1 Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625
Hannover, Germany
2 Department of Anatomy and Embryology, Academic Medical Center, University of
Amsterdam, 1105 AZ Amsterdam, The Netherlands
3 Department of Cell and Molecular Biology, Karolinska Institute, 17177
Stockholm, Sweden
* Author for correspondence (e-mail: kispert.andreas{at}mh-hannover.de)
Accepted 6 April 2005
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SUMMARY |
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Key words: T-box, Heart, Myocardium, Anterior heart field, Bmp
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Introduction |
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Chamber formation is a localized process
(Christoffels et al., 2000;
Meilhac et al., 2004
). A
ventral region of myocardium of the linear heart tube that comes to lie at the
outer curvature of the looping heart initiates a chamber-specific program of
gene expression that directs a `ballooning' growth to form the ventricular
chambers. Likewise, the atrial chamber myocardium differentiates and expands
from the dorsolateral portion of the heart tube. Increased rates of
proliferation and subsequent trabeculation, a high conduction velocity and
fast contractions characterize chamber (early working) myocardium. Patterned
expression of several genes encoding transcription factors and signalling
molecules provide evidence for the presence of anteroposterior (AP) and
dorsoventral (DV) patterning in the early heart tube that may control these
localized differentiation processes
(Christoffels et al., 2000
).
Myocardium outside of these distinct regions, in the inflow tract (IFT), the
atrioventricular canal (AVC), the outflow tract (OFT) and the connecting inner
curvatures, does not initiate the chamber-specific program of gene expression
and retains its primary character. Endocardium lining these regions undergoes
an epithelial-mesenchymal transition to form the endocardial cushions. These
cushions are pivotal to the formation of the septa of atria and ventricles,
and to the formation of the valves
(Eisenberg and Markwald,
1995
).
Several T-box (Tbx) genes have been implicated in the regulation of
vertebrate heart development. Tbx genes encode a family of proteins sharing a
highly conserved DNA-binding region, the T-box. T-box proteins act as
transcription factors that exert distinct transcriptional activation and
repression functions depending on the molecular context of the conserved
DNA-binding site. Members of the gene family are conserved throughout metazoan
evolution. In mammals, 18 T-box genes have been identified. Gene targeting
experiments in mice have revealed their crucial functions during gastrulation
and the development of various organ systems (for reviews, see
Papaioannou, 2001;
Tada and Smith, 2001
). In
addition, mutations in several T-box genes cause congenital human diseases
demonstrating the importance of the gene family both in development and
disease (for a review, see Packham and
Brook, 2003
). Functional analyses suggest that four of the six
T-box genes identified in vertebrate heart development, namely Tbx1, Tbx2,
Tbx5 and Tbx20 are important regulators of formation and
maturation of the heart. Functional relevance of cardiac expression of
Tbx3 and Tbx18 has not yet been reported (reviewed by
Plageman and Yutzey,
2004a
).
Tbx20 is a member of the Tbx1-subgroup of T-box transcription
factors. Tbx20 expression was reported in the allantois, dorsal part
of the retina, motoneurons, lateral plate mesoderm, cardiac crescent,
primitive heart tube and four-chambered heart of mouse and chick embryos
(Carson et al., 2000;
Iio et al., 2001
;
Kraus et al., 2001a
). More
detailed analyses have revealed differential expression in the developing
tetrapod heart. After widespread activation in the linear and looping heart,
expression becomes gradually more enriched in AVC, OFT and tricuspid and
mitral valves (Brown et al.,
2003
; Stennard et al.,
2003
; Takeuchi et al.,
2003
; Lincoln et al.,
2004
; Plageman and Yutzey,
2004b
; Yamagishi et al.,
2004
). Cardiac expression is found both in the myocardium and
endocardium, and in endocardial cushion tissues
(Carson et al., 2000
;
Kraus et al., 2001a
;
Stennard et al., 2003
). Bmp2
is a crucial inducer of cardiogenic cell fate. Tbx20, Tbx2 and
Tbx3, but not Tbx5, are induced by Bmp2 in avian cardiogenic
mesoderm, suggesting that Tbx20 acts at least partially downstream of
Bmp2 signaling (Yamada et al.,
2000
; Plageman and Yutzey,
2004b
).
Tbx20 acts as a transcriptional repressor on conserved T-box DNA-binding
sites in cardiac promotors (Plageman and
Yutzey, 2004b). Presence of both transactivation and
transrepression domains in the C terminus of the Tbx20 protein was reported,
providing evidence for a context-dependent control of gene transcription.
Collaboration with other cardiac transcription factors might also contribute
to functional specificity. Indeed, physical interaction with the cardiac
transcription factors Gata4, Gata5 and Nkx2.5 was reported
(Stennard et al., 2003
).
Tbx20 expression is also found in developing hearts of lower
vertebrates and invertebrates, suggesting conservation of a central
cardiogenic program. The Drosophila orthologs midline and
H15 are expressed in the dorsal heart tube. They are required in a
redundant fashion for the normal alignment of cardioblasts and associated
pericardial cells in the dorsal vessel
(Miskolczi-McCallum et al.,
2005; Qian et al.,
2005
). During zebrafish embryogenesis, the expression of the
ortholog hrt is found in the anterior lateral plate mesoderm, the
heart field and the endothelium of the dorsal aorta
(Ahn et al., 2000
;
Griffin et al., 2000
).
Functional studies using morpholino antisense oligonucleotides revealed a
requirement for hrt in cardiovascular development. hrt
morphant hearts do not undergo looping. Chamber formation and gene expression
are perturbed (Szeto et al.,
2002
). A similar cardiac phenotype was observed in Tbx20
morphant Xenopus embryos (Brown et
al., 2005
).
In this paper, we address the role of Tbx20 in cardiac development using a gene targeting approach in the mouse. Mice homozygous for the mutant allele die at E10.5 as a result of hemodynamic failure due to severe cardiovascular malformations. A linear heart tube is established but looping morphogenesis and chamber differentiation fail. We demonstrate that the expression domains of Tbx2, and of other markers for primary myocardium and endocardium lining the primary myocardium, are expanded in the mutant heart. We suggest that Tbx20 promotes progression from the linear to the looping and multi-chambered heart by repressing Tbx2 in the myocardial precursor cells destined to form the chambers, thus allowing chamber-specific differentiation to occur.
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Materials and methods |
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Genotyping of Tbx20 mutant mice
Genotypic characterization of ES cells, embryos and adult mice was done by
Southern blot analysis of restriction-digested genomic DNA. DNA was derived
from ES cells, embryonic yolk sacs and adult tails, and hybridized with probes
distinguishing wild-type and mutant alleles
(Fig. 1A). The 5'-probe
is a 374 bp KpnI/EcoRV fragment subcloned from the genomic
region adjacent but outside the targeting vector. This probe recognizes a 4.8
kb KpnI fragment in the wild type and an 8.2 kb KpnI
fragment in the mutant (Fig.
1B). The 3'-probe, a 582 bp BamHI/KpnI
fragment, detects a 10 kb HincII fragment in the wild type and an 8
kb HincII fragment in the targeted allele
(Fig. 1C).
After initial genotyping of E9.5 embryos by RFLP-Southern analysis, Tbx20 homozygous embryos were identified by phenotype. Genotyping on E7.5-E8.5 embryos was also carried out using a ß-galactosidase assay on yolk sac tissue, taking advantage of the Tbx20 expression in this tissue.
Collection of embryos
For timed pregnancies, plugs were checked in the morning after mating, noon
was taken as embryonic day (E) 0.5. Embryos harvested from heterozygous
intercrosses were dissected in phosphate-buffered saline (PBS), fixed in 4%
paraformaldehyde (PFA)/PBS overnight, dehydrated in methanol and stored at
20°C.
Histological analysis
Embryos for histological staining were fixed in 4% PFA, paraffin-wax
embedded and sectioned to 5 µm. Sections were stained with Hematoxylin and
Eosin. Whole-mount histochemistry for ß-galactosidase activity was
carried out as described (Echelard et al.,
1994). For detection of endothelial endocardium. anti-PECAM1
(CD31) monoclonal antibody (Pharmingen) was used at a dilution of 1:100 as
primary antibody, and 1:200-diluted HRP-coupled goat-anti-rat IgG was used as
a secondary antibody. The detection reaction was performed using
diaminobenzidine and hydrogen peroxide as substrates.
Proliferation and apoptosis assays
Cell proliferation in tissues of E8.5-E8.75, and E9.5, embryos was
investigated by the detection of incorporated BrdU on 5-µm sections of
paraffin wax-embedded specimens, similar to published protocols
(Bussen et al., 2004). Four
sections each of five embryos of each genotype at E8.5 were used for
quantification. The BrdU-labeling rate was defined as the number of
BrdU-positive nuclei relative to the total number of nuclei as detected by
DAPI counterstain in the heart region. Detection of apoptotic cells in 5-µm
paraffin sections of E8.5 and E9.5 embryos was based on the modification of
genomic DNA using terminal deoxynucleotidyl transferase (TUNEL assay), and
indirect detection of positive cells by Fluorescein-conjugated
anti-Digoxigenin antibody. The procedure followed exactly the recommendation
of the manufacturer (Serologicals Corporation) of the ApopTag kit used.
In situ hybridization analysis
Whole-mount in situ hybridization was performed, following a standard
procedure, with Digoxigenin-labeled antisense riboprobes
(Wilkinson, 1992). Stained
specimens were transferred into 80% glycerol prior to documentation.
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Results |
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Mice heterozygous for the mutant Tbx20 allele appear normal and are fertile. Mice homozygous mutant for Tbx20 show severe growth retardation at E9.5 and die at approximately E10.5. Dysmorphic hearts, an enlarged pericardial cavity, edemas and absence of blood circulation indicate that lethality is due to cardiovascular defects (Fig. 2E and data not shown). We here focus on the role of Tbx20 in cardiac development. The possible requirement for vasculogenesis will be considered elsewhere in more detail.
Mutant and wild-type hearts are morphologically indistinguishable at the linear heart tube stage (E8.0-E8.25; data not shown). At E8.25-E8.5, heart looping and chamber formation is initiated in the wild type (Fig. 2A,C). In the mutant, the heart tube fails to loop. Instead, two constrictions appear, separating a putative embryonic ventricle from a posterior inflow tract and an anterior outflow tract region (Fig. 2B,D). By E9.5, the wild-type heart has further elongated and looped, and atrial and ventricular chambers are being formed. By contrast, the mutant heart tube does not elongate further and the architecture of the heart remains unchanged from E8.5 onwards (Fig. 2G,I). Histological analysis confirmed the morphological findings and revealed the presence of myocardium, endocardium, endocardial cushion tissue, and cardiac jelly in the mutant heart (Fig. 2J-Q). Endocardial cushion is accumulated at the anterior constriction compromising the continuity of the endocardial lining of the tube (Fig. 2N). The mutant heart tube shows slow but rhythmic contractions that initiate at the posterior inflow tract region and propagate anteriorly (data not shown).
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We next wished to analyze whether anteroposterior (AP) patterning was
established in the mutant heart at E8.5. We used a set of marker genes whose
restricted expression along the linear heart tube defines such patterning.
-Myosin heavy chain (
MHC) (Myhca;
Mhy6 Mouse Genome Informatics) is expressed in a gradient
from the inflow to the outflow tract. ß-Myosin heavy chain
(ßMHC) (Myhcb; Mhy7 Mouse Genome
Informatics) is expressed in a reverse gradient from the outflow tract to the
inflow tract. Ventricular myosin light chain (Mlc2v; My12
Mouse Genome Informatics) expression is found in a bilaterally
restricted segment that includes the future left ventricle
(Christoffels et al., 2000
).
Tbx5 expression is high posteriorly in the inflow tract region and
declines to low levels in the outflow tract region
(Chapman et al., 1996
;
Bruneau et al., 1999
). Finally,
Gata4 is expressed in the posterior heart region and the endoderm
(Molkentin et al., 1997
).
Polarized expression of these markers is normal in the mutant heart at E8.5
and E9.5 (Fig.
3G'-K' and data not shown), suggesting that AP
patterning of the linear heart tube is established and maintained in the
mutant. Pitx2 expression is restricted to the left limb of the inflow
tract at E8.5 (Campione et al.,
2001
). Expression is unchanged in the mutant (arrow in
Fig. 3L') indicating the
presence/establishment of left-right signaling in the mutant heart.
The heart tube of Tbx20/ embryos does not elongate, but anterior and secondary heart field markers are not affected
The myocardium of the linear heart tube hardly proliferates, and the 4- to
5-fold elongation of the linear heart tube between E8 and E10.5 primarily
results from the recruitment of splanchnic mesoderm of the secondary
(including anterior) heart field, which proliferates rapidly
(Kelly and Buckingham, 2002;
Cai et al., 2003
).
Tbx20 is co-expressed with Mlc2a, a marker for the primary
heart field, but seems to slightly extend anteriorly and posteriorly into the
secondary heart field, which suggests a direct control of heart tube
elongation (Fig. 3C-F,
Fig. 4A-D, and data not shown).
The heart tube does not elongate in the Tbx20 mutant embryo.
Wnt11 expression, a marker for the OFT region of wild-type hearts at
E9.5 (arrow in Fig. 4E)
(Kispert et al., 1996
), is not
found in the anterior portion of the mutant heart, the putative OFT
(Fig. 4E').
Hand2 (Dhand) is expressed in the entire heart tube at E8.5
but becomes upregulated in the RV and OFT from E9.5 onwards (arrow in
Fig. 4F)
(Thomas et al., 1998
).
Dhand expression is not detected in the mutant heart tube
(Fig. 4F'). This suggests
that the ascending limb of the heart tube that gives rise to the RV and OFT of
the E9.5 heart has not been added from the anterior heart field. Hence, the
OFT of the Tbx20 mutant heart at E8.5 and E9.5 is a mere functional
term for a region of cells fated to contribute to a more upstream (posterior)
region in the wild type. We analyzed markers for the anterior (Fgf8,
Fgf10, Foxh1, Mef2C) (Lin et al.,
1997
; Kelly et al.,
2001
; von Both et al.,
2004
) and secondary heart field (islet 1; Isl1
Mouse Genome Informatics) (Cai et al.,
2003
) to assess whether a specific requirement for Tbx20
in this process can be unraveled. We studied marker expression in E8.5
embryos, shortly after looping has been initiated in the wild-type heart, to
exclude secondary changes. Expression of all of these markers was unaltered in
Tbx20 mutants at E8.5 (Fig.
4G'-M') suggesting that Tbx20 does not
primarily regulate the formation and differentiation of the secondary heart
field.
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The failure of chamber differentiation in the embryonic heart tube
indicates that the underlying DV patterning may be affected. Cited1
(also known as Msg1) and Hand1 (Ehand) are
expressed specifically at the ventral side of the linear heart tube and,
subsequently, at the outer curvature of the ventricular portion of the looped
heart tube (Dunwoodie et al.,
1998; Cserjesi et al.,
1995
; Biben and Harvey,
1997
; Thomas et al.,
1998
; Christoffels et al.,
2000
). Expression of neither Cited1 nor Hand1 is
detected in the heart of stage-matched mutant embryos
(Fig. 5E',F').
The T-box transcription factor Tbx3 is hardly detectable at E8.5,
but is expressed in the AVC in the E9.5 wild-type heart
(Hoogaars et al., 2004). The
heart tube of Tbx20 mutants is devoid of any Tbx3 signal
(Fig. 5G'). Finally,
Tbx18 expression is found in the septum transversum and the
proepicardial organ at E9.5 (Kraus et al.,
2001b
). Expression is unaltered in the mutant, suggesting that
proepicardial development is unaffected
(Fig. 5H').
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Tbx2 is expressed throughout the linear heart of Tbx20/ embryos
Tbx2 is both sufficient and necessary to prevent differentiation
of chamber myocardium (Christoffels et
al., 2004a; Harrelson et al.,
2004
). Therefore, we wondered whether lack of chamber
differentiation in the Tbx20 mutant heart is associated with
deregulation of Tbx2. In E8.5 wild-type hearts, Tbx2
expression is found in the myocardium of the IFT (more strongly in its
anterior part), in the forming AVC with a sharp border towards the forming
ventricle, and in the underlying septum transversum mesenchyme
(Habets et al., 2002
). At
E9.5, Tbx2 is expressed in the myocardium of the AVC and OFT
(Christoffels et al., 2004a
;
Harrelson et al., 2004
). In
the Tbx20 mutant, Tbx2 expression is strongly upregulated in
the cardiac crescent at E7.75-E8.0 (arrow in
Fig. 6A'). From E8.25
onwards, Tbx2 is strongly expressed throughout the linear heart tube,
i.e. in the IFT region, the embryonic ventricle and the OFT region
(Fig. 6B'-F').
Anti-PECAM immunohistochemistry showed the presence of endothelial endocardium
in the mutant heart at E8.5 and E9.5. The endothelial lining was found to be
discontinuous at the upper constriction. suggesting reduced or absent blood
circulation in Tbx20 mutant embryos
(Fig. 6G',H'). We
next investigated whether the mutant endocardium would also be reprogrammed to
a type of endocardium lining primary cardiac tissue by analyzing vinexin
(Sh3d4 Mouse Genome Informatics) expression. In the
wild-type heart, vinexin
expression is restricted to the endocardium
of the anterior part of the IFT and AVC at E8.5, and to the OFT and AVC
endocardium at E9.5 (Kawauchi et al.,
2001
). In the Tbx20 mutant, expression is found
throughout the endocardial layer of the linear heart tube at E8.5 and E9.5
(Fig. 6I'-K'). Recently, evidence has accumulated that cardiac Tbx2 is induced by
Bmp2, a secreted protein of the Dpp/Bmp signaling family
(Yamada et al., 2000
). We
reasoned that derepression of Tbx2 in the Tbx20 mutant heart
may be triggered by spread of Bmp2 expression from the IFT/AVC region
anteriorly into the primitive ventricle and OFT. Analysis of Bmp2
expression in Tbx20 mutant hearts at E8.5 and E9.5 revealed a weak
but consistent expression of Bmp2 in the myocardium of the primitive
ventricle, but downregulation in the inflow tract, and absence in the outflow
tract region (Fig.
6L'-N'). This marker analysis suggests that the
Tbx20 mutant heart, in particular the primitive ventricle, has
acquired a primary type of myocardium and endocardium, possibly by Bmp2
induction of Tbx2.
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Cellular proliferation was determined by the BrdU incorporation assay on transverse sections of E8.5-E8.75 wild-type and Tbx20 mutant embryos (Fig. 7E-G). Proliferation in the (primitive) ventricular heart region, as judged by the BrdU-labeling index, was significantly reduced from 0.133±0.0089 in the wild type to 0.03±0.0032 in the mutant (P<0.005; Fig. 7G). By contrast, proliferation in extracardiac regions was obviously unchanged in the mutant embryos (Fig. 7E,F). This suggests that the arrest of heart development in the Tbx20/ embryos is accompanied and probably partly caused by a reduction of cellular proliferation rates. At E9.5, Tbx20/ embryos are characterized by a complete arrest of cellular proliferation in all tissues. We assume that the general arrest in cell proliferation at this stage is due to the severe vascular defects of the Tbx20/ embryos.
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Discussion |
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Tbx20 regulation of Tbx2 and formation of cardiac chambers
Chamber formation relies on an integrated patterning program that directs
localized differentiation programs along the AP and DV axes of the linear
heart tube (reviewed by Moorman and
Christoffels, 2003). A linear heart tube with normal AP polarity
is established in Tbx20 mutant embryos. However, DV, i.e. inner-outer
curvature, patterning revealed by Hand1 and Cited1
expression is absent, and the program for chamber myocardial differentiation
is not initiated.
Conceivably, Tbx20 directly controls DV patterning and subsequent
activation of the chamber differentiation program. Loss of Hand1
expression may contribute to the phenotypic defects in
Tbx20/ hearts. Hand1, a marker for
DV patterning, is required for the formation of the ventrally derived
ventricular outer curvature (Biben and
Harvey, 1997; Christoffels et
al., 2000
; Riley et al.,
1998
). Alternatively, Tbx20 assures progression from the
linear heart tube by preventing the activation or maintenance of the primary
myocardial program, specifically in the primitive ventricle. We favor this
possibility, and suggest that Tbx20-mediated repression of
Tbx2 is pivotal to the normal program of chamber formation.
Tbx2 has a well-established role in maintaining the primary
myocardial phenotype. Tbx2 is expressed in regions of the looped and
multi-chambered heart retaining the primary myocardial phenotype
(Gibson-Brown et al., 1998;
Yamada et al., 2000
;
Habets et al., 2002
;
Christoffels et al., 2004a
;
Harrelson et al., 2004
). Loss
of Tbx2 expression leads to expansion of chamber myocardium into the
AVC, and subsequent defects in formation of septa and valves
(Harrelson et al., 2004
). Most
importantly, ectopic expression of Tbx2 in the myocardium of the
linear heart tube completely prevents chamber formation
(Christoffels et al., 2004a
).
Thus, loss of Tbx20 phenocopies misexpression of Tbx2 in the
linear heart tube. This suggests that ectopic expression of Tbx2 in
Tbx20 mutant hearts accounts for the arrest in cardiac
development.
Similar linear heart tube phenotypes have been described for
Nkx2.5 and Tbx5 mutants
(Lints et al., 1993;
Tanaka et al., 1999
;
Bruneau et al., 2001
). Cardiac
expression of Nkx2.5 and Tbx5 is unaltered in
Tbx20/ hearts, negating a role for these
genes in mediating Tbx20 function. Expression of Tbx20 is
unaltered in Tbx5 mutants
(Stennard et al., 2003
). This
and the different signature of molecular markers in all three mutants strongly
suggests that Tbx20, Nkx2.5 and Tbx5 act in distinct
cardiogenic programs of chamber formation in the mouse.
Tbx5 and Nkx2.5 synergistically activate the expression
of Nppa in the forming chamber myocardium
(Bruneau et al., 2001;
Hiroi et al., 2001
). The
expression of Nppa is completely abolished in Tbx20 mutant
hearts, although expression of the potential activators Tbx5 and Nkx2.5 is
maintained. Habets et al. have recently revealed the ability of Tbx2 to
counteract the synergistic activation of Nppa by Tbx5/Nkx2.5
(Habets et al., 2002
). Thus,
ectopic Tbx2 in the Tbx20/ heart may compete
with Tbx5 in binding to enhancer elements driving expression of Nppa
and possibly other chamber myocardial specific genes. In addition, Tbx2 is a
direct repressor of connexin 40 and connexin 43 in chamber myocardium
(Chen et al., 2004
;
Christoffels et al., 2004a
),
which explains the repression of these genes in the Tbx20 mutant
heart.
It is unclear how ectopic activation of Tbx2 expression in the
Tbx20/ heart is mediated on the molecular
level. Tbx20 has recently been shown to act as a transcriptional
repressor on T-sites in cardiac promotors
(Plageman and Yutzey, 2004b),
opening the possibility that Tbx20 directly represses Tbx2.
However, such a function has not been experimentally confirmed, and is not
easy to reconcile with the overlapping expression of Tbx2 and
Tbx20 in the AVC and OFT from E8.5 onwards. Alternatively, ectopic
expression of Tbx2 could be achieved indirectly. Tbx2 is
induced by Bmp signaling in cardiogenic mesoderm
(Yamada et al., 2000
).
Bmp2 is co-expressed with Tbx2 in the AVC. Thus, ectopic
expression of Tbx2 in the primitive ventricle of
Tbx20/ embryos could be achieved by
activation or derepression of its activator Bmp2. Indeed, our
analysis has shown that weak but consistent Bmp2 expression is found
in the primitive ventricle in Tbx20 mutant hearts. Regulation of
Bmp2 by Tbx20 is likely to be complex. Bmp2 is
co-expressed with Tbx20 in the AVC from E8.5 onwards
(Keyes et al., 2003
). However,
Bmp2 expression in the primitive IFT of the Tbx20 mutant
heart is downregulated. In addition, Tbx2 expression is also found in
the outflow tract region of the Tbx20/ heart
at E8.5, whereas Bmp2 is not. Conceivably, combinatorial action of
Tbx20 with other transcription factors will define the regionally
restricted expression of potent signaling molecules such as Bmp2 in
the developing heart.
We observed that endocardium of the Tbx20 mutant heart is also
reprogrammed to a type normally lining primary myocardium. At this point it is
unclear whether Tbx20 controls endocardial fate directly.
Alternatively, myocardial expression of Bmp2 and/or Tbx2 may
induce a fate change in the underlying endocardium. Analysis of transgenic
embryos ectopically expressing Tbx2
(Christoffels et al., 2004a)
will allow us to discriminate between these possibilities.
Tbx2 is closely related to Tbx3. Both proteins share an identical
DNA-binding region and act as transcriptional repressors on conserved
DNA-binding sites. Tbx2 and Tbx3 are co-expressed in the
primary myocardium of the AVC, and can similarly be induced by Bmp2
signaling (Yamada et al.,
2000; Plageman and Yutzey,
2004b
). Cardiac defects have not been described in mice homozygous
for a null allele of Tbx3
(Davenport et al., 2003
).
These experimental findings point to a redundant function of Tbx3
with Tbx2 in cardiac development. However, our results suggest that
both genes are differentially regulated and might thus exert distinct
functions in heart development. Tbx2 is upregulated in Tbx20
mutant hearts whereas Tbx3 expression is lost. Hence, ectopic Bmp2
expression might activate Tbx2 only. Conceivably, Tbx3
expression is regulated by other signaling systems or requires higher levels
of Bmp2 signaling than Tbx2.
At this point, we cannot exclude that other Tbx2-independent cardiac functions of Tbx20 exist. Analysis of the phenotypic consequences of Tbx20 loss in a Tbx2 mutant background will be a valuable approach to reveal additional requirements for Tbx20 in heart development.
Tbx20 and the secondary heart field
Detailed recent analyses suggest that, in the mouse, the right ventricle
and the outflow tract, as well as the atria and sinus venosus, originate by
continuous recruitment and myocardial differentiation of splanchnic mesodermal
cells (Kelly et al., 2001;
Cai et al., 2003
). Mutations
in genes that effect the recruitment, migration, differentiation or
proliferation of cells from this secondary heart field show severe hypoplasia
of the right ventricle, outflow tract and atria
(Lin et al., 1997
;
Srivastava et al., 1997
;
Cai et al., 2003
;
von Both et al., 2004
).
Similar defects are seen in the Tbx20/
heart, suggesting that Tbx20 may at least partly regulate secondary
heart field development. However, a primary role for Tbx20 in
secondary heart field development seems unlikely for several reasons. First,
early Tbx20 expression overlaps with that of Mlc2a, which is
considered to mark the primary heart field, but only marginally with that of
Isl1, a marker for the secondary heart field
(Stennard et al., 2003
;
Cai et al., 2003
). Second,
markers for the secondary heart field including Isl1, Foxh1, Mef2c
and Fgf10 are unchanged in Tbx20 mutant hearts, excluding
direct regulation of any of these genes by Tbx20. Last, the short
linear heart tube observed in Tbx20 mutant embryos and secondary
heart field mutants such as Isl1, are morphologically similar but
molecularly different. Markers for DV patterning and ventricular and atrial
differention are not expressed in Tbx20 mutant hearts. By contrast,
DV patterning (Hand1 expression) and ventricular differentiation
(Hey2 expression) take place normally in Isl1 and
Foxh1 mutant hearts (Cai et al.,
2003
; von Both et al.,
2004
).
However, even if ventricular development is arrested at E8.5 in
Tbx20/ embryos, the secondary heart field
should still add cells at the poles, resulting in elongation of the heart tube
at the arterial and venous ends after E8.5. We think that cells from the
secondary heart field are prevented from their normal fate for two reasons.
First, Tbx2 is ectopically expressed throughout the linear heart tube
of Tbx20 mutants. Tbx2 expression now abuts and possibly
also extends into the secondary heart field region. Ectopic Tbx2
might downregulate proliferation of mesenchymal cells in the secondary heart
field region, and/or prevent their myocardial differentiation at the border of
secondary heart field and myocardium. This hypothesis gains support from
cardiomyocyte-restricted overexpression of Tbx2 in transgenic mouse
embryos. These embryos had short heart tubes as well, supporting the notion
that Tbx2 expression at the border of the secondary heart field
interferes with the recruitment of mesenchymal cells. In some cases, we
observed transgenic Tbx2 expression extending into the anterior heart
field, as if these cells had turned on the Mhcb promoter, but had
failed to move in (Christoffels et al.,
2004a). Therefore, downregulation of Tbx2 in cells at the
myocardial-secondary heart field border may be required for their subsequent
recruitment to the poles of the heart tube. Second, it is likely that impaired
vascular development in Tbx20 mutant embyros dramatically affects
cell proliferation, thus preventing expansion of the pool of splanchnic
mesodermal cells in the secondary heart field.
A conserved program in vertebrate cardiogenesis?
In all vertebrates analyzed to date, Tbx20 is expressed in early
cardiogenic mesenchyme, in the linear heart tube, during heart looping and
chamber formation. Analyses of a Tbx20 mouse mutant in this study,
and of morphants of the zebrafish and Xenopus orthologs
(Szeto et al., 2002;
Brown et al., 2005
), suggest
that, in vertebrates, Tbx20 has no unique early function in the
induction of cardiac cell fate from the lateral plate mesoderm and the
formation of a linear heart tube, but only in the transition to the
multi-chambered heart. The late requirement for a T-box transcription factor
is reminiscent of the situation in mesoderm formation. There, brachyury
(T) is expressed in the mesoderm forming region with the onset of
gastrulation, but is only required for mesoderm formation and axial elongation
significantly later (Herrmann and Kispert,
1994
). In either case, redundancy with another Tbx family member
may account for this lack of an early requirement. Alternatively, these T-box
transcription factors may need to interact with auxiliary factors that become
expressed only later in development. The analyses of zebrafish hrt
and Xenopus Tbx20 morphant phenotypes have shown that the heart tube
acquires AP patterning, but fails to loop and forms abnormal chambers.
Although not addressed in those studies, it is tempting to assume that DV
patterning of the heart tube and chamber differentiation fails, similar to the
situation in the mouse. Interestingly, it was noted that the hrt
morphant heart contracted abnormally and slowly. This resembles the change of
contraction velocity and rhythm we observed in the
Tbx20/ heart tube. In zebrafish heart
development, hrt may regulate tbx5 negatively, as
hrt is both sufficient and required to repress tbx5
expression in the developing heart (Szeto
et al., 2002
). In the mouse, we observed unchanged Tbx5
expression but upregulated Tbx2 instead. Simplistically, one could
suggest that tbx5 functionally replaces Tbx2 in the
zebrafish. However, this is unlikely. tbx5 has been shown to be
required for looping and maintaining the heart tube in the zebrafish
(Garrity et al., 2002
), a role
that is similar to the requirement for murine Tbx5 in posterior heart
development (Bruneau et al.,
2001
). As Tbx2 has not yet been described in the
zebrafish, the functional significance of tbx5 derepression in the
hrt morphant heart remains unclear. Notably, in Xenopus
Tbx20 morphants cardiac Tbx5 expression is unchanged, similar to
the situation in the mouse. However, a synergistic role for Tbx5 and
Tbx20 in Xenopus heart development was suggested
(Brown et al., 2005
). Such a
mechanism seems unlikely for mouse cardiogenesis because the cardiac
phenotypes of Tbx5 and Tbx20 mutants differ significantly
(Bruneau et al., 2001
) (this
study). Together, these findings may provide evidence for the divergence of
Tbx20-controlled molecular pathways in zebrafish, Xenopus
and mouse, compatible with the increase in cardiac complexity achieved in
tetrapod evolution.
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ACKNOWLEDGMENTS |
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