Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 West Holcombe Boulevard, Houston, TX 77030, USA
* Author for correspondence (e-mail: jmartin{at}ibt.tamhsc.edu)
Accepted 10 October 2005
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SUMMARY |
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Key words: Bone morphogenetic protein, Epithelial-mesenchymal transition, Cardiac morphogenesis, Mouse
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Introduction |
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In mouse embryos, the signals that induce EMT are less well understood.
Data from the in-vitro collagen gel assays suggested that Tgfß2 is the
signal required for EMT; however, mice homozygous for a null allele of
Tgfß2 still make cushions, indicating that other signals are required in
vivo to induce EMT in mammals (Bartram et
al., 2001; Camenisch et al.,
2002
; Sanford et al.,
1997
). Notch signaling, through regulation of the Snai1
(previously Snail) repressor, has recently been shown to be required
for cushion formation through downregulation of vascular/endothelial-cadherin
(VE-cadherin) in cushion endocardium
(Timmerman et al., 2004
). In
zebrafish, Wnt signaling has been implicated in the EMT required for valve
formation (Hurlstone et al.,
2003
). However, mice with an endocardial-specific deletion of
ß-catenin have cushions, although defective, suggesting that Wnt
signaling is not essential for EMT in mammals
(Liebner et al., 2004
).
In addition to providing a signal to the endocardium for AV cushion
formation, the AV myocardium is phenotypically distinct from the chamber
myocardium. Chamber myocardium is coupled intercellularly, has a fast
contraction pattern and expresses the chamber-specific genes Cx40
(Gja5 - Mouse Genome Informatics), Anf (Nppa -
Mouse Genome Informatics) and chisel (Smpx - Mouse Genome
Informatics). By contrast, the smooth-walled myocardium of the AV canal
retains an embryonic phenotype and fails to express genes that are found in
the chamber myocardium (Habets et al.,
2002).
T-box (Tbx) genes have been implicated in regional patterning of the
myocardium (Plageman and Yutzey,
2005). Tbx2 has been shown to repress expression of
chamber-specific genes in the AV myocardium. Mice with a Tbx2
loss-of-function have expansion of chamber-specific genes into the AV
myocardium (Harrelson et al.,
2004
). Moreover, Tbx2 has been shown to bind to an
element in the Anf gene that represses Anf expression in AV
myocardium through competition with Tbx5
(Habets et al., 2002
). In
other experiments, Tbx20 has been shown to directly repress
Tbx2, indicating that a Tbx gene regulatory network
functions to regionally pattern the myocardium
(Cai et al., 2005
;
Singh et al., 2005
;
Stennard et al., 2005
;
Takeuchi et al., 2005
).
Previous data revealed that Bmp2 is expressed in the AV myocardium
at 9.5 and 10.5 days post coitum (dpc) and in the cushion mesenchyme at later
stages (Lyons et al., 1990;
Sugi et al., 2004
). Moreover,
gene inactivation studies in mice revealed that Bmp2 was necessary
for early myocardial development (Zhang
and Bradley, 1996
). Bmp2 has been suggested to have a
role in valve morphogenesis in mouse embryos, based on data from the collagen
gel invasion assay (Sugi et al.,
2004
). In that work, Bmp2 could substitute for myocardium to
induce endocardial EMT. Moreover, noggin treatment of explants efficiently
inhibited EMT. However, other data indicated that Bmp2 was insufficient to
induce EMT on its own but could enhance Tgfß transformation activity
(Yamagishi et al., 1999
).
Here, we provide strong genetic evidence that Bmp2 induces AV cushion EMT in mammals. We inactivated Bmp2 specifically in the AV myocardium and found that Bmp2 mutant embryos failed to form AV cushions. Expression of Has2 was decreased in Bmp2 mutant embryos with reduced formation of the cardiac jelly. Furthermore, inactivation of the Bmp type 1A receptor, Bmpr1a, in endocardium also resulted in loss of AV cushion formation, indicating that Bmp2 signaled directly to the endocardial cushions. Our findings also indicate that the basic helix-loop-helix (bHLH) transcription factor Twist1 is a downstream effector of Bmp2 in EMT. We also found that Bmp2 has a planar signaling function to regulate patterning of the AV myocardium through regulation of Tbx2 expression. Taken together, our data indicate that Bmp2 has a crucial function in coordinating cushion morphogenesis with AV myocardial patterning.
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Materials and methods |
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Whole-mount in-situ hybridization
Whole-mount and section in-situ hybridization was performed as previously
described (Lu et al., 1999).
Details about probes will be provided upon request. In all in-situ
experiments, at least three mutants and three control embryos were analyzed
for each experiment.
lacZ staining and histology
For histology, embryos were fixed overnight in Bouin's fixative or buffered
formalin, dehydrated through graded ethanol and embedded in paraffin. Sections
were cut at 7-10 µm and stained with H&E. Staining for lacZ
was as previously described (Lu et al.,
1999).
Immunohistochemistry
Tissue sections were deparaffinized, rehydrated and boiled for 5 minutes in
10 mmol/l sodium citrate for antigen retrieval. After washing in PBS, the
nuclear factor of activated T-cells (NFAT) c1 (Cat. No.: 56602; BD Pharmingen,
San Jose, CA 95131, USA) primary antibody (1:250 dilution), was applied and
incubated overnight at 4°C. The sections were washed and incubated
consecutively with biotin-labeled secondary antibody (sc-2017; Santa Cruz, CA
95060 USA). The secondary antibodies were detected using the avidin-biotin
method. To detect Phospho-Smad1/Smad5/Smad8, sections were blocked in
PBS/0.05% BSA and primary antibody (#9511'; Cell Signaling, MA 01915,
USA) was applied with 1:200 dilution for overnight at 4°C. Sections were
washed and incubated with secondary antibody (Cat. No.: DPVR-15DAB). Color was
developed with diaminobenzidine (DAB) provided by the kit of secondary
antibody and the sections were counterstained with hematoxylin. For control
staining, preimmune serum was used instead of the primary antibody.
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Results |
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Inactivation of Bmp2 in the AV myocardium
To directly investigate Bmp2 function in cardiac development, we
constructed a Bmp2 conditional null allele, the
Bmp2floxneo allele, which contained LoxP sites
surrounding exon 3 encoding the mature Bmp2 peptide. Deletion of exon
3 has been shown to result in a Bmp2 null allele
(Ma and Martin, 2005). To
inactivate Bmp2 in the heart, we used the
Nkx2.5cre knock-in allele, which directs cre activity to
the anterior splanchnic mesoderm of the primary heart field and within the
mature heart (Fig. 2A,B)
(Liu et al., 2004
;
Moses et al., 2001
).
To generate Bmp2 cardiac-specific mutant embryos, we crossed the
Nkx2.5cre+/- mice to the
Bmp2floxneo+/- mice to obtain Nkx2.5cre;
Bmp2floxneo compound heterozygotes. Intercrosses between these
mice and the Bmp2floxneo homozygous mice resulted in
recovery of 25% of embryos that were Nkx2.5cre;
Bmp2floxneo/floxneo(f/f), hereafter referred to as
Bmp2 CKO. This crossing strategy required that both
Bmp2floxneo alleles undergo cre-mediated recombination to
generate a Bmp2 null cell. Because there is a delay in the timing of
cre-mediated recombination as intracellular cre protein accumulates, we
predicted that the two-allele recombination strategy would result in a slight
delay in Bmp2 cardiac inactivation
(Nagy, 2000). This would
provide a stable genetic system to study Bmp2 in either the sinus
venosus or the AV canal.
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Embryonic lethality and AV cushion defects in the absence of Bmp2 in the AV myocardium
Analysis of litters at embryonic time points revealed that Bmp2
CKO mutant embryos were indistinguishable from wild-type littermates at 8.5
and 9.0 dpc. At 9.5 dpc, Bmp2 CKO mutant embryos had abnormal
morphology of the AV canal constriction but were still recovered at Mendelian
frequencies (Table 1). By 10.5
dpc, all Bmp2 CKO mutants exhibited pericardial effusion and growth
retardation, indicating that these embryos suffered from heart failure at this
stage.
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Inactivation of Bmpr1a in the endocardium disrupts endocardial cushion formation
In order to establish more firmly that Bmp2 signaled directly to the
underlying endocardium, we generated mouse embryos that were deficient in the
competence to receive Bmp signals within the endocardium. We inactivated the
type 1A Bmp receptor, Bmpr1a, in the endocardium using the Tie2
cre transgenic line (Kisanuki et al.,
2001). Bmpr1a is a major type 1 receptor for
Bmp2 and Bmp4 (von
Bubnoff and Cho, 2001
). Because germline mutation of
Bmpr1a is early embryonic lethal, we used the Bmpr1a
conditional null (Bmpr1aflox) allele
(Mishina et al., 2002
;
Mishina et al., 1995
). We
generated embryos that carried Tie2cre and the
Bmpr1aflox and Bmpr1anull
(Bmpr1an/f) alleles. We examined Tie2cre;
Bmpr1an/f mutant embryos at 9.5 dpc and found that embryos
deficient for Bmpr1a in the cushion endocardium failed to form
cushions (Fig. 4A,B). It is
notable that one-third of Tie2cre; Bmpr1an/f
mutants made cushions. This probably results from variability of cre activity;
however, it is possible that other type 1 receptors also have a function in
the cushion endocardium.
To determine the extent of Bmpr1a inactivation in the Tie2cre; Bmpr1an/f mutants, we examined Smad 1/5/8 immunostaining in the AV endocardium of wild-type and Bmpr1a mutant embryos. In the wild type, most endocardial cells stained positive for P-Smad 1/5/8 (Fig. 4C), while in the Tie2cre; Bmpr1a mutant embryos approximately 50% fewer strong positive cells were detected (Fig. 4D). We noted that the main reduction in P-Smad 1/5/8 immunoreactivity in Tie2cre; Bmpr1a mutants was in endocardium located toward the atrium (Fig. 4D, n=3). The significance of this observation is presently unclear. These data indicate that while endocardial inactivation of Bmpr1a results in incomplete loss of endocardial Bmp responsiveness, the AV cushion phenotype is strong, revealing that AV cushion EMT is very sensitive to small decreases in Bmp signaling. Moreover, our findings support the hypothesis that other type 1 receptors may have overlapping function in the AV canal endocardium.
|
Twist has been shown to negatively regulate expression of
E-cadherin in breast cancer cells, prompting us to examine expression of
VE-cadherin in the AV canal endocardium of Tie2cre;
Bmpr1an/f mutant embryos
(Kang and Massague, 2004).
During EMT, VE-cadherin is normally downregulated
(Fig. 4G,I); however, in the
Tie2cre; Bmpr1an/f mutant endocardium
VE-cadherin expression persisted when compared with the wild-type embryo
(Fig. 4H,J). Notch signaling
has been shown to be required for EMT in the AV canal
(Timmerman et al., 2004
). In
the Tie2cre; Bmpr1a mutants, we found that expression of
Notch1 was similar to wild-type embryos
(Fig. 4K,L). We also examined
expression of Snai1, a zinc finger transcription factor that has been
implicated in EMT and has been shown to be a target of Notch signaling in the
AV endocardium (Timmerman et al.,
2004
). By contrast to Twist1, Snai1 expression was
unaffected by loss of Bmpr1a in AV endocardium
(Fig. 4M,N). Taken together,
these data indicate that Bmp2 signals directly to the cushion endocardium
through the type 1 receptor, Bmpr1a.
|
As for the Tie2cre; Bmpr1an/f mutant
embryos, expression of Twist1, normally detected in the AV cushion
endocardium and mesenchyme of wild-type embryos was not detected in the
Bmp2 CKO mutant endocardium (Fig.
5E-H). Bmp signaling regulates the Msx1 homeobox gene in
other developmental fields (Liu et al.,
2005; Vainio et al.,
1993
). Expression of Msx1 was detected in the AV
endocardium of wild-type embryos but was absent in the Bmp2 CKO
mutant embryos (Fig. 5I-L). The
inhibitory Smad, Smad6, is a negative regulator of Bmp signaling that
is also transcriptionally regulated by Bmp signaling. Furthermore,
Smad6 has been shown to be a negative regulator of valve development
(Desgrosellier et al., 2005
;
Galvin et al., 2000
;
Ishida et al., 2000
). In
wild-type embryos, Smad6 was expressed in the AV endocardium, while
in the Bmp2 CKO mutants, Smad6 expression was absent
(Fig. 5M,N). This finding
uncovers a negative feedback loop that functions to limit the extent of
endocardial EMT.
During activation of the AV endocardium, VE-cadherin expression is
downregulated in wild-type embryos, but in the Bmp2 CKO mutant
VE-cadherin persists (Fig.
5O,P). A Notch-Snai1 signaling pathway has been implicated in
VE-cadherin downregulation in AV endocardium
(Timmerman et al., 2004). In
the Bmp2 CKO mutants, Notch 1 and Snai1 expression
was reduced compared with wild-type embryos, indicating that the Notch and
Bmp2 signaling pathways cooperate in AV cushion morphogenesis
(Fig. 5Q-V). Taken together,
our data indicate that Bmp2 induces EMT-promoting genes, such as
Twist1 and Snai1, and other genes that restrain EMT such as
Smad6. Moreover, these data indicate that Bmp2 CKO mutants,
in contrast to Bmpr1a-deficient embryos, have defects in Notch-Snai1
signaling in the AV cushion endocardium (see Discussion).
Bmp2 regulates Tbx2 in the AV myocardium to control chamber-specific gene expression
Previous studies in chick embryos had suggested that Bmp2 regulated
Tbx2 expression in the developing heart, prompting us to examine
Tbx2 expression in Bmp2 CKO mutants
(Yamada et al., 2000). In
wild-type embryos, Tbx2 was expressed specifically in the AV
myocardium, while in the Bmp2 CKO mutant embryos Tbx2
expression was undetectable (Fig.
6A,B). By contrast, expression of Tbx5 and Tbx20
were unaffected by Bmp2 deletion
(Fig. 6C-F). These data
indicate that Bmp2 promotes expression of Tbx2 and so is important to
maintain the correct ratio of Tbx genes in the AV myocardium.
In the AV canal, Tbx2 is known to repress chamber-specific gene expression. Consistent with this, expression of the chamber-specific genes Anf, chisel, and connexin 40 was limited to the ventricular and atrial myocardium in wild-type embryos (Fig. 7A,C,E,G,I,K). By contrast, in the Bmp2 CKO mutant embryos, expression of all three chamber-specific genes was expanded into the AV myocardium (Fig. 7B,D,F,H,J,L). Markers of the AV myocardium, such as Tgfß2 and Lef1, were lost in the Bmp2 CKO mutant embryos (Fig. 7M-P). From these findings, we conclude that Bmp2 signaling directs regionalized myocardial patterning.
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Discussion |
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Bmp2 is a crucial inducer of EMT in the AV cushion
The early lethality of the germline Bmp2 null mice has hampered
definitive insight into the role of Bmp2 in AV cushion development
(Zhang and Bradley, 1996).
Recently reported experiments, using the collagen gel invasion assay,
suggested that Bmp2 could substitute for myocardium to induce EMT in mouse
embryos (Sugi et al., 2004
).
However, other experiments, also using the collagen gel invasion assay, showed
that Bmp2 functioned only in combination with Tgfß3 to promote EMT in
chick embryos (Yamagishi et al.,
1999
). Furthermore, chick embryos treated with a retrovirus
expressing Noggin caused defects in the OFT but did not affect the AV canal
(Allen et al., 2001
). Our data
indicate that Bmp2 is the myocardial-derived signal that induces EMT in
mammals. One likely explanation for the different results obtained from these
studies is species-specific differences between mice and chicks
(Camenisch et al., 2002
).
|
It is important to note that Alk2 has also been shown to be
required for EMT in the in-vitro explant system using chick embryos
(Desgrosellier et al., 2005).
A constitutively active Alk2 was able to induce EMT, while Alk2
antisera could effectively inhibit EMT. Furthermore, mouse embryos deficient
for Alk2 in cushion endocardium had a severe defect in AV cushion
morphogenesis (Wang et al.,
2005
). Our data indicate that the Notch-Snai1 pathway was intact
in the Tie2cre; Bmpr1a n/f mutant embryos, while this
pathway was disrupted in the Alk2 and Bmp2 mutant embryos
(this work) (Wang et al.,
2005
). This indicates that Bmpr1a and Alk2 may
activate distinct pathways in cushion endocardium. However, other genes such
as Msx1, were reduced in both the Bmpr1a and Alk2
mutants. Taken together, these findings indicate that signaling through
Alk2 and Bmpr1a activates distinct but overlapping target
pathways. Alternatively, as some (approximately 33%) Tie2cre;
Bmpr1a n/f mutants made AV cushions, it is possible that there is partial
redundancy between type 1 receptors in AV endocardium, similar to that
observed in chondrocytes (Yoon et al.,
2005
). Future studies will be required to investigate these
questions in more detail.
Previous work investigating Bmp ligands in cardiac development uncovered
considerable functional redundancy. For example, Bmp7 mutants have no
phenotype in the heart as a result of redundancy with other Bmp ligands
(Luo et al., 1995). Bmp6;
Bmp7 double mutants have delayed formation of the OFT cushions
(Kim et al., 2001
), while the
Bmp5; Bmp7 double mutants have severe defects in cushion formation
(Solloway and Robertson,
1999
). Moreover, we found evidence for overlapping function
between Bmp4 and Bmp7 in OFT development
(Liu et al., 2004
). By
contrast, we have found that Bmp2 function is essential in the AV
canal. It will be important in the future to investigate the functional
relationship of the distinct classes of Bmp ligands in cushion
morphogenesis.
Tgfß and Bmp signaling in AV cushion morphogenesis
Tgfß signaling is a crucial signal in avians for EMT in the AV
cushions, as determined by the collagen invasion assay
(Barnett and Desgrosellier,
2003). However, although Tgfß2 is clearly important for
normal cushion development in mice, EMT can still occur in the Tgfß2 null
mice (Sanford et al., 1997
).
Our data, and the work of Sugi et al.
(Sugi et al., 2004
), indicate
that much of the function of Tgfß has been co-opted by Bmp signaling in
mammals. In mice, current evidence suggests that Tgfß2 expression is
regulated by Bmp signaling. Inactivation of Bmpr1a in myocardium
using an MHC cre transgene resulted in downregulation of Tgfß2 expression
in the AV canal (Gaussin et al.,
2002
). This suggested a cell-autonomous requirement for Bmp
signaling in myocardium to maintain Tgfß2 expression, which was important
for the later aspects of AV cushion development. Furthermore, Bmp2 induces
Tgfß2 in explants cultured in collagen gel
(Sugi et al., 2004
), and
Tgfß2 expression is off or reduced in the AV cushions of Bmp2
CKO mutant embryos (this work). Future experiments will be required to firmly
establish the genetic relationship between Bmp signaling and Tgfß2
transcriptional regulation.
|
Particularly relevant to the present study is the recent observation that
Twist1 promotes EMT in cancer cells and facilitates tumor metastasis.
Twist1 regulates EMT through a mechanism involving repression of
E-cadherin transcription (Kang
and Massague, 2004; Yang et
al., 2004
). Our data indicate that Twist1 expression was
lost in both the Bmp2 CKO and Tie2cre; Bmpr1a n/f
mutant embryos. Moreover, we observed a modest defect in VE-cadherin
downregulation in the Bmp2 and Bmpr1a mutant AV canals.
These findings suggest that Twist1 is an effector of the
Bmp-signaling pathway in the AV canal that promotes endocardial EMT.
We observed only mild defect in VE-cadherin downregulation, despite loss of
Twist1 in the Bmp2 CKO mutant AV canal. This is probably a
result of compensatory function of the closely related Twist2 gene.
There are two Twist genes in mammals, Twist1 and
Twist2, which have redundant functions in cytokine regulation
(Bialek et al., 2004;
Li et al., 1995
;
Sosic et al., 2003
). Further
experiments will be required to investigate this possibility.
It is notable that the Snai1 transcription factor has recently
been shown to be required for AV canal EMT as part of the Notch
signaling pathway (Timmerman et al.,
2004). In Drosophila, Twist and Snai1 are both
required for correct dorsoventral patterning and mesoderm induction as part of
the Dorsal/Twist/Snai1 pathway
(Stathopoulos and Levine,
2002
). In vertebrates, both Snai1 and Twist1
have been shown to promote EMT through inhibition of E-cadherin
expression. Our findings suggest that Bmp and Notch signaling cooperate to
influence VE-cadherin expression. It will be interesting to
investigate the functional relationship of Notch and Bmp signaling in AV canal
EMT in future experiments.
Bmp2 regulates myocardial differentiation
We provide evidence that Bmp2 is upstream of Tbx2 in the
AV myocardium. Our observation that myocardial patterning in the
Tie2cre; Bmpr1a mutants is normal argues against the
hypothesis that an endocardial-derived signal regulates myocardial patterning.
Further experiments will be required to investigate whether Bmp2 directly
regulates Tbx2 expression.
Our findings can be interpreted in the context of recent Tbx20
inactivation studies that led to conflicting interpretations of the
Bmp2-Tbx2 regulatory relationship. Tbx20 mutant embryos had
expanded Tbx2 expression throughout the myocardium despite diminished
Bmp2 expression, suggesting that Tbx2 regulation is
Bmp2-independent (Cai et al.,
2005; Stennard et al.,
2005
). Another, independently generated, Tbx20 null
allele resulted in ectopic Bmp2 with resulting expanded Tbx2
(Singh et al., 2005
). Our data
support the conclusion that Bmp2 promotes Tbx2 expression in the AV
myocardium.
It is interesting to note that Tbx20 and Tbx2 are co-expressed in the AV myocardium, indicating an AV-canal-specific mechanism that allows Tbx2 to escape Tbx20-imposed repression. Our data suggest that Bmp2-signaling counters the repressive activity of Tbx20 in the AV myocardium. Because Tbx20 is expressed normally in the Bmp2 CKO mutant embryos, Bmp2 may regulate Tbx20 post-transcriptionally. Bmp2 may also induce genes that functionally inhibit Tbx20 activity. The precise mechanism underlying this inhibition of Tbx20 is presently unclear and awaits future experiments.
In conclusion, our findings reveal that Bmp2 is required for EMT in the mammalian AV canal (see Fig. 8). We have uncovered a genetic pathway involving the type 1 Bmp receptor, Bmpr1a, which functions in EMT. Our data also revealed that Bmp2 signaling is required for normal deposition of the cardiac jelly. Within the myocardium, Bmp2 functions upstream of Tbx2 to control regionalized myocardial patterning.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Allen, S. P., Bogardi, J. P., Barlow, A. J., Mir, S. A., Qayyum, S. R., Verbeek, F. J., Anderson, R. H., Francis-West, P. H., Brown, N. A. and Richardson, M. K. (2001). Misexpression of noggin leads to septal defects in the outflow tract of the chick heart. Dev. Biol. 235,98 -109.[CrossRef][Medline]
Armstrong, E. J. and Bischoff, J. (2004). Heart
valve development: endothelial cell signaling and differentiation.
Circ. Res. 95,459
-470.
Barnett, J. V. and Desgrosellier, J. S. (2003). Early events in valvulogenesis: a signaling perspective. Birth Defects Res. C Embryo Today 69,58 -72.[CrossRef][Medline]
Bartram, U., Molin, D. G., Wisse, L. J., Mohamad, A., Sanford,
L. P., Doetschman, T., Speer, C. P., Poelmann, R. E. and Gittenberger-de
Groot, A. C. (2001). Double-outlet right ventricle and
overriding tricuspid valve reflect disturbances of looping, myocardialization,
endocardial cushion differentiation, and apoptosis in TGF-beta(2)-knockout
mice. Circulation 103,2745
-2752.
Bialek, P., Kern, B., Yang, X., Schrock, M., Sosic, D., Hong, N., Wu, H., Yu, K., Ornitz, D. M., Olson, E. N. et al. (2004). A twist code determines the onset of osteoblast differentiation. Dev. Cell 6, 423-435.[CrossRef][Medline]
Cai, C. L., Zhou, W., Yang, L., Bu, L., Qyang, Y., Zhang, X.,
Li, X., Rosenfeld, M. G., Chen, J. and Evans, S. (2005).
T-box genes coordinate regional rates of proliferation and regional
specification during cardiogenesis. Development
132,2475
-2487.
Camenisch, T. D., Molin, D. G., Person, A., Runyan, R. B., Gittenberger-de Groot, A. C., McDonald, J. A. and Klewer, S. E. (2002). Temporal and distinct TGFbeta ligand requirements during mouse and avian endocardial cushion morphogenesis. Dev. Biol. 248,170 -181.[CrossRef][Medline]
Chen, Z. F. and Behringer, R. R. (1995). twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev. 9,686 -699.[Abstract]
Crabtree, G. R. and Olson, E. N. (2002). NFAT signaling: choreographing the social lives of cells. Cell 109,S67 -S79.[CrossRef][Medline]
Desgrosellier, J. S., Mundell, N. A., McDonnell, M. A., Moses, H. L. and Barnett, J. V. (2005). Activin receptor-like kinase 2 and Smad6 regulate epithelial-mesenchymal transformation during cardiac valve formation. Dev. Biol. 280,201 -210.[CrossRef][Medline]
Eisenberg, L. M. and Markwald, R. R. (1995).
Molecular regulation of atrioventricular valvuloseptal morphogenesis.
Circ. Res. 77,1
-6.
Galvin, K. M., Donovan, M. J., Lynch, C. A., Meyer, R. I., Paul, R. J., Lorenz, J. N., Fairchild-Huntress, V., Dixon, K. L., Dunmore, J. H., Gimbrone, M. A. et al. (2000). A role for smad6 in development and homeostasis of the cardiovascular system. Nat. Genet. 24,171 -174.[CrossRef][Medline]
Gaussin, V., Van de Putte, T., Mishina, Y., Hanks, M. C.,
Zwijsen, A., Huylebroeck, D., Behringer, R. R. and Schneider, M. D.
(2002). Endocardial cushion and myocardial defects after cardiac
myocyte-specific conditional deletion of the bone morphogenetic protein
receptor ALK3. Proc. Natl. Acad. Sci. USA
99,2878
-2883.
Gitelman, I. (1997). Twist protein in mouse embryogenesis. Dev. Biol. 189,205 -214.[CrossRef][Medline]
Habets, P. E., Moorman, A. F., Clout, D. E., van Roon, M. A.,
Lingbeek, M., van Lohuizen, M., Campione, M. and Christoffels, V. M.
(2002). Cooperative action of Tbx2 and Nkx2.5 inhibits ANF
expression in the atrioventricular canal: implications for cardiac chamber
formation. Genes Dev.
16,1234
-1246.
Harrelson, Z., Kelly, R. G., Goldin, S. N., Gibson-Brown, J. J.,
Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (2004).
Tbx2 is essential for patterning the atrioventricular canal and for
morphogenesis of the outflow tract during heart development.
Development 131,5041
-5052.
Howard, T. D., Paznekas, W. A., Green, E. D., Chiang, L. C., Ma, N., Ortiz de Luna, R. I., Garcia Delgado, C., Gonzalez-Ramos, M., Kline, A. D. and Jabs, E. W. (1997). Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome. Nat. Genet. 15,36 -41.[CrossRef][Medline]
Hurlstone, A. F., Haramis, A. P., Wienholds, E., Begthel, H., Korving, J., Van Eeden, F., Cuppen, E., Zivkovic, D., Plasterk, R. H. and Clevers, H. (2003). The Wnt/beta-catenin pathway regulates cardiac valve formation. Nature 425,633 -637.[CrossRef][Medline]
Ishida, W., Hamamoto, T., Kusanagi, K., Yagi, K., Kawabata, M.,
Takehara, K., Sampath, T. K., Kato, M. and Miyazono, K.
(2000). Smad6 is a Smad1/5-induced smad inhibitor.
Characterization of bone morphogenetic protein-responsive element in the mouse
Smad6 promoter. J. Biol. Chem.
275,6075
-6079.
Kang, Y. and Massague, J. (2004). Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 118,277 -279.[CrossRef][Medline]
Kim, R. Y., Robertson, E. J. and Solloway, M. J. (2001). Bmp6 and Bmp7 are required for cushion formation and septation in the developing mouse heart. Dev. Biol. 235,449 -466.[CrossRef][Medline]
Kisanuki, Y. Y., Hammer, R. E., Miyazaki, J., Williams, S. C., Richardson, J. A. and Yanagisawa, M. (2001). Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230,230 -242.[CrossRef][Medline]
Krug, E. L., Runyan, R. B. and Markwald, R. R. (1985). Protein extracts from early embryonic hearts initiate cardiac endothelial cytodifferentiation. Dev. Biol. 112,414 -426.[CrossRef][Medline]
Li, L., Cserjesi, P. and Olson, E. N. (1995). Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis. Dev. Biol. 172,280 -292.[CrossRef][Medline]
Liebner, S., Cattelino, A., Gallini, R., Rudini, N., Iurlaro,
M., Piccolo, S. and Dejana, E. (2004). Beta-catenin is
required for endothelial-mesenchymal transformation during heart cushion
development in the mouse. J. Cell Biol.
166,359
-367.
Liu, W., Selever, J., Wang, D., Lu, M. F., Moses, K. A.,
Schwartz, R. J. and Martin, J. F. (2004). Bmp4 signaling is
required for outflow-tract septation and branchial-arch artery remodeling.
Proc. Natl. Acad. Sci. USA
101,4489
-4494.
Liu, W., Selever, J., Murali, D., Sun, X., Brugger, S. M., Ma, L., Schwartz, R. J., Maxson, R., Furuta, Y. and Martin, J. F. (2005). Threshold-specific requirements for Bmp4 in mandibular development. Dev. Biol. 283,282 -293.[CrossRef][Medline]
Lu, M. F., Pressman, C., Dyer, R., Johnson, R. L. and Martin, J. F. (1999). Function of Rieger syndrome gene in left-right asymmetry and craniofacial development. Nature 401,276 -278.[CrossRef][Medline]
Luo, G., Hofmann, C., Bronckers, A. L., Sohocki, M., Bradley, A. and Karsenty, G. (1995). BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev. 9,2808 -2820.[Abstract]
Lyons, K. M., Pelton, R. W. and Hogan, B. L.
(1990). Organogenesis and pattern formation in the mouse: RNA
distribution patterns suggest a role for bone morphogenetic protein-2A
(BMP-2A). Development
109,833
-844.
Ma, L. and Martin, J. F. (2005). Generation of a Bmp2 conditional null allele. Genesis 42,203 -206.[CrossRef][Medline]
Mishina, Y., Suzuki, A., Ueno, N. and Behringer, R. R. (1995). Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9,3027 -3037.[Abstract]
Mishina, Y., Hanks, M. C., Miura, S., Tallquist, M. D. and Behringer, R. R. (2002). Generation of Bmpr/Alk3 conditional knockout mice. Genesis 32, 69-72.[CrossRef][Medline]
Moses, K. A., De Mayo, F., Braun, R. M., Reecy, J. L. and Schwartz, R. J. (2001). Embryonic expression of an Nkx2-5/Cre gene using ROSA26 reporter mice. Genesis 31,176 -180.[CrossRef][Medline]
Nagy, A. (2000). Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99-109.[CrossRef][Medline]
Plageman, T. F. and Yutzey, K. E. (2005). T-box genes and heart development: Putting the "T" in heart. Dev. Dyn. 232,11 -20.[CrossRef][Medline]
Runyan, R. B. and Markwald, R. R. (1983). Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue. Dev. Biol. 95,108 -114.[CrossRef][Medline]
Sanford, L. P., Ormsby, I., Gittenberger-de Groot, A. C.,
Sariola, H., Friedman, R., Boivin, G. P., Cardell, E. L. and Doetschman,
T. (1997). TGFbeta2 knockout mice have multiple developmental
defects that are non-overlapping with other TGFbeta knockout phenotypes.
Development 124,2659
-2670.
Schultheiss, T. M., Burch, J. B. and Lassar, A. B. (1997). A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11,451 -462.[Abstract]
Singh, M. K., Christoffels, V. M., Dias, J. M., Trowe, M. O.,
Petry, M., Schuster-Gossler, K., Burger, A., Ericson, J. and Kispert, A.
(2005). Tbx20 is essential for cardiac chamber differentiation
and repression of Tbx2. Development
132,2697
-2707.
Solloway, M. J. and Robertson, E. J. (1999).
Early embryonic lethality in Bmp5; Bmp7 double mutant mice suggests functional
redundancy within the 60A subgroup. Development
126,1753
-1768.
Sosic, D., Richardson, J. A., Yu, K., Ornitz, D. M. and Olson, E. N. (2003). Twist regulates cytokine gene expression through a negative feedback loop that represses NF-kappaB activity. Cell 112,169 -180.[CrossRef][Medline]
Stathopoulos, A. and Levine, M. (2002). Dorsal gradient networks in the Drosophila embryo. Dev. Biol. 246, 57-67.[CrossRef][Medline]
Stennard, F. A., Costa, M. W., Lai, D., Biben, C., Furtado, M.
B., Solloway, M. J., McCulley, D. J., Leimena, C., Preis, J. I., Dunwoodie, S.
L. et al. (2005). Murine T-box transcription factor Tbx20
acts as a repressor during heart development, and is essential for adult heart
integrity, function and adaptation. Development
132,2451
-2462.
Sugi, Y., Yamamura, H., Okagawa, H. and Markwald, R. R. (2004). Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice. Dev. Biol. 269,505 -518.[CrossRef][Medline]
Takeuchi, J. K., Mileikovskaia, M., Koshiba-Takeuchi, K., Heidt,
A. B., Mori, A. D., Arruda, E. P., Gertsenstein, M., Georges, R., Davidson,
L., Mo, R. et al. (2005). Tbx20 dose-dependently regulates
transcription factor networks required for mouse heart and motoneuron
development. Development
132,2463
-2474.
Timmerman, L. A., Grego-Bessa, J., Raya, A., Bertran, E.,
Perez-Pomares, J. M., Diez, J., Aranda, S., Palomo, S., McCormick, F.,
Izpisua-Belmonte, J. C. et al. (2004). Notch promotes
epithelial-mesenchymal transition during cardiac development and oncogenic
transformation. Genes Dev.
18, 99-115.
Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M. and
Martin, G. R. (1999). Cre-mediated gene inactivation
demonstrates that FGF8 is required for cell survival and patterning of the
first branchial arch. Genes Dev.
13,3136
-3148.
Vainio, S., Karavanova, I., Jowett, A. and Thesleff, I. (1993). Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75,45 -58.[CrossRef][Medline]
von Bubnoff, A. and Cho, K. W. (2001). Intracellular BMP signaling regulation in vertebrates: pathway or network? Dev. Biol. 239,1 -14.[CrossRef][Medline]
Wang, J., Sridurongrit, S., Dudas, M., Thomas, P., Nagy, A., Schneider, M. D., Epstein, J. A. and Kaartinen, V. (2005). Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart. Dev. Biol. 286,299 -310.[CrossRef][Medline]
Yamada, M., Revelli, J. P., Eichele, G., Barron, M. and Schwartz, R. J. (2000). Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. Dev. Biol. 228,95 -105.[CrossRef][Medline]
Yamagishi, T., Nakajima, Y., Miyazono, K. and Nakamura, H. (1999). Bone morphogenetic protein-2 acts synergistically with transforming growth factor-beta3 during endothelial-mesenchymal transformation in the developing chick heart. J. Cell Physiol. 180, 35-45.[CrossRef][Medline]
Yang, J., Mani, S. A., Donaher, J. L., Ramaswamy, S., Itzykson, R. A., Come, C., Savagner, P., Gitelman, I., Richardson, A. and Weinberg, R. A. (2004). Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117,927 -939.[CrossRef][Medline]
Yoon, B. S., Ovchinnikov, D. A., Yoshii, I., Mishina, Y.,
Behringer, R. R. and Lyons, K. M. (2005). Bmpr1a and Bmpr1b
have overlapping functions and are essential for chondrogenesis in vivo.
Proc. Natl. Acad. Sci. USA
102,5062
-5067.
Zhang, H. and Bradley, A. (1996). Mice
deficient for BMP2 are nonviable and have defects in amnion/chorion and
cardiac development. Development
122,2977
-2986.