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First published online 11 June 2008
doi: 10.1242/dev.018986
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Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.,
Author for correspondence (e-mail:
kling{at}cellbio.duke.edu)
Accepted 25 April 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: Noggin, Chordin, Nodal, BMP, Left-right asymmetry, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
This L-R asymmetric organ morphogenesis depends on earlier L-R axis
formation. In mouse, this is established around the node, the location of the
Spemann organizer at the end of gastrulation, and is propagated from the
midline to the lateral plate mesoderm (LPM)
(Shiratori and Hamada, 2006).
Nodal, a member of the transforming growth factor beta (TGFβ) superfamily
of secreted ligands, is a crucial left-side determinant. Nodal
expression occurs peripheral to the node and then in the left LPM
(Collignon et al., 1996).
Perinodal Nodal expression is required for Nodal expression
in the left LPM (Brennan et al.,
2002; Saijoh et al.,
2003). An initially low level of Nodal in the LPM can induce
Nodal expression itself, via a positive-feedback mechanism
(Saijoh et al., 2000). Nodal
also activates downstream target genes in the left LPM, such as
Lefty2, an inhibitor of Nodal itself
(Saijoh et al., 2000), and
Pitx2, which regulates left-side-specific morphogenesis
(Logan et al., 1998;
Yoshioka et al., 1998).
Inappropriate Nodal expression in the LPM thus leads to severe
laterality defects, underscoring the importance of determining how the
asymmetric expression of Nodal is regulated.
Bone morphogenetic proteins (BMPs), another class of the TGFβ
superfamily, play an important role in regulating Nodal expression in
the LPM. In chick embryos, a Cerberus-like factor, Caronte, is
expressed in the left paraxial mesoderm and left LPM, where it promotes
Nodal expression; it appears to do so by inhibiting Bmp2, Bmp4 and
Bmp7 in the LPM, suggesting a negative role for BMP in regulating
Nodal (Yokouchi et al.,
1999; Rodriguez-Esteban et
al., 1999). However, beads coated with BMP in the right LPM
activated Nodal, whereas beads coated with the BMP antagonist noggin placed in
the left LPM blocked Nodal expression
(Piedra and Ros, 2002). Thus,
data from the chick system have suggested both positive and negative roles for
BMP in regulating asymmetric Nodal expression.
How these results from the chick model relate to the roles of BMP in
mammalian L-R formation is unclear. Molecular regulation of L-R axis formation
may differ between chick and mouse (Meyers
and Martin, 1999), and there does not appear to be a mammalian
Caronte ortholog. In mouse, Bmp2 and Bmp4 are
expressed symmetrically in the LPM
(Fujiwara et al., 2002) and are
present at the right time and place to influence LPM Nodal
expression. Experiments in mouse embryos have also suggested either positive
or negative roles for BMP signals in regulating Nodal. Without
embryonic Bmp4, Nodal is not expressed, and embryos cultured with the
BMP antagonist noggin (Nog) do not express Nodal in the LPM
(Fujiwara et al., 2002). These
results suggest a positive role. Nevertheless, embryos lacking the BMP
signaling component genes Smad5 or Alk2 (Acvr1 -
Mouse Genome Informatics) show bilateral Nodal expression in the LPM
(Chang et al., 2000;
Kishigami et al., 2004),
suggesting a negative role in the mouse. This conclusion is consistent with
data from other vertebrates; for example, exogenous introduction of a
constitutively active BMP receptor causes diminished Nodal expression
in the Xenopus embryo (Ramsdell
and Yost, 1999). Similarly, ectopic expression of bmp2b
throughout the zebrafish embryo causes diminished Nodal expression
(Chocron et al., 2007).
Overall, it is clear that BMP signaling plays an important role in regulating
asymmetric Nodal expression during mammalian development, but both
its function in doing so and the manner in which it is regulated remain
unresolved.
The organizer BMP antagonists Nog and chordin (Chrd) regulate BMP signaling
prior to ligand-receptor binding (Balemans
and Van Hul, 2002). Chrd;Nog double-mutant mouse embryos
exhibit defects in the formation of all three embryonic axes
(Bachiller et al., 2000), but
there has been no analysis of the role of these factors in establishing the
L-R axis. Here we use genetics and molecular embryology to study the roles of
BMP signaling and BMP antagonism in regulating Nodal expression
during mammalian L-R axis establishment.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Chrd is homozygous viable in this genetic background, without the
phenotypes of the specific backgrounds previously reported
(Bachiller et al., 2003).
Nodaltm1Rob (Collignon
et al., 1996) and Bmp4tm2Blh
(Lawson et al., 1999)
heterozygotes were maintained in an ICR genetic background. Triple and
quadruple mutants were generated by crossing
Chrd-/-;Nog+/-;Bmp4+/-
with Chrd-/-;Nodal+/-.
Embryos were collected at E8.0-8.5 or E9.0-9.5, or after culture, fixed at
4°C overnight in 4% paraformaldehyde in PBS, washed in PBS and stored at
-20°C in methanol. After gene expression analysis, genomic DNA was
prepared from each embryo and genotyped by PCR as described: Nog and
Chrd (Anderson et al.,
2002); Bmp4
(Stottmann et al., 2006);
Nodal (Collignon et al.,
1996).
Gene expression assays and immunostaining
Whole-mount in situ hybridization (WMISH) was performed according to
standard procedures (Yamamoto et al.,
2004) with the following probes: eGFP, Lefty2, Lefty1
(Nakamura et al., 2006);
Lfng (Kume et al.,
2001); Cryptic (Shen
et al., 1997); Nodal
(Collignon et al., 1996);
Pitx2c (Liu et al.,
2001); Nog (McMahon
et al., 1998); and Chrd
(Klingensmith et al., 1999).
lacZ staining was performed as described
(Stottmann et al., 2001).
Quantitative (q) RT-PCR used total RNA prepared from 20 pieces of left or
right 4-5s LPM using Trizol (Invitrogen) and glycogen carrier (Ambion), with
reverse transcription employing a Taqman RT-PCR Kit (Applied Biosystems) after
DNase I treatment (Ambion). qPCR was performed using the MyiQ Real-Time PCR
System and SYBR Green mix (Bio-Rad) with the following primers (forward and
reverse): Nog, 5'-TTTTGGCCACGCTACGTGAA-3' and
5'-CTAGCAGGAACACTTACACT-3'; Chrd,
5'-TTCCCAGAGAATCAGAGCTG-3' and
5'-TCTGGAAGGGTTCTAGTCTC-3'; Nodal,
5'-ACTTTGCTTTGGGAAGCTGA-3' and
5'-ACCTGGAACTTGACCCTCCT-3'; Bmp4,
5'-AGACCCTAGTCAACTCTGTT-3' and
5'-CTCTACCACCATCTCCTGAT-3'; β-actin,
5'-AAGAGCTATGAGCTGCCTGA-3' and
5'-CACAGGATTCCATACCCAAG-3'. Whole-mount immunostaining was
performed as described with anti-phospho-Smad1/5/8 antibody (Cell Signaling)
(Yang and Klingensmith, 2006)
or anti-acetylated tubulin (Sigma) (Nakaya
et al., 2005). Node areas were calculated by NIH Image software
and statistical treatment used the 
2 test.
Whole embryo and explant culture
For whole embryo culture, headfold-stage embryos were isolated and parietal
endoderm removed. Liposomes composed of expression vectors and Lipofectamine
2000 (Invitrogen) were injected between the endoderm and LPM (see Fig. S3 in
the supplementary material). Expression vectors were eGFP, with the
eGFP gene in the pCAGGS vector
(Okabe et al., 1997), and
Nog, Bmp4 or Nodal, in which each coding region was inserted
into pEF-BOS (Mizushima and Nagata,
1990). For control embryos, GFP vector was injected
alone. For experimental embryos, equal concentrations of eGFP and secreted
product vectors were used. Liposome solution was prepared as follows: 12.5
µl OPTI-MEM (Invitrogen) was mixed with 1 µl Lipofectamine 2000 or with
1 µg expression vector(s). These solutions were combined and incubated for
5 minutes at room temperature; 0.1 µl solution was injected. Embryos were
cultured with rotation for 14 hours in a humidified atmosphere of 5%
CO2/95% air at 37°C in Dulbecco's Modified Eagle's Medium
(DMEM) with 50% rat serum. Embryos with direct eGFP fluorescence in the
desired regions were selected for WMISH with probes for GFP and a
relevant marker. For Nodal signaling experiments, 3s ICR embryos were cultured
with DMSO (0.1% in DMEM) or SB431542 (Sigma) in 0.1% DMSO (100 µg/ml) for 3
hours until 5s.
Node explants (including peripheral tissues) were isolated from 1-3s ICR embryos and cultured for 4 hours at 37°C and 5% CO2 in DMEM containing 10% FCS. Control culture was performed with BSA (200 ng/ml). Experimental culture was performed with recombinant human BMP2 (200 ng/ml) (R&D Systems). Twenty explants from either treatment were used to isolate total mRNA. qRT-PCR was performed using the following primers (forward and reverse): Lfng, 5'-CACCATTGGCTACATTGTAG-3' and 5'-CAAACATGCCATAGCTTCAGG-3'; Dll1, 5'-AAGTGCCAGTCACAGAGCTC-3' and 5'-TGCAGACAGAACATACACCG-3'; Notch1, 5'-AGTCAGGCAGATGTACAACC-3' and 5'-AGGAACTGGGTAGTGGTCAT-3'; Rbpjk, 5'-ACCTTCACCTACACACCAGA-3' and 5'-GACGATGTGACACTGGTAGA-3'.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Nakamura et al., 2006).
Accordingly, we assayed the spatiotemporal distribution of BMP signaling
activity during early somite stages. We used immunohistochemistry to visualize
binding of antibodies to phosphorylated Smads 1, 5 and 8 (p-Smad1/5/8; Smad 8
is also known as Smad9 - Mouse Genome Informatics) in mouse embryos.
Activation of the BMP receptor complex by BMP ligand binding causes Smad1/5/8
phosphorylation and subsequent signal transduction
(Kishigami and Mishina, 2005).
As previously shown (Yang and
Klingensmith, 2006), the distribution of active BMP signaling
during gastrulation and neurulation largely coincides with expression of
Bmp4 and other BMP genes (Furuta
et al., 1997; Solloway and
Robertson, 1999). At 2s, p-Smad1/5/8 expression was observed
bilaterally in all embryos. At 3s, 75% of embryos (9/12) still expressed
bilaterally, but 25% (3/12) showed weaker expression in left LPM. Strikingly
asymmetric p-Smad1/5/8 distribution was observed in all embryos at 4-5s, with
elevated levels in the right LPM relative to the left
(Fig. 1A-C). By contrast,
Bmp4 is expressed bilaterally in the LPM at 4-5s
(Fig. 1D,E)
(Fujiwara et al., 2002).
Expression of Bmp2 is bilateral like Bmp4
(Fujiwara et al., 2002), and
Bmp7 is in the node and midline
(Solloway and Robertson,
1999). In summary, we observed higher levels of BMP signaling
activity in the right LPM than in the left, although none of the known BMP
ligands active at this time shows a similarly biased expression pattern. This
left-sided decrease in BMP signaling occurs at or just before the time when
Nodal begins to be expressed in the left LPM.
Our results suggest that asymmetric activation of BMP signaling in the
mammalian LPM does not depend on BMP gene expression patterns. It might
instead be created by asymmetric BMP antagonist activity. We therefore
examined expression of Chrd and Nog in this context. At
embryonic day 8 (E8.0), Nog is expressed robustly in the node,
notochord and lateral neural folds
(McMahon et al., 1998), and at
lower levels in heart, allantois and LPM. At 4-5s, elevated expression was
observed in the left LPM relative to the right
(Fig. 1F-H). Chrd was
expressed broadly at low levels, with higher levels in node and notochord;
however, we also detected increased Chrd hybridization in left LPM
relative to the right (Fig.
1I-K). Embryo sections confirmed that these expression domains are
in the LPM itself, rather than in adjacent germ layers (see Fig. S1 in the
supplementary material). We independently assayed BMP antagonist expression by
quantitative RT-PCR (qPCR) using RNA isolated from left or right LPM at 4-5s,
finding that both Nog and Chrd are expressed at higher
levels in the left LPM (Fig.
1L). Together, these results indicate that the BMP antagonists
Nog and Chrd are expressed at elevated levels in left LPM
(Fig. 1M), implicating them in
producing asymmetric BMP signaling activity during early L-R axis
formation.
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) (Fig. 2A-D;
see Table S1 in the supplementary material). By contrast,
Chrd-/-;Nog+/- and
Chrd-/-;Nog+/+ embryos all exhibited normal
heart looping (Fig. 2D). These
results suggest that the BMP antagonists Nog and Chrd function redundantly in
L-R axis formation. We therefore investigated the expression of Nodal
and other asymmetrically transcribed genes in
Chrd-/-;Nog-/- embryos. Nodal
expression was greatly diminished or absent in 82% of embryos (n=11)
(Fig. 2E,F). A few embryos (9%)
expressed Nodal in right LPM (Fig.
2I). Expression of Lefty2 and Pitx2, targets of
Nodal in the left LPM (Hamada et
al., 2002), was also diminished or absent in most
Chrd-/-;Nog-/- embryos
(Fig. 2F,H,I; see Fig. S2 in
the supplementary material). Expression of Cryptic (Cfc1 -
Mouse Genome Informatics), a Nodal signaling component, was unchanged (see
Fig. S2 in the supplementary material). These results suggest that
Nog and Chrd are required for mammalian L-R axis
establishment by promoting Nodal expression in the left LPM.
Noggin and chordin are required for Nodal expression around the node and for node morphology
A key factor in initiating Nodal expression in the left LPM is
Nodal expression around the node
(Brennan et al., 2002;
Saijoh et al., 2003). Given
that Chrd and Nog are expressed in the node
(Klingensmith et al., 1999),
and that LPM expression of Nodal is absent in most
Chrd-/-;Nog-/- mutants, we also examined
perinodal expression of Nodal. This assay revealed three populations:
29% of Chrd-/-;Nog-/- mutants (7/24)
expressed perinodal Nodal at essentially the same level as in wild
type (denoted ++); 58% of mutants (14/24) showed greatly reduced
expression (+); and 12% showed no detectable Nodal expression around
the node (-) (Fig. 3A-E). These
data reveal that Chrd and Nog play an important role in
promoting perinodal Nodal expression, but are not necessarily
essential for its expression.
We correlated the level of Nodal expression around the node with the status of Nodal expression in the LPM in the Chrd-/-;Nog-/- mutants, revealing two populations. Mutants showing essentially normal levels of Nodal around the node (the `++' class) showed weak but detectable expression of Nodal in the LPM (3/7). By contrast, embryos showing weak (+) or absent (-) Nodal expression around the node (17/17) showed no expression of Nodal in the LPM (Fig. 3F). Thus, Chrd and Nog promote Nodal expression around the node and subsequent expression in LPM.
These results imply that ectopic BMP activity around the node would downregulate local Nodal expression. We observed ectopic anti-p-Smad1/5/8 staining around the nodes of Chrd-/-;Nog-/- mutants relative to wild-type embryos at the stages we assayed, including presomitic bud-stage embryos (data not shown) and 4-5s embryos (see Fig. S3 in the supplementary material). This indicates that BMP signaling is indeed increased perinodally in the absence of Chrd and Nog. To directly test the consequences of ectopic BMP around the node on Nodal expression, we cultured explants isolated from 1-3s wild-type embryos with recombinant human BMP2 protein. Whereas the control carrier protein BSA had no affect, ectopic BMP markedly decreased perinodal Nodal expression (see Fig. S3 in the supplementary material).
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; Raya et al.,
2003). To assess Notch signaling in
Chrd-/-;Nog-/- embryos, we assayed expression
of lunatic fringe (Lfng), a positive transcriptional target in the
presomitic paraxial mesoderm (Morales et
al., 2002). We observed sharply reduced levels of Lfng
expression just lateral and anterior to the node at the early somite stage in
the mutants (see Fig. S3 in the supplementary material), suggesting that BMP
reduces the activity of the Notch pathway. To test this, we cultured explants
from the node region of wild-type embryos with BMP2. Expression of the Notch
signaling targets Dll1 and Lfng was reduced (see Fig. S3 in the supplementary
material). We also observed dysmorphic nodes in
Chrd-/-;Nog-/- embryos, of abnormal shape and
with fewer cilia (Fig. 3G-I).
Along with L-R heart looping defects, similarly dysmorphic nodes and absent
perinodal Nodal expression have been observed previously in embryos
lacking the Notch signaling components Dll1
(Przemeck et al., 2003) and
Baf60c (Smarcd3 - Mouse Genome Informatics)
(Takeuchi et al., 2007).
Collectively, these findings indicate that BMP antagonism in the node via Chrd
and Nog is necessary for expression of Nodal around the periphery of
the node, and suggest that the loss of this expression in
Chrd-/-;Nog-/- embryos results from decreased
activity of the Notch pathway.
Ectopic noggin in left LPM rescues local Nodal expression in Chrd;Nog mutants
Some Chrd-/-;Nog-/- mutants with significant
perinodal Nodal expression nevertheless lacked Nodal
expression in the left LPM (Fig.
3B,F). Moreover, in wild-type embryos, both Chrd and
Nog are elevated in left LPM, whereas BMP signal transduction through
Smad1/5/8 is reduced there relative to the right LPM. These findings raise the
possibility that beyond promoting Nodal expression around the node,
Nog and Chrd have an additional direct function to promote
Nodal expression in the LPM. Accordingly, we investigated BMP
signaling activity in the LPM of Chrd-/-;Nog-/-
embryos by assaying p-Smad1/5/8 staining. We observed increased staining, with
bilaterally equivalent levels in left and right LPM
(Fig. 4A,B), indicating that
Chrd and Nog are required for the left-sided reduction in BMP signaling.
To directly test whether BMP antagonism in the left LPM per se can promote Nodal expression therein, we introduced a Nog expression vector into the left LPM of Chrd-/-;Nog-/- embryos at the late headfold stage (schematized in Fig. S4 in the supplementary material). GFP was included with Nog to mark transfected cells. In control injections, we introduced the GFP vector alone. Embryos were then cultured to 5-6s and assayed by in situ hybridization for Nodal and GFP. The results are summarized in Fig. 4F. Injection of Nog and GFP vectors into the left LPM of Chrd-/-;Nog-/- embryos resulted in Nodal expression in left LPM in 40% of embryos (10/25), whereas introduction of GFP vector alone resulted in 4% of mutants (1/23) expressing Nodal in the left LPM (Fig. 4Fa), a highly significant difference (P<0.001).
We then considered how the expression of Nodal in the left LPM related to the expression of Nodal around the node in the mutants injected with Nog and/or GFP vectors (Fig. 4Fb). Among embryos showing robust Nodal expression around the node (++), 4/6 also showed Nodal expression in the LPM when Nog was ectopically expressed there. By contrast, only 1/5 of such embryos expressed Nodal in the left LPM when only GFP was injected. In Chrd-/-;Nog-/- embryos with low perinodal Nodal expression (+), 6/15 showed Nodal in the left LPM when Nog was injected into this tissue (Fig. 4E). None (0/14) expressed Nodal in the left LPM when only GFP was introduced (Fig. 4D). This difference is significant (P=0.007). When the mutant embryos showed no detectable Nodal around the node, however, ectopic Nog in the left LPM never resulted in Nodal expression there (0/4). These data indicate that BMP antagonism in the left LPM promotes Nodal expression in this tissue, but BMP antagonism might not be sufficient for Nodal expression in the left LPM. Instead, it is likely to depend on synergistic influences from the node region, such as Nodal itself. Thus, BMP antagonism appears to create a permissive environment for Nodal expression in the left LPM.
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Chordin and noggin synergize with Nodal to promote left-side determination by impeding endogenous Bmp4
Nodal promotes its own expression in the left LPM via a positive-feedback
loop (Shiratori and Hamada,
2006), and our results indicate that BMP antagonism also promotes
Nodal expression in the LPM. To genetically assess the relevance of
our embryo culture experiments to Nodal regulation, we produced
embryos mutant for combinations of Chrd, Nog, Nodal and Bmp4
null alleles, then assayed Nodal expression. Embryos of the control
genotypes Nodal+/-, Chrd-/- and
Chrd-/-;Nog+/- were indistinguishable
from wild type, showing normal robust Nodal expression in the node
and left LPM (Fig. 5A,B,C,F).
By contrast, Chrd-/-;Nodal+/- and
Chrd-/-;Nog+/-;Nodal+/-
embryos fell into two distinct classes (see Table S2 in the supplementary
material). One had robust Nodal expression around the node, as in
wild-type embryos (denoted ++). Among these, 66% of
Chrd-/-;Nodal+/- embryos and only
12.5% of
Chrd-/-;Nog+/-;Nodal+/-
embryos showed Nodal expression in the left LPM
(Fig. 5D,G). Embryos in the
second class showed markedly reduced perinodal Nodal expression (+),
of which 42% of Chrd-/-;Nodal+/-
embryos expressed Nodal in the left LPM but at reduced levels,
whereas the rest showed no expression (Fig.
5E). Strikingly, no
Chrd-/-;Nog+/-;Nodal+/-
embryos showed any expression of Nodal in the LPM
(Fig. 5H). Thus Chrd,
Nog and Nodal have a positive genetic interaction that promotes
robust Nodal expression in the left LPM.
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),
raising the possibility that it is an endogenous BMP ligand antagonized by
Chrd and Nog in the left LPM. We therefore reduced the gene dosage of
Bmp4 in the triple
Chrd-/-;Nog+/-;Nodal+/-
mutants to see whether the phenotypic defects were lessened. Indeed, many
quadruple mutant
Chrd-/-;Nog+/-;Nodal+/-;Bmp4+/-
embryos showed rescue of Nodal expression in the left LPM, with 42%
expressing robust levels of perinodal Nodal (++) and 25% expressing
low levels (+) (Fig. 5J,K). By
contrast, when Bmp4 dosage was normal, only 13% of
Chrd-/-;Nog+/-;Nodal+/- embryos
showed Nodal expression in LPM when perinodal expression was robust
(Fig. 5G), and none showed such
expression when perinodal expression was weak
(Fig. 5H). These results
suggest that endogenous left-side elevated Chrd and Nog
expression permits activation of a Nodal positive-feedback loop in
the left LPM by antagonizing local Bmp4 activity.
Elevated BMP antagonist expression in the left LPM is induced by Nodal
Given the significance of Nodal as a left-side determinant, we considered
whether the asymmetry in BMP antagonist expression is initially established by
left-side-specific Nodal signaling. Nog expression in the left LPM
correlates spatially and temporally with Nodal expression (see Fig.
S6 in the supplementary material); moreover, we never observed asymmetric
Nog expression in the LPM of
Chrd-/-;Nog-/- embryos
(Fig. 6B,D), in contrast to
wild type (Fig. 1) and
Nog-/- homozygotes
(Fig. 6A,C). Also,
Nodal is expressed in the left LPM of Nog-/- (see
Fig. S5 in the supplementary material) but not of
Chrd-/-;Nog-/- embryos
(Fig. 2F,I). These data suggest
that elevated BMP antagonist expression in the left LPM might be a product of
left-side-specific Nodal signaling.
To explore this possibility, we cultured wild-type embryos with a specific
inhibitor of Nodal signaling, SB431542
(Inman et al., 2002). It
inhibits activation of TGFβ signaling by blocking the phosphorylation
ability of the activin type I receptors Alk4, 5 and 7 (Acvr1b, Tgfbr1 and
Acvr1c, respectively - Mouse Genome Informatics) without affecting BMP
signaling activation (Inman et al.,
2002). SB431542 can block both endogenous and exogenous signaling
activation via Smad2 phosphorylation in embryos
(Ho et al., 2006). We first
evaluated expression of the Nodal target gene Lefty2 in embryos
subjected to SB431542 or to the carrier, DMSO. Embryos cultured with DMSO
alone showed normal Lefty2 expression in left LPM
(Fig. 6E), whereas embryos
cultured with SB431542 lacked Lefty2 expression
(Fig. 6F). Thus, Nodal
signaling is inhibited by SB431542 treatment in this assay. The DMSO-treated
embryos showed normal elevation of Nog expression in the left LPM
(Fig. 6G). By contrast, embryos
cultured with SB431542 lacked left-side elevation of Nog expression,
it being reduced to approximately the same basal level as in the right LPM
(Fig. 6H). We also assayed BMP
antagonist expression by qPCR in left and right LPM of 4-5s embryos treated
with DMSO or SB431542. Both Nog and Chrd expression levels
in the left LPM were dramatically decreased by SB431542 treatment (see Fig. S7
in the supplementary material).
Lastly, we investigated the consequences of ectopic Nodal in the
right LPM on BMP antagonist expression. Control embryos injected with the
GFP expression vector showed normal Lefty2 expression in
left LPM and left-side elevation of Nog expression
(Fig. 6I,K). As expected from
previous results (Nakamura et al.,
2006), embryos injected with the Nodal vector into the
right LPM showed reversed, right-side expression of Lefty2
(Fig. 6J). Thus, these
conditions created a L-R reversed Nodal signaling context. In such embryos,
Nog expression was elevated on the right side rather than on the left
(Fig. 6L). Altogether, these
results reveal that the left-side-specific elevation of Nog
expression is produced by Nodal-dependent left-side determination, and suggest
that increased BMP antagonist expression in the left LPM is induced by Nodal
signaling.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Chordin and noggin promote Nodal expression around the node
A crucial leftward flow created by rotating monocilia in the node leads to
the asymmetric distribution of extracellular cues, with subsequent transfer of
asymmetry cues to the left LPM (Shiratori
and Hamada, 2006). Perinodal expression of Nodal is
required for Nodal expression in left LPM
(Brennan et al., 2002;
Saijoh et al., 2003), and may
itself be a component of the laterality signal transferred from the node to
the LPM (Oki et al., 2007).
Chrd;Nog mutant embryos form nodes, but of abnormal morphology and
reduced cilia density. They have a variable reduction of Nodal
expression around the node, ranging from nearly normal to none detectable.
These data indicate that BMP antagonists promote perinodal expression of
Nodal but are not essential for it.
The initial L-R defects in Chrd;Nog mutants might be explained by
one or more of the following mechanistic possibilities. The sparse cilia or
dysmorphic shape in Chrd;Nog nodes might be inadequate to generate
sufficient levels of essential leftward cues. Or, an unknown BMP-like molecule
might directly repress perinodal Nodal expression, and the lack of
local BMP antagonism in the mutants would leave it unopposed. A third
possibility is that Chrd;Nog mutants lack sufficient activity of a
positive regulator of Nodal expression in this domain. Data suggest
this might be the Notch pathway, which is known to promote Nodal
expression around the node (Krebs et al.,
2003; Raya et al.,
2003). We observed reduced Lfng and Dll1
expression, genes that are positively regulated by Notch signaling, in the
vicinity of the node. The phenotypes we observed of heart looping defects,
dysmorphic nodes and reduced perinodal Nodal expression are similar
to those of embryos lacking the Notch signaling components Dll1
(Przemeck et al., 2003) and
Baf60c (Takeuchi et al.,
2007). These findings imply an antagonistic relationship between
BMP and Notch signaling in L-R patterning at the node. A precedent for such a
relationship has been documented in the cerebellar rhombic lip
(Machold et al., 2007).
BMP signaling represses Nodal expression in LPM
We observed higher levels of endogenous BMP signaling activity in the right
LPM, i.e. in the side lacking Nodal expression. Our site-specific
manipulation experiments demonstrated that BMP signaling in the LPM leads to
local repression of Nodal expression. Introduction of a Bmp4
expression vector into portions of the left LPM resulted in a coincident
reduction in Nodal expression, whereas exogenous Nog
similarly introduced into the right LPM caused ectopic Nodal
expression in the right LPM. These results strongly support the conclusion
that the right-sided elevation of endogenous BMP signal activity functions to
repress Nodal expression in the right LPM.
Additional evidence from Xenopus, zebrafish, chick and mouse
studies also supports a negative regulatory role for BMP signaling on
Nodal expression in the LPM (see Introduction). Nevertheless, some
data suggest a positive role for BMP signaling in regulating Nodal
expression in the LPM. Of greatest relevance is a previous report in mouse
that Bmp4 is a positive regulator of Nodal expression in the left LPM
(Fujiwara et al., 2002), which
is the opposite conclusion to ours. Fujiwara et al. found that when
Bmp4 was absent in extraembryonic as well as embryonic tissues, node
morphology was abnormal and Nodal expression was absent. When only
embryonic expression was missing, node morphology was restored but
Nodal was still absent, in both node and LPM. However, because
Bmp4 is never expressed in the node or its vicinity, it seems very
unlikely that Bmp4 has a local role in regulating Nodal expression
around the node. It is more likely that the node was functionally abnormal
owing to defects in primitive streak or mesodermal development, despite
looking morphologically intact.
|
)
cultured wild-type embryos with Nog after Nodal expression was
established around the node; this resulted in a lack of Nodal in the
LPM. By contrast, our data demonstrate that BMP activity has a negative role
directly in the LPM to downregulate Nodal. Moreover, our genetic
crosses demonstrate that Bmp4 per se has an antagonistic relationship
with Chrd, Nog and Nodal in expression of Nodal in
the LPM. We speculate that the discrepancy between the results of these
studies might be owing to experimental differences in the timing or
specificity of manipulations. For example, because the exposure to Nog
recombinant protein in these cultured embryos was ubiquitous rather than
specific to the left LPM, we suggest that the lack of Nodal
expression might have been due to an indirect effect of defective production
or transport of the laterality signal from the node to the LPM, rather than to
a direct effect on the LPM.
BMP antagonism in the left LPM relieves the repressive effects of BMP on Nodal expression
Our analysis revealed asymmetric Nog and Chrd expression
in the LPM. Elevation of expression of these genes on the left side is
consistent with the endogenous right-side elevation of BMP signal activity we
observed in wild-type embryos. By contrast, Chrd;Nog embryos showed
bilaterally equivalent BMP signal distribution in the LPM. Introduction of
exogenous Nog directly into the LPM of Chrd;Nog embryos
rescued Nodal expression in the LPM in some embryos. Thus,
Nog and Chrd promote Nodal expression in both the
node and LPM, and function synergistically to help establish the L-R axis
sequentially in the node and LPM.
The functional significance of this asymmetric BMP antagonist distribution was further supported by the genetic interaction of Chrd, Nog and Nodal. Chrd-/-;Nog+/-;Nodal+/- embryos showed diminished Nodal expression in LPM even in those embryos having robust Nodal expression around the node. Removal of one functional allele of Bmp4 in this compound mutant partially rescued Nodal expression in the left LPM, implying that endogenous Nog and Chrd function in the left LPM to protect Nodal expression by antagonizing local Bmp4.
Robust Nodal expression in the LPM is established by a
positive-feedback loop mechanism dependent on Nodal itself
(Saijoh et al., 2000). An
early step toward Nodal expression in the left LPM appears to be the
transfer of a small amount of Nodal from the node
(Oki et al., 2007). We showed
that asymmetric Nog expression in LPM is induced by Nodal.
Accordingly, the physiological significance of the endogenous asymmetric BMP
antagonist expression in the LPM might be to maintain this Nodal
positive-feedback loop. Meanwhile, left-side expression of Nodal is
suggested to suppress Nodal expression on the right side through
induction of Nodal inhibitors (Nakamura et
al., 2006). Our finding of right-side elevated BMP signal
distribution and its repressive effect on Nodal expression might
function synergistically with this mechanism.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/14/2425/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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