results in the chick embryo suggest that at least three signaling mechanisms
(triggered by Shh, Wnt/ß-catenin and PKA) operate independently to
restrict Nodal transcription to the left side of Hensen's node.
Taken together, our results allow us to propose a model for the involvement
of Wnts and PKA in LR determination in the chick embryo
(Fig. 5). We speculate that the
sum of Wnt activities in or around Hensen's node results in the activation of
Nodal transcription on the left side of the node, in a process
mediated by ß-catenin. The control of Nodal by Wnt genes appears
to be conserved during evolution, as the promoter of the mouse Nodal
gene contains LEF-1/TCF sequences, which are known to mediate transactivation
by ß-catenin (Norris and Robertson,
1999), and in
Xenopus, the Nodal-related genes Xnr1 and
Xnr3 also have Wnt-responsive regulatory sequences (McKendry et al.,
1997
; Hyde and Old,
2000
). Interestingly, the role
of Wnt signals as left determinants may not be limited to Wnts that signal
through ß-catenin. Wnt-11, which does not signal through ß-catenin,
has also some activity as a left determinant during LR determination in the
chick (C. R.-E., J. C., Y. K. and J. C. I. B, unpublished), further suggesting
that a complex network of Wnt signals controls Nodal expression. On
the right side of the node, the activation of Nodal by Wnts may be
antagonized by at least two mechanisms: (1) the presence of high levels of
N-cadherin, which can presumably inhibit ß-catenin-mediated
transactivation of Wnt targets; and (2) the presence of the endogenous PKA
inhibitor PKI, which may prevent the activation of Nodal by PKA. In
summary, the results presented here demonstrate a role for Wnt signaling and
PKA in LR development of the chick embryo, and underscore the importance of a
tight regulation of Nodal expression, as the key regulator of LR
development in vertebrates.
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ACKNOWLEDGMENTS |
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