1 Victor Chang Cardiac Research Institute, St Vincent's Hospital, 384 Victoria
Street, Darlinghurst 2010, New South Wales, Australia
2 Laboratório de Cardiologia Celular e Molecular, Instituto de Biofisica
Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 20941-000,
Brazil
3 Cardiovascular Research Institute, University of California, San Francisco, CA
94143-0130, USA
4 Faculties of Medicine and Life Sciences, University of New South Wales,
Kensington 2056, New South Wales, Australia
Author for correspondence (e-mail:
r.harvey{at}victorchang.unsw.edu.au)
Accepted 21 February 2005
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SUMMARY |
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Key words: T-box, Tbx20, Heart, Nkx2-5, Tbx2, Chamber myocardium, Dilated cardiomyopathy, mice
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Introduction |
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Of 18 T-box factor genes identified in mammals, at least six of them
(Tbx1/2/3/5/18/20) are expressed in the developing heart
(Plageman and Yutzey, 2004).
T-box proteins are characterized by the presence of a sequence-specific
DNA-binding domain called the T-box
(Smith, 1999
). During
embryogenesis, T-box genes are expressed in restricted and sometimes
overlapping domains throughout gastrulation and/or organogenesis, and in some
cases roles in controlling cell fate and migration have been demonstrated
(Chapman and Papaioannou,
1998
; Naiche and Papaioannou,
2003
; Russ et al.,
2000
). T-box factors can act up- or downstream of signaling
factors of the TGF-ß (Suzuki et al.,
2004
), fibroblast growth factor
(Brown et al., 2004
;
Hu et al., 2004
;
Sakiyama et al., 2003
;
Yamagishi et al., 2003
), sonic
hedgehog (Suzuki et al., 2004
;
Yamagishi et al., 2003
) and
wingless-related (Takeuchi et al.,
2003
) superfamilies.
Haploinsufficiencies for several human T-box genes have been linked to
congenital anomaly syndromes (Bongers et
al., 2004; Packham and Brook,
2003
). Two of these involve cardiac malformations. Di George
syndrome, also occurring as part of chromosome 22q11 deletion syndrome, is
characterized by dysmorphogenesis of the cardiac outflow tract (OFT), as well
as thymic, splenic and craniofacial abnormalities
(Yamagishi and Srivastava,
2003
). Holt Oram syndrome is characterized by congenital
abnormalities of the upper limbs and heart, the latter involving atrial and
ventricular septal defects, tetralogy of Fallot and atrioventricular
conduction block (Gruber and Epstein,
2004
). Targeted mutation of causative genes in mice has reproduced
many aspects of the human cardiac disease phenotypes, thus providing valuable
models for understanding underlying mechanisms
(Bruneau et al., 2001
;
Lindsay et al., 2001
;
Merscher et al., 2001
;
Yamagishi and Srivastava,
2003
).
Tbx20 is an ancient member of the T-box gene subfamily related to
Tbx1 (Plageman and Yutzey,
2005). The Drosophila gene is expressed in early
cardioblasts of the dorsal vessel of the fly, a primitive heart-like organ
(Griffin et al., 2000
). During
fish development, Tbx20 (hrT) is expressed in cardiac
progenitors, developing heart and endothelial cells of the dorsal aorta
(Ahn et al., 2000
;
Griffin et al., 2000
), and
Tbx20 orthologs with analogous expression patterns have since been
identified from frog, chicken, mice and humans
(Carson et al., 2000
;
Horb and Thomsen, 1999
;
Kraus et al., 2001
;
Meins et al., 2000
;
Stennard et al., 2003
;
Yamagishi et al., 2004
).
During mouse development, Tbx20 is expressed in the cardiac crescent
prior to heart tube formation, then in myocardium and endocardium of the
looping heart (Kraus et al.,
2001
; Stennard et al.,
2003
).
As for other T-box factors (Bruneau et
al., 2001; Casey et al.,
1999
; Habets et al.,
2002
; Harrelson et al.,
2004
; He et al.,
1999
; Hoogaars et al.,
2004
; Hsueh et al.,
2000
; Kispert et al.,
1995
; Lamolet et al.,
2001
; Paxton et al.,
2002
; Stennard et al.,
2003
; Tada and Smith,
2000
), Tbx20 can regulate transcription of target genes positively
and negatively, depending on the particular isoform expressed and,
potentially, cellular context (Plageman
and Yutzey, 2004
; Stennard et
al., 2003
). The T-box DNA-binding domain of Tbx20 can associate
specifically, albeit weakly, with the consensus DNA half site sequence defined
for the brachyury (T) protein, the founding member of the T-box family, and
can interact in solution and function synergistically with homeodomain factor
Nkx2-5 and zinc finger factor Gata4
(Stennard et al., 2003
).
Morpholino oligonucleotide knockdown of Tbx20 in fish produces
small and dysmorphic hearts showing upregulation of Tbx5, ectopic
expression of blood markers caudally and an abnormally patterned aorta
(Szeto et al., 2002).
Tbx20 downregulation in frogs leads to absent
(Horb and Thomsen, 1999
) or
dysmorphic (Brown et al., 2005
)
hearts. Frog Tbx20 physically interacts with Tbx5 and cardiac defects in
embryos are more frequent and severe if both proteins are concomitantly
inhibited (Brown et al., 2005
).
Enforced expression experiments in frog embryos show that mouse Tbx20 can
induce mesodermal and endodermal cell fates and their coordinated cell
migration (Stennard et al.,
2003
).
We report here the loss-of-function phenotype of murine Tbx20. Tbx20 null embryos showed grossly abnormal cardiac development and arrested yolk sac vascular remodeling. Our analysis has highlighted a role for Tbx20 as a transcriptional repressor during the primary lineage split in myocardium into chamber and non-chamber fates. Furthermore, hierarchical interaction between different T-box genes was revealed as a central element of early heart patterning and morphogenesis. Tbx20 also acts in adult heart function and homeostasis, with implications for human cardiomyopathies.
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Materials and methods |
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Embryo culture
Explants from C57BL/6J E8.5 embryos were cultured in DMEM media containing
0.5% (v/v) heat-inactivated fetal bovine serum, 10 mmol/l glutamine, 100
units/ml penicillin and streptomycin (GibcoBRL) in 1% agarose-coated wells for
24 hours with or without recombinant human Heregulin ß2 (10-9
mol/l) (Fiddes et al.,
1995).
Gene targeted mice
Tbx20lacZ/+ mice were generated by Ozgene Pty Ltd
(Perth, Australia). Nkx2-5lacZ mice were generated using a
vector similar to one described (Biben et
al., 2000), in which a lacZ cassette was inserted in
frame into exon 1 (M.S., C.B. and R.P.H., unpublished).
Transthoracic echocardiography
Two-dimensional echocardiographic images were obtained using a Sonos 5500
ultrasonograph with 12 MHz probe (Philips Medical Systems) as described
(Fatkin et al., 2000).
Statistical significance of data was determined by ANOVA and Student's
t-test.
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Results |
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Cardiac gene expression in Tbx20 null embryos
The early transcription factor program was significantly compromised in
Tbx20lacZ/lacZ hearts. Expression of T-box factor gene
Tbx5 was reduced at E8.5, although the caudal-high, graded pattern
seen in normal hearts was preserved
(Bruneau et al., 1999)
(Fig. 3A). Expression had
recovered somewhat by E9.5 (Fig.
3B), suggesting delayed activation. The cranial limit of
Tbx5 expression at E9.5 is normally at the level of the
interventricular sulcus (Bruneau et al.,
1999
). In mutants, it was at the sulcus between the inflow and
outflow chamber-like swellings (Fig.
3B), suggesting that these swellings represent precursors of the
normal systemic (left) ventricle and pulmonary (right) ventricle/OFT,
respectively. Consistent with this model, Hey1, encoding a basic
helix-loop-helix factor acting downstream of Notch signaling and expressed
predominantly in endocardium of the OFT in normal E9.5 hearts
(Iso et al., 2003
), was
expressed only in the outflow ventricle-like chamber in mutants
(Fig. 3C).
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As noted above, the outflow chamber in mutant hearts probably corresponds
to precursors of the pulmonary ventricle and outflow tract of normal embryos,
which are derived from the anterior SHF. To assess this further, we
constructed transgenic mice bearing the human placental alkaline phosphatase
gene (hPLAP) driven by an enhancer of the Mef2c gene, previously
shown to accurately mark cells of the anterior SHF and their pulmonary
ventricle and outflow derivatives (Dodou
et al., 2004) (Fig.
3I). Tbx20lacZ/lacZ embryos carrying the
transgene showed strong hPLAP staining in the outflow chamber extending
caudally to the sulcus at E9.0, strongly supporting our hypothesis that this
chamber is SHF-derived. Weaker staining was also seen in the caudal
ventricle-like chamber, suggesting a contribution to this chamber from the
anterior SHF. A minority of anterior SHF cells do contribute to the left
ventricle in wild-type embryos (Cai et
al., 2003
), although the Mef2c enhancer used in these
experiments is normally downregulated in those cells (D.J.M. and B.L.B.,
unpublished).
Excessive cell death was not detected in mutant myocardium or endocardium (data not shown). We therefore measured the mitotic index (proportion of cells expressing phosphohistone H3) in three zones of E9.0 hearts corresponding in wild-type embryos to the sinuatrium, systemic ventricle and pulmonary ventricle/OFT. The myocardium of the outflow ventricle-like chamber in mutants (n=2) had a mitotic index 6 to 7-fold less than the equivalent region in controls (n=2; P<0.0001, chi-squared test) (see Table S1 in the supplementary material). The index in the inflow ventricle-like chamber was also reduced, although less so (2.2 and 3.3-fold; P=0.056 and 0.008), while indices in the sinuatrium and head mesoderm were normal.
Expanded cardiac pre-pattern in Tbx20 and Tbx20/Nkx2-5 homozygotes
The Myl2 gene, which encodes myosin light chain 2v, is expressed
in ventricles and the atrioventricular canal (AVC), but not atria, betraying a
molecular pre-pattern in the forming heart
(Fig. 4A). How this pre-pattern
is established is unknown, although maximal Myl2 expression requires
the homeodomain factor Nkx2-5 (Lyons et
al., 1995). Myl2 was expressed in
Tbx20lacZ/lacZ hearts at a level diminished compared with
wild-type controls at E9.25, but nonetheless considerably higher than seen in
Nkx2-5GFP/GFP embryos, which lack Nkx2-5 function
(Biben et al., 2000
)
(Fig. 4A). Expression
encroached partially into the outflow ventricle-like chamber of Tbx20
mutants (Fig. 4B), consistent
with the notion discussed above that this chamber is SHF-derived and composed
of progenitors that would normally form the pulmonary ventricle and OFT.
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To investigate the apparent expansion of the Myl2 expression domain further, we compared expression of Myl2 and its relative Myl7 (encoding myosin light chain 2a) at earlier stages. Despite the obvious delay in heart tube formation in mutants, dimensions of the cardiac myogenic field, as highlighted by Myl7, appeared comparable to those of controls at E8.5 (Fig. 4D,E). Myl2 expression was highly regional within the Myl7 domain. However, expression in mutants, unlike that in wild types, extended into the apparent sinuatrial region. Furthermore, before and during the onset of heart tube formation at E7.75 and 8.0, respectively, Myl2 expression was qualitatively different and expanded caudally and medio-anteriorly in mutants relative to somite-matched controls (Fig. 4F and data not shown). At E7.75, approximately three times the number of cells expressed Myl2 in the mutants, demonstrating primary dysregulation of patterning processes (see Discussion).
Spatial specification of the atrial domain appeared nonetheless normal in
mutants. Although diminished, a morphological sinuatrium and AVC had formed by
E9.5 (Fig. 2C,D). Furthermore,
the regional expression of Hey1 was spatially correct in the outflow
domain (see above) and sinuatrial region, albeit downregulated significantly
in this latter domain (Fig.
4G). Expression of Aldh1a2, which overlaps the sinuatrial
region of normal hearts and is essential for atrial specification
(Niederreither et al., 2001),
was also normal in Tbx20lacZ/lacZ embryos
(Fig. 4H).
Chamber formation in Tbx20 mutants
The myogenic layer of the early heart tube undergoes an initial regional
specialization to form working myocardium of the ventricles and atrial
appendages (Christoffels et al.,
2000), an event that depends on transcription factors Nkx2-5
(Palmer et al., 2001
), Tbx5
(Bruneau et al., 2001
) and
Foxh1 (von Both et al., 2004
).
Expression levels of Nppa and Smpx, markers of chamber
myocardium (Christoffels et al.,
2000
; Palmer et al.,
2001
), were severely reduced in mutant hearts at E8.5 and 9.5
(Fig. 4I-J), demonstrating lack
of chamber differentiation. Hand1, expressed predominantly in the
forming left ventricle, was also dramatically downregulated
(Fig. 4K).
In the looping heart, non-chamber myocardium retains the slow conduction
features evident in the primary heart tube and is destined to form elements of
the central conduction system
(Christoffels et al., 2004a).
Tbx2, encoding another member of the T-box family, is expressed in
non-chamber myocardium, most prominently in the AVC
(Habets et al., 2002
), where
it has been proposed to repress formation of chamber myocardium
(Christoffels et al., 2004b
;
Harrelson et al., 2004
). By
contrast to the highly regional expression of Tbx2 in the forming AVC
in normal embryos at E8.5, Tbx2 was strikingly upregulated and
ectopically expressed throughout the entire Tbx20lacZ/lacZ
mutant heart (Fig. 4L). The
pattern extended considerably more caudally in lateral plate mesoderm than in
wild-type embryos, identical to the patterns of Myl7 and
Tbx20-lacZ at this stage (Fig.
4E and data not shown). We examined Tbx2 expression in
Nkx2-5GFP/GFP embryos, in which formation of chamber
myocardium is also blocked (Palmer et al.,
2001
). Tbx2 was expressed in the normal pattern in these
embryos, although slightly diminished in level
(Fig. 4L), suggesting that
upregulation of Tbx2 in all or most myogenic progenitor cells in
Tbx20lacZ/lacZ embryos is not a default state arising from
loss of chamber myocardium (see Discussion), and that Tbx20, directly or
indirectly, represses Tbx2 and plays a major role in its regional
expression. As expected,
Tbx20lacZ/lacZ/Nkx2-5GFP/GFP doubly homozygous
embryos also showed upregulation of Tbx2 across the heart
(Fig. 4L).
Bmp2 is expressed in myocardium of the AVC and OFT in the looping
heart and has been proposed to positively regulate Tbx2 and establish
its regional pattern (Yamada et al.,
2000). We therefore assessed expression of Bmp2 and
Bmp4 in the early looping hearts of E8.0 embryos. Bmp2
expression was in fact severely downregulated in
Tbx20lacZ/lacZ hearts at this stage
(Fig. 4M), demonstrating that
Tbx2 upregulation in Tbx20lacZ/lacZ hearts occurs
independently of Bmp2. The pattern of Bmp4 expression was normal
(data not shown).
Tbx20 is repressed by neuregulin 1
The data above show that Tbx20 is required for chamber
differentiation, although it is unclear whether this is direct or indirect.
For example, loss of chamber myocardium could result from the depressed Nkx2-5
expression (Palmer et al.,
2001) or, importantly, ectopic activation of Tbx2, a repressor of
chamber-specific gene expression
(Christoffels et al., 2004b
).
Paradoxically, Tbx20 may itself be a chamber repressor the long Tbx20a
isoform, which carries strong transcriptional activation and repression
domains in its C-terminal region (Stennard
et al., 2003
), acts as a repressor of the chamber-specific gene
Nppa1 in vitro (Plageman and
Yutzey, 2004
). In embryos, Tbx20 expression is at first
enhanced in forming chamber myocardium at the outer curvature, then
downregulated from E9.0, initially in the more differentiated cells of
trabeculae (Stennard et al.,
2003
), suggesting that it is non-essential for the later stages of
chamber differentiation. To clarify this issue, we asked whether
Tbx20 expression increased or decreased after treatment of myocardium
in situ with a pro-chamber stimulus. Neuregulin 1 (Nrg1), a member of the
epidermal growth factor family of signaling ligands, is expressed in the
endocardium of the early heart tube
(Garratt et al., 2003
) and,
along with its co-receptors ErbB2 and 4, expressed in myocardium, is essential
for formation of trabeculae, a morphological feature of chamber myocardium.
Excess Nrg1 induces trabecular overgrowth in vivo and enhances
myofibrillogenesis in vitro (Hertig et
al., 1999
). We explanted the cardiac region of wild-type E8.5
embryos and cultured them with and without Nrg1 (1 nmol/l) in low serum (0.5%)
medium for 24 hours. Overall, cardiac-specific gene expression was reduced in
cultured explants, for some genes dramatically
(Fig. 5 and data not shown), a
possible consequence of cardiac unloading. However, expression of the chamber
markers Nppa and Cited1 in explants was restored to
approximately normal levels and the correct pattern by Nrg1. The
pan-myocardial marker Actc1 (encoding
-cardiac actin) was also
slightly increased (Fig.
5A,B,D). Notably, however, Tbx20 expression was
significantly repressed by Nrg1 in a dose-dependent manner, and remaining
expression was mostly in endocardium (Fig.
5C and data not shown). These findings support the expression data
suggesting a non-essential role for Tbx20 in the later stages of chamber
differentiation. The simplest interpretation is that chamber loss in
Tbx20lacZ/lacZ hearts is indirect, although an early
direct role for Tbx20 in setting up the chamber program cannot be excluded.
Additionally, Nrg1 may be the agent that actively represses Tbx20
during formation of chamber myocardium in vivo.
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Humans with NKX2.5 mutations show secundum atrial septal defect
(ASD) at high penetrance (Schott et al.,
1998). Nkx2-5 heterozygous mutant mice also show ASD but
only rarely (1% on C57BL/6 background), although they do manifest a spectrum
of less severe atrial septal abnormalities including shortened septum primum,
patent foramen ovale and atrial septal aneurysm
(Biben et al., 2000
). They may
therefore be sensitized to mutation or downregulation of other genes involved
in atrial septation. Tbx20 may be one such gene, since it is
expressed in the inter-atrial septum primum
(Fig. 1G). Indeed, anatomical
dissection revealed frank ASD in 16% (n=4/24) of
Tbx20lacZ/+/Nkx2-5GFP/+ mice, while none were
found in Tbx20lacZ/+ and Nkx2-5GFP/+
mice (P<0.05) (Table
1).
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Despite reduced contractile function, compensatory myocardial hypertrophy
was not evident in any of the mutant hearts. Specifically, there was no change
in heart weights or heart weight/body weight ratios
(Table 2), and no overt signs
of myofiber hypertrophy. Moreover, while northern analysis showed upregulation
of Nppa, a general marker of myocardial stress, in
Tbx20lacZ/+ hearts, multiple markers of cardiac
hypertrophy were either normally expressed or diminished in mutant genotypes
(Fig. 7G). Nppa was
significantly downregulated in Nkx2-5GFP/+ and
Tbx20lacZ/+/Nkx2-5GFP/+ hearts, probably
because Nkx2-5 plays a direct role in Nppa transcription
(Durocher et al., 1996).
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Discussion |
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Morphology and gene expression in Tbx20 mutant hearts
In the absence of Tbx20, heart tube development was severely
retarded. By E9.5, a small, forward looped, hourglass-shaped heart with two
distinct ventricle-like chambers separated by a pronounced sulcus and a
diminished sinuatrium had formed. Our morphological findings are generally
consistent with the consequences of Tbx20 morpholino knockdown
experiments in fish and frogs, which showed small, unlooped and dysmorphic
hearts with poor chamber discrimination
(Brown et al., 2005;
Szeto et al., 2002
).
While the cardiac progenitor field appeared to form normally in
Tbx20 mutants, there was initially a profound delay in incorporation
of progenitors into the forming primary heart tube. We have previously shown
that injection of Tbx20 mRNA into Xenopus embryos induces
mesodermal and endodermal cell fates and their coordinated migration via cell
non-autonomous mechanisms (Stennard et
al., 2003), and numerous other T-box factors have been implicated
in control of cell migration at gastrulation
(Russ et al., 2000
;
Tada and Smith, 2000
;
Yamamoto et al., 1998
).
While Tbx20 could control cell migration of cardiac progenitors directly,
the effects may in part be indirect due to blocked or delayed differentiation
of myocardium or endocardium. Indeed, in Tbx20lacZ/lacZ
mutants, the early cardiac regulatory program involving transcription factors
Nkx2-5, Gata4 and Mef2c and cardiac inducing factor Bmp2 was significantly
compromised. Furthermore, expression of Tbx20-lacZ in foregut flags
the possibility that Tbx20 could also regulate differentiation of
endoderm, a well-known source of factors that induce and support
differentiation (and migration) of the cardiomyocyte lineage
(Nascone and Mercola,
1996).
Development of SHF derivatives in Tbx20lacZ/lacZ
mutants was also defective. Abnormal deployment or differentiation of SHF
cells is likely to underlie conotruncal and other congenital heart defects in
humans, with haploinsufficiency for the T-box factor gene, TBX1,
expressed in SHF cells and associated endoderm, thought to be the major
determinant of a spectrum of heart as well as branchial region defects
associated with chromosome 22q11 deletion syndrome
(Yagi et al., 2003). The
outflow ventricle-like chamber in Tbx20lacZ/lacZ hearts is
likely to be derived from the SHF, and this is supported by strong expression
of the Mef2c-SHF-hPLAP transgene in this chamber. However, cell
proliferation in the outflow chamber was severely compromised and the
structure remained bulbous and did not undergo elongation or looping as in
normal hearts. Furthermore, expression of Tnc, encoding a matrix
protein with broad regulatory functions on cell adhesion, migration and
proliferation through interactions with other matrix components and cell
surface receptors (Jones and Jones,
2000
), was not maintained in the outflow region. Thus,
Tbx20 is essential for cell proliferation and gene expression in SHF
cells once they enter the outflow region of the heart, effects that may be
mediated by alterations to the extracellular matrix. This would affect clonal
growth patterns in the forming heart that support correct cardiac looping and
outflow tract morphogenesis (Meilhac et
al., 2004
). It is noteworthy that other T-box genes have been
implicated in control of cell proliferation
(Hatcher et al., 2001
;
Xu et al., 2004
).
Chamber formation and transcriptional repression in the developing heart
Chamber muscle becomes evident early in heart development from the regional
expression of several genes, most notably Nppa and Smpx, and
formation of trabeculae (Christoffels et
al., 2000; Palmer et al.,
2001
). Transcription factors Tbx5, Nkx2-5 and Foxh1 are essential
for its specification (Bruneau et al.,
2001
; Lyons et al.,
1995
; von Both et al.,
2004
), and Tbx5 and Nkx2-5 directly regulate chamber-specific
genes in vitro (Bruneau et al.,
2001
; Stennard et al.,
2003
). In Tbx20lacZ/lacZ hearts, expression of
chamber-specific markers was severely downregulated, indicating that they do
not differentiate chamber muscle. The Nrg1 pathway, which is necessary (but
not sufficient) for chamber differentiation, repressed Tbx20
expression in situ, suggesting that loss of chamber myocardium in
Tbx20lacZ/lacZ hearts is indirect, probably a consequence
of ectopic activation of Tbx2. It is still feasible, however, that Tbx20 plays
a direct positive role at the earliest stages of chamber formation.
A key finding of this work is that Tbx2 was ectopically expressed
in all or most committed myocyte progenitors in Tbx20 mutant hearts.
Tbx2 is expressed normally in non-chamber myocardium, and in the AVC
it is thought to compete with Tbx5 for interaction with Nkx2-5 on the
cis-regulatory elements of chamber-specific genes, thus inhibiting
their expression (Habets et al.,
2002). The global expression of Tbx2 in mutant hearts
could merely reflect the loss of chamber myocardium and expansion of
non-chamber myocardium. However, two facts argue against this possibility.
First, Tbx2 was markedly upregulated (3-fold by RT-PCR quantitation)
as well as ectopically expressed. Second, Tbx2 was expressed normally
in the hearts of Nkx2-5GFP/GFP embryos, in which chamber
differentiation is also blocked at the level of a controlling transcription
factor. We conclude that Tbx20 directly or indirectly represses Tbx2
in myocardium, and that Tbx20 plays a defining role in specification
of chamber and non-chamber myocardium, a lineage digression in the early heart
upon which all subsequent morphogenesis depends.
Our data suggest a model in which chamber formation in the heart involves
`default repression', a feature of virtually all well-studied, conserved,
signal-induced transcriptional regulatory systems acting in development
(Barolo and Posakony, 2002).
Default repression occurs when a developmental process is actively repressed
in the absence of its inducing signal to prevent cryptic activation by other
positive factors involved in specificity. Thus, specification of the
Tbx2 pattern in the AVC and other regions of non-chamber myocardium
must involve regional and presumably signal-dependent inhibition of the
repressive role of Tbx20 on Tbx2 expression, a possible role for Bmps
(Yamada et al., 2000
).
A repressive role for Tbx20 was also evident in regulation of the
Myl2 cardiac pre-pattern. Myl2 is expressed at only very low
levels in hearts of Nkx2-5 null embryos
(Biben et al., 2000;
Lyons et al., 1995
), yet was
`reactivated' in Tbx20lacZ/lacZ/Nkx2-5GFP/GFP
embryos, which lack both Nkx2-5 and Tbx20 function. This
finding inextricably implicates transcriptional repression involving Tbx20 in
the regulation of Myl2. The Myl2 pattern was also broader
relative to morphological landmarks in Tbx20lacZ/lacZ and
Tbx20lacZ/lacZ/Nkx2-5GFP/GFP hearts. Consistent
with these findings, expanded expression of the cmlc2 gene (an
Myl2 ortholog) into the atria was noted after morpholino knockdown of
zebrafish Tbx20, and the ventricle-specific gene vmhc was
also activated in this region (Szeto et
al., 2002
). These patterns probably reflect loss of repressive
roles for Tbx20, and it is noteworthy that expansion of the expression domains
of developmental genes, as seen here, is also one hallmark of loss of default
repression (Barolo and Posakony,
2002
). Our data suggest multiple repressive functions for Tbx20 in
the core cardiac regulatory program. We envisage a genetic circuitry for heart
development based on interactions between multiple cardiac T-box factors and
their co-factors, involving overlapping steps of repression and
de-repression.
A role for cardiac transcription factors in adult heart pathology
Our studies have also revealed a key role for Tbx20 in adult cardiac
function. Tbx20 haploinsufficiency led to LV dilation, decreased wall
thickness and contractile dysfunction, indicative of DCM. Gross dilation was
also seen in the RV in some
Tbx20lacZ/+/Nkx2-5GFP/+ mice. In the atrial
compartment, ASD was evident in 16% of
Tbx20lacZ/+/Nkx2-5GFP/+ mice, and there was
left atrial dilation in all mutant genotypes analyzed, although most severely
in the Tbx20lacZ/+/Nkx2-5GFP/+ mice. The
specific roles for Tbx20 in adult cardiac structure and function remain to be
determined. ASD is developmental in origin and our data highlight
TBX20 as a candidate ASD gene in humans. In relation to ventricular
defects, we found no deficit in expression of developmental genes such as
Tbx5, Nkx2-5, Smpx and Gja1 in
Tbx20lacZ/+ mice (data not shown). Nevertheless, it will
be important to explore the timing of onset of LV DCM to determine whether it
is also developmental in origin or reflects specific adult functions for
Tbx20. In humans, a large number of disease genes for familial DCM have been
identified, including those for sarcomeric, cytoskeletal, nuclear and calcium
handling proteins (Fatkin and Graham,
2002). However, known disease genes account for only a small
proportion of all familial cases. Mutations in cardiac transcription factor
genes may prove to be another cause of DCM in humans.
The onset of LV dilation and contractile dysfunction in
Tbx20lacZ/+ mice in the absence of hypertrophy, fingers
Tbx20 as an essential gene in the adult cardiac adaptive response. In
most models of adult cardiomyopathy, hypertrophy is a component of the
pathophysiological response, although it can be bypassed if structural
proteins, potential sensors of biomechanical stress, are absent
(Brancaccio et al., 2003;
Knoll et al., 2002
). Recent
work highlighting the developmental transcription factor Gata4 as a
convergence point for cardiac hypertrophy pathways
(Liang and Molkentin, 2002
),
has supported the long-held view that developmental pathways are reactivated
in hypertrophy, although mechanistic understanding is still limited and
distinctions between adaptive and pathological hypertrophy are unclear
(Fatkin and Graham, 2002
).
Further analysis of the Tbx20 model should advance our understanding
of the important link between development and adaptive responses in the adult
organ.
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ACKNOWLEDGMENTS |
---|
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/2451/DC1
* Present address: The Wellcome Trust/Cancer Research UK, Gurdon Institute of
Cancer and Developmental Biology, University of Cambridge, Tennis Court Road,
Cambridge CB2 1QR, UK
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