1 Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan
2 Division of Molecular Biology, Institute for Molecular and Cellular Biology,
Osaka University, and Core Research for Evolutional Science and Technology
Corporation, Osaka 565-0871, Japan
3 Department of Developmental Neurobiology, Institute of Development, Aging and
Cancer, Tohoku University, Sendai 980-8575, Japan
Author for correspondence (e-mail:
ogura{at}idac.tohoku.ac.jp).
Accepted 11 August 2003
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SUMMARY |
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Key words: Heart development, Ventricular septum, Tbx5, Tbx20, dHand/eHand
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Introduction |
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One of most well-characterized Tbx genes is Tbx5. This gene is
expressed in developing forelimb buds and on the dorsal side of the retina. We
and another group have reported that Tbx5 is a crucial determinant of
wing (forelimb) (Takeuchi et al.,
1999; Rodriguez-Esteban et
al., 1999
). In addition, Tbx5 regulates pattern formation
of the eye and also the retinotectum projection along its dorsoventral axis
(Koshiba-Takeuchi et al.,
2000
). The roles played by Tbx5 during heart development,
however, remain unclear, although mutations of human TBX5 have been
found in individuals with Holt-Oram syndrome (OMIM 142900)
(Basson et al., 1997
;
Li et al., 1997
;
Basson et al., 1999
). In such
individuals, characteristic defects of the upper limb and heart are observed
(Holt and Oram, 1960
). Precise
analysis suggests the haploinsufficiency of TBX5 in Holt-Oram
syndrome. Similar haploinsufficiency was reported for other T-box genes, such
as human TBX3 and TBX1
(Jerome and Papaioannou, 2001
;
Lindsay et al., 2001
;
Merscher et al., 2001
;
Bamshad et al., 1997
;
Epstein, 2001
), indicating
that the levels of T-box proteins are crucial for normal functioning
(Bruneau et al., 2001
;
Hatcher and Basson, 2001
).
Recently, mouse Tbx5 was knocked out to generate heterozygous and
homozygous mice (Bruneau et al.,
2001). Homozygous Tbx5del/del mice do not
survive past embryonic day 10.5 (E10.5) because of the arrest in heart
development at E9.5. By contrast, heterozygous Tbx5del/+
mice show several morphological alterations in both the heart and the
forelimb. In such deformed hearts, large atrial septum defects (ASDs) and
ventricular septum defects (VSDs) are observed as a variety of complex cardiac
malformations. In addition, abnormalities of the cardiac conduction system are
found. These lines of evidence highlight the multiple roles of Tbx5
in heart development and the haploinsufficiency of Tbx5, providing a
valuable model of congenital heart diseases
(Bruneau et al., 2001
).
Expression patterns of Tbx5 were reported previously
(Bruneau et al., 1999;
Yamada et al., 2000
). In both
chick and mouse, Tbx5 is expressed in the precardiac mesoderm. This
expression then becomes restricted to the posterior part of the looping heart
tube. Later, Tbx5 expression is restricted to the atria and the left
ventricle, and a ventricular septum is formed at the boundary of
Tbx5-expressing and non-expressing domains. Hence, this gene would
provide a novel and valuable marker to explore the mechanism of ventricular
specification. Recently, interesting heart phenotypes of transgenic mice have
been reported. For example, the Tbx5 gene was overexpressed
ubiquitously in the primitive heart tube under the control of a ß-myosin
heavy chain promoter. Persistent expression resulted in heart looping defects,
abnormalities of early chamber development and loss of ventricular-specific
gene expression, indicating an essential role for Tbx5 in early heart
development (Horb and Thomsen,
1999
; Hatcher et al.,
2001
; Liberatore et al.,
2000
). Nonetheless, the premature death of such transgenic mice
prohibited precise analysis of cardiac development.
Vertebrates exhibit different heart morphologies: fish have one ventricle/one atrium, whereas birds and mammals have two ventricles/two atria. Considering the left ventricle-specific expression of Tbx5, this gene could be useful for exploring the evolution of vertebrate hearts and could provide valuable insights on the ventricle specification, onset of congenital heart diseases and evolution of vertebrate heart morphology. For this purpose, we modified our in ovo electroporation techniques to optimize efficient expression of transgenes in the developing heart and analyzed the functional roles of the Tbx5 gene during both chick and mouse cardiac development in detail. In addition, we report that chick Tbx20 is expressed in the right ventricle, showing a mutually exclusive pattern to Tbx5. Our data provide important insights on a combinatorial expression patterns of Tbx genes in the developing heart and their putative interaction.
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Materials and methods |
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Whole-mount in situ hybridization and isolation of probes
In situ hybridization was performed as described previously
(Wilkinson, 1993). Probes for
ANF, BMP2, Tll1 and VEGF were amplified by RT-PCR using
primers derived from the published sequences (GenBank Accession Number X57702,
X75914, U75331 and S79680, respectively). Zebrafish probes for Tbx5 and Tbx20
were also amplified by RT-PCR based on the published sequences (GenBank
Accession Number AF152607 and AF253325, respectively).
Transient transgenic assay
Transgenic mice were generated by pronuclear injection of plasmids into
fertilized eggs as described previously
(Saijoh et al., 1999). The
injected embryos were transferred into pseudopregnant recipients and allowed
to develop in utero. Embryos were examined for the presence of the transgene
by PCR. Four primers were used for PCR analysis:
5'-GAGTTCCCCAAGTGAATGAAA-3' and
5'-GCAGACATTCAGTGGACT-3' for the ß-MHC construct
(Liberatore et al., 2000
), and
5'-TGGTGAACCGCATCGAGCTGAAG-3' and
5'-CGTCCTCGATGTTGTGGCGGATC-3' for the MLC-2
construct
(Ross et al., 1996
).
Transfection assay
Zebrafish Tbx20 full-length sequence was amplified by RT-PCR, then
subcloned in pCAGGS expression vector
(Niwa et al., 1991). Human
ANF promoter was amplified by PCR using primers from the published
sequence (1539 bp upstream fragment from the initiation codon of
ANF). This region was been reported to contain three Tbx5-binding
sites (Bruneau et al., 2002). Transfection was performed based on the
Polyethylenimine (PEI)-mediated gene delivery method
(Boussif et al., 1995
).
Luciferase and ß-gal assays were carried out as described previously
(Ogura and Evans, 1995
).
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Results |
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Recently, physical interactions between several heart-specific
transcription factors have been reported to play pivotal roles during cardiac
development (Bruneau, 2002;
Nemer and Nemer, 2001
). Tbx5
associates directly with Nkx2.5 to promote cardiomyocyte differentiation
(Bruneau et al., 2001
;
Hiroi et al., 2001
). Nkx2.5
interacts with GATA4 to regulate several cardiac genes
(Durocher et al., 1997
;
Lee et al., 1998
). In
addition, physical interactions among Tbx2, Tbx5 and Nkx2.5 are crucial for
the cardiac development (Habets et al.,
2002
). These lines of evidence indicate that the tight crosstalks
among heart-specific factors control the orchestrated cardiac development. As
expected, two key factors, Nkx2.5 and GATA4 genes, are
expressed ubiquitously in the developing heart ventricles
(Fig. 1C,D).
To explore the roles played by Tbx5, we exploited our in ovo
electroporation technique to misexpress this gene in the developing chick
heart. With our system, the configurations of the electrodes and DNA injection
enabled us to obtain rapid and targeted expression of transgenes in the heart
(Takeuchi et al., 1999;
Koshiba-Takeuchi et al., 2000
;
Ogura, 2002
). Using this
approach, we misexpressed a Tbx5-GFP fusion gene inserted in the RCAS
retroviral vector. As reported previously, we did not detect any functional
difference between this fusion gene and the wild-type Tbx5
(Takeuchi et al., 1999
;
Koshiba-Takeuchi et al.,
2000
). In addition, we repeated electroporation of EGFP
gene alone in chick heart, but we did not detect any morphological alteration.
When the Tbx5-GFP was electroporated into the precardiac mesoderm of
embryos at stage 5, we observed robust GFP signals in the entire heart at E5
(Fig. 1G). At this stage, the
position of ventricular septum is already evident as a small indentation on
the surface of the normal heart (arrowhead in
Fig. 1E). By contrast, when
Tbx5 was misexpressed ubiquitously, as confirmed by the GFP signals
(Fig. 1G), such an indentation
was not formed, making the contour of the heart round and smooth
(Fig. 1F,H). At E8, we obtained
robust GFP signals uniformly in the entire ventricle, whereas the normal heart
did not show any GFP fluorescence (Fig.
1I), indicating that the Tbx5 gene was misexpressed in
the entire heart. As the indentation that is formed on the surface of a normal
heart (arrowhead in Fig. 1H)
corresponds to the position of the ventricular septum, this morphological
change indicates that septum formation was disrupted in the
transgene-electroporated heart (Fig.
1H).
To analyze further, we checked the expression of the chick atrial
ANF gene in both the normal and electroporated hearts
(Fig. 1J). In the normal heart,
the ANF gene is expressed in the left ventricle without any signal in
the right as observed in mouse (Zeller et
al., 1987). The position of the arrowhead in
Fig. 1J indicates the boundary
between the left and right ventricles. Contrary to the normal heart,
ANF was induced strongly in the entire ventricle when Tbx5
was misexpressed in the developing precardiac field. This strongly suggests
that the formation of the right ventricle was disturbed by the extensive
misexpression of Tbx5 (n=8/11). As reported previously
(Bruneau et al., 2001
),
ANF gene is one of direct targets of Tbx5. As ANF is
expressed in the entire ventricle, this also suggests that Tbx5 was
successfully misexpressed in the developing heart ventricle. In mammals,
ANF gene is induced by cardiac stress as first reported by Burnett et
al. (Burnett et al., 1986
),
ubiquitous induction of ANF gene in Tbx5-misexpressed heart might be
due to cardiac stress. Nonetheless, we did not observe cardiac overload in
these chick hearts. Rather, hemodynamic observation suggests that circulation
is severely disturbed and slow, thereby cardiac overload is not a primary
cause of ANF induction, although we do not exclude that possibility
that subsequent hypoxic stress might partially contribute to this
induction.
To check the effects of Tbx5 misexpression on the right ventricle-specific marker gene, we examined the expression of Tbx20 in the Tbx5-misexpressed heart. As shown in Fig. 1B, in the normal heart, Tbx20 is expressed in the right ventricle with its left limit located at the small indentation (arrowhead in Fig. 1K). By contrast, when Tbx5 was misexpressed, Tbx20 expression disappeared from the whole ventricle, which shows again the round and smooth contour (Fig. 1K) (n=4/11). These results strongly suggest that misexpression of Tbx5 in the entire ventricle induces the ANF expression, and represses Tbx20 in the right ventricle, thereby converting the ANF-off/Tbx20-on profile of the right ventricle to the ANF-on/Tbx20-off profile.
To analyze further the morphological changes induced by misexpression of Tbx5, we made a series of continuous sections of the normal and electroporated hearts at E8.0. In the normal heart, the ventricular septum formed at the boundary of the left and right ventricles (Fig. 2A-E) (n=6). At this stage, extensive trabecular formation was also evident in both the right and left ventricles that are connected to the pulmonary artery and aorta, respectively (Fig. 2A,B). Both left and right atrio-ventricular canals formed normally (Fig. 2B-D).
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We observed another type of morphological alteration in electroporated hearts, in which the Tbx5-positive domain was expanded rightward by electroporation (Type 2 in Fig. 2K-O) (n=17/23). In this case, the left ventricle expanded, and the right ventricle shrank (Fig. 2K-O), with a distinct VSD (arrowhead in Fig. 2N). The relative sizes of the two ventricles suggested that the ventricular septum was shifted to the right. Conal septation and rotation defects were also observed (Fig. 2K-M). The atrial septum was formed, but it was membranous and thin (Fig. 2N). Trabecular formation and the thickness of the ventricular wall were not affected. Observed cardiac defects are illustrated in Fig. 2P (Type 2).
We also misexpressed the Tbx5 gene in a restricted domain of developing hearts. In this case, we placed two electrodes in a cross configuration (Fig. 3A) to target electric pulses to a restricted part of the precardiac field. In addition, we used RCAS virus-incompetent chick embryos to prevent expansion of transgene expression. Even in such hosts, stable and long-lasting expression of Tbx5-GFP was obtained. When an RCAS-Tbx5-GFP construct was injected at stage 5, discrete GFP fluorescence signals were observed in a limited part of the prospective right ventricle at E4.0 (Fig. 3B,C). At E7.0, an abnormal indentation (red arrowhead in Fig. 3D) was observed on the right ventricle, corresponding to the domain of the restricted GFP signals (red arrowhead in Fig. 3E). Experimental design is illustrated in Fig. 3F. When Tbx5-GFP was misexpressed in a restricted region, an ectopic boundary of Tbx5-positive and -negative regions was formed in the developing right ventricle, as illustrated in Fig. 3F. In this case, endogenous Tbx5 expression was stronger in the caudal part of the precardiac mesoderm at stage 5, whereas the electroporated Tbx5 was misexpressed in the restricted rostral area that gives rise to the right ventricle. At stage 10, endogenous caudal expression is maintained in the developing heart tube, while the expression of the introduced Tbx5 transgene became evident at its rostral end. At stage 25, three domains were formed from the left side of heart to the right: (1) an endogenous Tbx5-positive domain (the prospective left ventricle), (2) a Tbx5-negative area (the prospective right ventricle) and (3) an ectopic Tbx5-positive area (Fig. 3F). Hence, this restricted expression results in the extra-boundary formation of Tbx5-positive and -negative regions in the prospective right ventricle.
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To analyze the morphological changes in detail, we made serial sections of
electroporated hearts at E6 and carried out in situ hybridization using
several ventricular septum markers. Contrary to the normal heart, an ectopic
septum-like structure (IVS* in Fig.
3J,K) was formed in the right ventricle (IVS in
Fig. 3J,K) (n=8/43).
To confirm that this ectopic structure is indeed the ventricular septum, we
performed in situ hybridization using several septum markers: BMP2,
Tll1 (tolloid-like 1) and VEGF (vascular endothelial
growth factor) (Lyons et al.,
1990b; Lyons et al.,
1995
; Clark et al.,
1999
; Tomanek et al.,
1999
; Miquerol et al.,
2000
). As expected, all of these markers were expressed
(Fig. 3L-N, respectively).
These lines of evidence strongly suggest that restricted expression of
Tbx5 in the prospective right ventricle induces an ectopic
ventricular septum at the new border of Tbx5 expression in the
developing right ventricle.
Misexpression experiments in chick hearts indicate that Tbx5 specifies the left ventricle, and that the ventricular septum is formed at the boundary of Tbx5-positve and -negative domains. To confirm this hypothesis, we checked the expression of several markers known to be expressed asymmetrically in the heart. As shown in Fig. 1J, chick ANF was induced in the developing right ventricle when Tbx5 was misexpressed. By contrast, Tbx20, which is expressed in the right ventricle, was repressed (Fig. 1K). Nonetheless, in the chick heart, other markers such as the dHAND and eHAND genes are expressed uniformly in both ventricles (data not shown). This prompted us to carry out misexpression studies in mouse hearts.
For this purpose, we used two expression constructs
(Fig. 4A) to target transgene
expression in developing mouse hearts. One is a mouse Tbx5 expression
construct in which the ß-MHC (myosin heavy chain) promoter was
used to misexpress this gene uniformly in the ventricle
(Liberatore et al., 2000;
Lyons et al., 1990a
). In
another construct, we used the MLC-2
(myosin light chain)
promoter, which was reported to drive transgene expression in the right
ventricle (Ross et al., 1996
).
In this construct, the chick Tbx5-EGFP fusion gene was used to
monitor expression. With these two constructs, we made several transgenic mice
in which the Tbx5 genes were misexpressed transiently. We did not
establish a stable transgenic line, as we speculated that misexpression of the
Tbx5 gene in the ventricle itself would induce severe abnormalities
of heart morphology, and hence cause premature death of the embryos.
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Next, we analyzed the morphological changes induced by the
MLC-2/chick Tbx5-EGFP construct. When we checked
transgene expression by GFP fluorescence signals, GFP expression was evident
on the right side of the heart tube as expected (yellow arrowheads in
Fig. 4D,F), indicating that the
chick Tbx5 gene was misexpressed in the prospective right ventricle.
Contrary to the electroporation experiments in the chick, the
Tbx5-EGFP gene was expressed in a gradient fashion in this transgenic
mouse, as confirmed by both GFP signals
(Fig. 4D,F) and whole-mount in
situ hybridization using chick Tbx5 as a probe
(Fig. 4H). Although
Tbx5-EGFP was strongly expressed at the right-most end of the
prospective right ventricle, this expression became faint at the middle of the
ventricle. We did not detect GFP fluorescence at the position of the
ventricular septum (Fig. 4D,H).
Interestingly, such transgenic hearts exhibit a consistent morphological
alteration, namely, swelling of the prospective right ventricle (red
arrowheads in Fig. 4E,G).
Later, a small protrusion was formed on the surface of the induced swelling in
the prospective right ventricle at E14.5 (blue arrowhead in
Fig. 4I).
To confirm the nature of these morphological alterations, we checked the
expression of several markers by in situ hybridization
(Fig. 5). As reported
previously, mouse eHAND is expressed in the prospective left
(Fig. 5A,J). By contrast,
dHAND genes is predominantly expressed in the prospective right,
albeit expanding to the bulbus cordis, the part of the prospective left
ventricles and the ventricular septum (Fig.
5D,M) (Srivastava et al.,
1997; Firulli et al.,
1998
; Srivastava et al.,
1995
). In addition, the mouse ANF gene is expressed in
the left ventricle (Fig. 5G,R) (Zeller et al., 1987
), as is
found in the chick. Hence these three genes provide good markers to confirm
the identity of the left and right ventricles. When we analyzed transgenic
hearts in which Tbx5 was misexpressed uniformly with the
ß-MHC promoter, we found that the domains of eHAND and
mouse Anf expression were expanded (n=3/3, n=4/4,
respectively), although the gross size of the heart decreased
(Fig. 5B,H). By contrast,
expression of dHAND was repressed, leaving the entire ventricle
dHAND-negative (Fig.
5E) (n=5/5).
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The same results were obtained when analyzed in tissue sections. This analysis also enabled us to examine the induced morphological changes in detail. As observed with whole-mount in situ hybridization, the eHAND, mouse Anf and dHAND genes are expressed in similar fashions (Fig. 5J,M,R). In addition, we found that the ventricular septum is developing at this stage (E10.5). In the deformed hearts dissected from the ß-MHC-Tbx5 transgenic mice, both eHAND and mouse Anf genes were induced almost to the right end of the developing ventricle (black arrowhead in Fig. 5K,Q), whereas expression of the dHAND gene was found to be repressed completely (Fig. 5N). Furthermore, septum formation was completely suppressed in such hearts (Fig. 5K,N,Q). Instead, a tiny bulge of ventricular wall was formed in a small eHAND/mouse Anf-negative region (black arrowheads in Fig. 5K,N,Q).
Conversely, the ventricular septum formed normally in hearts of the MLC2v-Tbx5 transgenic mice (Fig. 5L,O,R), although the right ventricle appeared to be enlarged. In this enlarged right ventricle, the mouse Anf gene was induced strongly (Fig. 5R). Likewise, expression of the eHAND gene was induced (red arrowhead in Fig. 5L), albeit expression disappeared near the septum (black arrowhead in Fig. 5L). In this eHAND-negative domain near the septum, expression of the dHAND gene was detected (red arrowhead in Fig. 5O), although this gene was completely suppressed in the remaining part of the right ventricle (Fig. 5O).
Taken together, these lines of evidence strongly suggest that the forced expression of the Tbx5 gene in the prospective right ventricle converts expression patterns of several right and left ventricular markers with extensive morphological alterations.
Our embryological data indicate that Tbx5 specifies the left
ventricle and the ventricular septum is formed at the boundary between
Tbx5-positive and Tbx5-negative regions. Interestingly,
another T-box gene, chick Tbx20 is expressed in a complementary
fashion, hence expressed in the Tbx5-negative right ventricle. As
misexpression of Tbx5 in the right ventricle represses Tbx20
expression, these two Tbx genes may be mutually exclusive. To understand
molecular interaction between Tbx5 and Tbx20, we carried out a set of
transfection assays using human ANF promoter-luciferase reporter
construct. As reported previously, Tbx5 and Nkx2.5 synergistically activate
this promoter (Bruneau, 2002;
Nemer and Nemer, 2001
;
Bruneau et al., 2001
;
Hiroi et al., 2001
). In
addition, another heart-specific transcription factor, GATA4 again
synergistically activates cardiac
-actin promoter with Nkx2.5
(Durocher et al., 1997
;
Lee et al., 1998
). In
addition, recently, it has been reported that Tbx2 abrogates the synergistic
activation of the ANF promoter by Tbx5 and Nkx2.5
(Habets et al., 2002
). These
lines of evidence suggest that the tight interactions and the crosstalks among
heart-specific transcription factors play essential roles for the chamber
formation of heart (Bruneau,
2002
; Nemer and Nemer,
2001
).
When a human ANF promoter-luciferase construct was transfected to COS7 cells along with several expression plasmids, Tbx5, Nkx2.5 and GATA4 activated this promoter about 4.7-, 3.7- and 3.5-fold, respectively (Fig. 6). As reported previously, when both Tbx5 and Nkx2.5 were co-expressed, synergistic activation was observed (about 11.6 fold). Interestingly, when both Tbx5 and GATA4 were co-expressed, robust synergistic activation was obtained (25.3-fold), indicating that the synergism between Tbx5 and GATA4 is more potent. By contrast, when full-length zebrafish Tbx20 was misexpressed, these synergistic actions between Tbx5/Nkx2.5 and Tbx5/GATA4 were abrogated (7.0- and 3.2-fold, respectively). As ANF gene is expressed in the left ventricle, our data indicate that the left side expression of this gene is regulated in two ways: (1) activation by Tbx5 in the left ventricle and (2) repression by Tbx20 in the right ventricle. These lines of evidence suggest that the specification of chick left/right ventricles and the position of ventricular septum are controlled by the distinct actions of two Tbx genes expressed in the mutually exclusive fashion. When we used chick Tbx20, exactly same data were obtained (data not shown).
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Discussion |
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In the chick and mouse hearts in which Tbx5 was misexpressed
ubiquitously, the ventricular wall was thinner than normal, and the trabecular
formation was coarse and rough. These phenotypic changes indicate that
Tbx5 regulates cardiac muscle differentiation. Recently, it was found
that the Tbx5 protein associates and interacts physically with the cardiac
homeoprotein Nkx2.5, which is essential in cardiac muscle development
(Bruneau et al., 2001;
Hiroi et al., 2001
). In
addition to this, we found that the interaction between Tbx5 and GATA4
synergistically activates the ANF promoter. These lines of evidence
strongly suggest that Tbx5 possesses multiple interfaces necessary for the
multiple protein-protein interactions. Hence, changes of the level of Tbx5
protein in differentiating cardiomyocytes might affect the transcriptional
control of cardiac genes by disturbing the balance of multiple interactions.
Both the loss and gain of Tbx5 function disturb the transcriptional
control of Tbx5 targets, probably through the abnormal balance between the
Tbx5, Tbx20, GATA4 and Nkx2.5 proteins.
Contrary to chick Tbx20, it has been reported that mouse
Tbx20 is expressed uniformly in all four heart chambers
(Kraus et al., 2001). Similar
differences in expression patterns of cardiac genes can be found in
dHAND and eHAND. Mouse dHAND (Hand2 Mouse
Genome Informatics) and eHAND (Hand1 Mouse Genome
Informatics) genes are expressed differently in the ventricles, whereas chick
HAND genes are expressed uniformly in the developing heart. In search
of putative right ventricle-specific markers, we cloned chick desmin,
dystrophin and SM22
genes, as mouse counterparts of these
genes are expressed in the right ventricle
(Kuisk et al., 1996
;
Kimura et al., 1997
;
Moessler et al., 1996
;
Li et al., 1996
). However,
these genes are expressed uniformly in chick heart (data not shown). These
lines of evidence suggest that the mechanism of ventricular specification
might be different in species. Because ANF gene is the direct target
of Tbx5, it might not be adequate to argue that chick ANF is the left
ventricle-specific marker. As described above, several genes are expressed in
different manners in mouse and chick hearts. Hence, it is important to isolate
novel left or right ventricle-specific markers. For this purpose, we are
performing cDNA subtraction and RDA (representational difference analysis) to
isolate region-specific markers in developing vertebrate hearts.
Recently, functions of zebrafish T-box gene tbx20 (previously
known as hrT) gene have been reported
(Szeto et al., 2002).
Interestingly, loss of tbx20 function resulted in upregulation of
Tbx5. Conversely, misexpression of tbx20 induced
downregulation of tbx5. These data indicate that Tbx20 regulates
tbx5 expression in zebrafish. Our chick data also indicate that Tbx5
represses Tbx20 when misexpressed
(Fig. 1K). These lines of
evidence suggest that the tight regulatory interaction between Tbx5 and Tbx20
is crucial in zebrafish and chick, but not in mouse right ventricle.
Comparative and comprehensive approaches using various molecular markers
should be carried out to uncover the mechanism of development and evolution of
different vertebrate hearts.
In addition, Tbx20 represses the synergistic action of Tbx5 and GATA4 on human ANF promoter, indicating that Tbx20 represses ANF expression in the right ventricle. In mouse heart, ANF expression is also restricted to the left ventricle, although mouse Tbx20 is expressed uniformly. This suggests that different mechanism might operate in the mouse left ventricle to sustain the action of Tbx5 or inhibit the function of Tbx20. As the levels of T-box proteins are crucial for normal development, the levels of Tbx5 and Tbx20 proteins might be important for the development of mouse left ventricle. In addition, we do not exclude the possibility that unknown factor(s) might be involved in this process.
Interestingly, the ANF and connexin 40 (cx40) genes were
found to be direct targets of the Tbx5/Nkx2.5 protein complex
(Bruneau et al., 2001;
Hiroi et al., 2001
).
Consistent with this, misexpression of Tbx5 in the right ventricle
induces robust expression of the ANF gene in both mice and chicks, as
described above (Figs 1 and
5). Although we did not examine
the expression of cx40 in our system, we found that the beating
pattern of electroporated chick hearts was abnormal: simultaneous contraction
of atria and ventricles instead of normal serial beating (data not shown).
This could be related to the abnormal conduction systems found in both the
heterozygous Tbx5del/+ mice and individuals with Holt-Oram
syndrome.
As observed in our gain-of-function approaches, Tbx5 misexpression
disturbs the normal differentiation of cardiac muscle. By contrast, multiple
anomalies found in both the heterozygous Tbx5del/+ mice
and individuals with Holt-Oram syndrome indicate the haploinsufficiency of
Tbx5 in cardiac development. These lines of evidence strongly suggest
that the level of Tbx5 expression in the developing cardiac muscle
cell is crucial. As reported, the Tbx5 protein interacts with Nkx2.5
(Bruneau et al., 2001;
Hiroi et al., 2001
), and
another T-box protein Tbr1 makes a complex with CASK, one of the
membrane-associated guanylate kinases (MAGUKs), to regulate transcription of
target genes (Hsueh et al.,
2000
). These physical interactions suggest that the balances
between Tbx proteins and other interacting partners are important for the
orchestrated processes of pattern formation. As genetic analysis of
Drosophila suggests the interaction between optomotor blind
(omb), one of the Drosophila T-box genes, and DPP/WG
signaling, the physical interactions between Tbx proteins and other factors
including signal transduction factors could be a general characteristic
(Srivastava and Olson, 2000
;
Conlon et al., 2001
). Solving
these putative interactions would be an important key to understanding the
roles played by Tbx genes during development.
As an opposite approach, we electroporated a dominant negative form of Tbx5 (EnR-Tbx5: fusion of Engrailed suppressor domain and chick Tbx5), expecting a leftward shift of the ventricular septum. Nonetheless, misexpression of EnR-Tbx5 led premature death of embryos from E1.5 to E3 (n=76/76), probably because this type of misexpression itself disturbs cardiac development at very early stages, such as heart tube looping and differentiation.
As shown in Fig. 2F-O, ventricular myocardium was thin when Tbx5 was misexpressed. This would suggest that misexpression of Tbx5 might induce apoptosis or inhibition of cell growth. Nonetheless, when we performed the TUNEL assay to detect apoptotic cell death in differentiating cells, we could not detect any difference between the normal and the Tbx5-misexpressed hearts (data not shown). Even between E4 and E5, at which the ventricular septum starts to form, apoptotic cell death was not evident, indicating that the loss of ventricular septum is not due to Tbx5-induced apoptosis. Rather, we speculate that retardation of myocardium growth is an indirect effect of abnormal heart development, as septum-less single ventricle hearts exhibited an abnormal beating pattern (data not shown). In addition, both ASD and VSD could cause severe circulation defects, resulting in loss of hemodynamic stimulation to the developing myocardium. We also do not exclude the possibility that overexpressed Tbx5 Inhibits proliferation of myocardiac cells.
Contrary to the phenotypes observed in chick hearts, misexpression of Tbx5 in mouse hearts did not affect growth of myocardium. As shown in Fig. 5L,O,R, misexpression of Tbx5 driven by the MLC2v promoter induced swelling of the right ventricle, leaving the size of the left ventricle normal. In addition, thickness of the ventricle was normal, even when Tbx5 was misexpressed in the entire ventricle by the ß-MHC promoter. These lines of evidence suggest that the abnormal phenotypes observed in both chick and mouse were not due to the proliferation/apoptosis defects. Nonetheless, we do not exclude that possibility that over or high doses of Tbx5 misexpression might affect the growth and/or differentiation of cardiomyocytes, as the balances with other transcription factors, such as Tbx20, GATA4 and Nkx2.5 are pivotal for the normal patterning of vertebrates hearts.
As mentioned previously, vertebrates exhibit different heart morphologies:
fish possess one ventricle/one atrium; amphibians have one ventricle/two
atria; reptiles have two incomplete ventricles/two atria; and birds/mammals
have two complete ventricles/two atria. Although we have not yet expanded our
analysis to other vertebrates, the Tbx5 and Tbx20 genes
could be good markers with which to explore the evolution of heart morphology
of various vertebrate animals. To explore further, we checked expression
patterns of zebrafish tbx20 and tbx5 in developing hearts
(right panel in Fig. 7).
Interestingly, zTbx20 is expressed in the Bulbus arteriosus (BA) and
the ventricle near the BA, whereas zTbx5 in the atrioventricular
junction (AVJ) and the ventricle near the AVJ. Expression of these Tbx genes
in the ventricle is complementary at both 48 and 120 hpf, yet showing gradient
expression without making a clear boundary observed in chick heart. These
observation are compatible with the mutually repressive actions of these Tbx
genes (Szeto et al., 2002) and
our observations that the ventricular septum is formed at the distinct
expression boundary of Tbx5.
Our data provide important insights on cardiac development, onset of human congenital heart diseases, and evolution of vertebrate hearts. Although we are far from a complete understanding, precise molecular analysis of the Tbx5 gene would provide valuable information for the comprehensive understanding of vertebrate pattern formation.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Cardiovascular Research, The Hospital for Sick Children,
555 University Ave., Toronto, ON M5G 1X8, Canada
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