1 Cardiovascular Research, The Hospital for Sick Children, Toronto, ON, M5G 1X8,
Canada
2 Developmental Biology, The Hospital for Sick Children, Toronto, ON, M5G 1X8,
Canada
3 The Heart and Stroke/Richard Lewar Centre of Excellence, University of
Toronto, Toronto, ON, M5S 1A8, Canada
4 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University
Avenue, Toronto, ON, M5G 1X5, Canada
5 Cardiovascular Research Institute and Department of Biochemistry and
Biophysics, University of California, San Francisco, CA 94143-0130, USA
6 Department of Molecular and Medical Genetics, University of Toronto, Toronto,
ON, M5S 1A8, Canada
7 Unité de recherche en développement et différenciation
cardiaques, Institut de recherches cliniques de Montréal (IRCM),
Montréal, QC, H2W 1R7, Canada
8 Programme de biologie moléculaire, Faculté des études
supérieures, Université de Montréal, Montréal, QC,
H3C 3J7, Canada
9 Mouse Imaging Centre, The Hospital for Sick Children, 555 University Avenue,
Toronto ON, M5G 1X8, Canada
10 Department of Medical Biophysics, University of Toronto, Toronto, ON, M5S 1A8,
Canada
* Author for correspondence (e-mail: bbruneau{at}sickkids.ca)
Accepted 17 March 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Heart, Tbx20, T-box, Transcription factors, Mouse, Embryo, Motoneurons, RNAi, Optical projection tomography
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the heart, T-box genes play important roles in the development of
specific cardiac structures and in the transcription of specific cardiac
genes. Tbx5 specifies the formation of the posterior segments of the
heart, the atria and left ventricle; decreased Tbx5 dose results in defective
septation and conduction system formation
(Ahn et al., 2002;
Brown et al., 2005
;
Bruneau et al., 2001
;
Garrity et al., 2002
;
Horb and Thomsen, 1999
).
Misexpression studies in the chick have suggested that expression of
TBX5 dictates the location of the interventricular septum
(Takeuchi et al., 2003
).
Tbx2 is required for the molecular and morphological distinction
between the cardiac chambers and the atrioventricular canal
(Christoffels et al., 2004
;
Harrelson et al., 2004
), while
Tbx1 is important for aortic arch formation and also contributes to
the development of the outflow tract (Hu
et al., 2004
; Jerome and
Papaioannou, 2001
; Lindsay et
al., 2001
; Merscher et al.,
2001
; Xu et al.,
2004
). Biochemical studies have suggested that specific activities
of T-box proteins are dictated in part by interactions between different T-box
proteins or between T-box proteins and other types of transcription factors:
Tbx5 interacts with the homeodomain transcription factor Nkx2-5 to activate
cardiac genes (Bruneau et al.,
2001
; Hiroi et al.,
2001
), while Tbx2/Tbx5 and Tbx3/Tbx5 counterbalances are important
for the proper expression of genes restricted to the cardiac chambers (atria
and ventricles) and for their exclusion from the atrioventricular canal
(Habets et al., 2002
;
Harrelson et al., 2004
;
Hoogaars et al., 2004
).
Tbx20 is a recently described T-box transcription factor that is expressed
most notably in the heart, retina and motoneurons in vertebrates
(Ahn et al., 2000;
Brown et al., 2003
;
Carson et al., 2000
;
Kraus et al., 2001
;
Meins et al., 2000
;
Plageman and Yutzey, 2004
;
Stennard et al., 2003
;
Takeuchi et al., 2003
).
Tbx20 knockdown by morpholino antisense RNA in zebrafish or
Xenopus results in abnormal cardiac morphogenesis
(Brown et al., 2005
;
Szeto et al., 2002
), but the
role of Tbx20 in mammalian heart formation, or its mechanism of action, have
not been elucidated. Furthermore, conflicting data exist regarding the
activity of Tbx20 as a transcription factor: depending on the target gene and
on the presence of other cardiac transcription factors, Tbx20 can either
activate or repress transcription
(Plageman and Yutzey, 2004
;
Stennard et al., 2003
;
Takeuchi et al., 2003
).
In order to investigate the roles of Tbx20 in mammalian embryonic
development, we have inhibited Tbx20 function by transgenic RNA
interference (RNAi) in embryonic stem (ES) cell-derived mouse embryos
(Kunath et al., 2003;
Lickert et al., 2004
). We find
that Tbx20 is essential for normal cardiac chamber formation,
especially that of the outflow tract and right ventricle, the anterior
derivatives of the secondary/anterior heart field (AHF)
(Cai et al., 2003
;
Kelly et al., 2001
;
Meilhac et al., 2004
).
Incomplete knockdown of Tbx20 results in hypoplastic right ventricle
and persistent truncus arteriosus, as well as severely compromised valve
formation. In the central nervous system, Tbx20 is required for
differentiation of motoneurons, in particular expression of Isl2 and
Hb9, genes that encode transcription factors essential for motoneuron
differentiation (Thaler et al.,
2004
). Tbx20 can interact with Isl1, which is crucial for AHF
formation (Cai et al., 2003
),
to activate the AHF enhancers of both Mef2c and Nkx2-5.
These results indicate that Tbx20 is a dose-sensitive regulator of terminal
differentiation events in cardiogenesis and neurogenesis, and that it does so
via interactions with and regulation of transcription factor networks.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridization and antibody staining
In situ hybridization was preformed according to standard protocols. For
fluorescent whole-mount in situ hybridization, embryos were hybridized with a
mixture of digoxigenin-labeled Mybpc3 and Actc probes, and
detection was performed using rhodamine tyramide amplification reagents
(Perkin-Elmer). Whole-mount immunofluorescence was carried out on embryos
fixed in Dent's fixative, using MF20 antiserum directly conjugated to Alexa
594 (Molecular Probes) and unconjugated rat anti-PECAM monoclonal antibody
(Pharmingen) visualized with an Alexa 488-conjugated secondary antibody
(Molecular Probes). The MF20 antibody developed by D. A. Fischman was obtained
from the Developmental Studies Hybridoma Bank developed under the auspices of
the NICHD and maintained by The University of Iowa (Department of Biological
Sciences, Iowa City, IA 52242). Western blot for Tbx20 was performed using
affinity-purified rabbit antiserum raised against Tbx20.
Optical projection tomography
Optical projection tomography (OPT) was performed essentially as described
(Sharpe, 2004;
Sharpe et al., 2002
), on
embryos fluorescently labeled by whole-mount in situ hybridization or
immunofluorescence. Analysis and visualization of OPT data was performed with
Amira V.3.0 (TGS).
Transactivation assays and reporter constructs
Transactivation assays were performed essentially as described
(Bruneau et al., 2001;
Durocher et al., 1997
). The
Nppa-luc, Nkx2-5-luc (FL construct) and Mef2c-lacZ reporters
have been previously described (Brown et
al., 2004
; Dodou et al.,
2004
; Durocher et al.,
1997
). Expression constructs for Tbx20 were generated by
introducing a full-length Tbx20 cDNA with an N-terminal Myc epitope
tag into pcDNA3.1. All other expression constructs were previously described
(Lickert et al., 2004
). The
Nkx2-5 2.5 kb lacZ reporter
(Lien et al., 1999
) (a kind
gift from Dr E. Olson, UT Southwestern, Dallas, TX) consisted of nucleotide
residues 9700 to 6187 relative to the transcriptional start site
upstream of the hsp68-lacZ reporter gene
(Kothary et al., 1989
).
Mutations in the putative Isl1-binding site were introduced by PCR.
Co-immunoprecipitation assays
HeLa cells were transfected with an Isl1 expression construct together with
FLAG-Gata4, Myc-Tbx20, Myc-Tbx1 or FLAG-Tbx5 expression constructs.
Co-immunoprecipitation was preformed as previously described
(Lickert et al., 2004).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Vectors expressing shRNAs directed against Tbx20 (without the
dsRed cassettes) were electroporated into a hybrid ES cell line (G4), derived
from crosses of 129SvJ and C57Bl/6 mice. Quantitative real-time RT-PCR was
used to identify ES cell clones with a significant reduction in Tbx20
mRNA levels (Tbx20 knockdown clones). For each shRNA sequence,
several knockdown clones were identified, with a range of efficiency of
reduction of Tbx20 mRNA (Fig.
3A). Aggregations with tetraploid embryos were performed to
generate entirely ES cell-derived embryos from several knockdown lines.
Because RNAi can be used to generate an epiallelic series
(Hemann et al., 2003;
Kunath et al., 2003
;
Lickert et al., 2004
), we
examined the effect of reducing Tbx20 to varying levels on mouse
embryo development. The following knockdown ES cell lines were used to
generate entirely ES cell-derived embryos: b12, which has a 96% reduction in
Tbx20 mRNA levels; a6 and b2, which have an 80-85% reduction in
Tbx20 mRNA levels; and a4 and b3, which have a 65% reduction in
Tbx20 mRNA levels. Western blot on protein extracted from E9.5
embryos (Fig. 3B) revealed that
line b12 had less than 5% wild-type Tbx20 protein, a6 8% and a4 40% wild-type
Tbx20 levels. In situ hybridization showed residual Tbx20 mRNA in the
heart of line a6-derived embryos, but no detectable Tbx20 mRNA in b12
embryos (Fig. 3C).
|
|
|
|
|
Tbx20 and motoneuron development
Tbx20 is expressed in post-mitotic motoneurons of the spinal cord
in mouse and chick (Fig. 1G,H;
Fig. 8A,C)
(Iio et al., 2001;
Kraus et al., 2001
). The
delayed lethality and pronounced decrease in neural Tbx20 mRNA levels
in a6 knockdown embryos allowed the examination of markers of motoneuron
development at E9.5 to assess a potential role for Tbx20 in these
cells (Fig. 8). Isl1, Isl2 and
Hb9 are LIM-homeodomain transcription factors that are collectively required
for differentiation of somatic and visceral motoneurons
(Arber et al., 1999
;
Pfaff et al., 1996
;
Thaler et al., 1999
;
Thaler et al., 2004
).
Expression of Isl2 and Hb9 was decreased in Tbx20
knockdown embryos, while expression of Isl1 was intact
(Fig. 8A). Dorsoventral
patterning of the spinal cord was not affected in Tbx20 knockdown
embryos, as Pax6 and Irx3 expression was normally patterned
(Fig. 8B). These results
suggest that Tbx20 is a crucial determinant of motoneuron differentiation, via
activation of motoneuron-specific transcription factors. The activation may be
indirect, as we did not detect complete overlap of Tbx20 expression
with that of Isl2 and Hb9 at E9.5
(Fig. 8A). However, earlier
(E9.0) the expression patterns of Tbx20, Isl1, Isl2 and Hb9
overlapped almost completely (Fig.
8C), suggesting that the effect of Tbx20 on Isl2 and
Hb9 might be direct at this stage.
|
|
A proximal enhancer required for Nkx2-5 expression in the AHF and
its derivatives has been shown to require Gata-binding sites
(Lien et al., 2002;
Lien et al., 1999
;
Searcy et al., 1998
).
Gata-binding sites at a more distal enhancer are also required for expression
throughout the heart tube (Brown et al.,
2004
). We examined the upstream regulatory regions of the proximal
Nkx2-5 enhancer and identified a conserved Isl1-binding site, located
adjacent to the Gata site required for AHF expression
(Fig. 9C). This arrangement of
Isl1- and Gata-binding sites is identical to that of the Mef2c AHF
enhancer (Dodou et al., 2004
).
We mutated the Isl1-binding site and assessed the activity of the enhancer
linked to a lacZ reporter gene. Unlike the intact
Nkx2-5-lacZ construct that expressed ß-gal primarily in the RV
and outflow tract (Fig. 9D,F),
mutation of the Isl1 enhancer disrupted expression of Nkx2-5-lacZ
(Fig. 9E,G).
Co-transfection in 10T1/2 cells of a Tbx20 expression construct with
Nkx2-5-luciferaseFL, which includes several cardiac enhancers and the
endogenous Nkx2-5 promoter (Brown
et al., 2004), resulted in strong activation of the
Nkx2-5 reporter gene (Fig.
9A). Co-transfection of a Gata4 expression construct along with
the Tbx20 expression construct resulted in synergistic activation of
Nkx2-5-luciferase (Fig.
9H). No synergistic activation was observed with activated
bone-morphogenic protein receptor (aAlk3), but the combination of Tbx20,
Gata4, and aAlk3 resulted in strong synergy, indicating that Tbx20 can
activate the Nkx2-5 gene via interactions with Gata4
(Fig. 9H). We also
co-transfected in 10T1/2 cells the Nkx2-5-luciferaseFL construct with
Tbx20 in combination with an Isl1 expression construct
(Fig. 9H). As with the
Mef2c enhancer, Tbx20 could readily activate the Nkx2-5
enhancer synergistically with Isl1. Co-immunoprecipitation experiments in HeLa
cells revealed physical interactions between Isl1 and Tbx20
(Fig. 9I). We conclude that
Tbx20 is a potent activator of transcription factors that regulate the AHF,
and that Tbx20 can activate AHF genes via interactions with Isl1, Nkx2-5 and
Gata4, which are crucial for AHF expansion and gene expression
(Cai et al., 2003
;
Dodou et al., 2004
;
Tanaka et al., 1999
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tbx20 and cardiac morphogenesis
Loss of Tbx20 resulted in severe defects in cardiac morphogenesis, most
notably of the anterior segment of the heart, the RV and outflow tract. In
particular, a pronounced lack of outflow tract was observed, both
morphologically and by genetic marker analysis. The dysmorphogenesis of the RV
appeared less pronounced in the most severe knockdown lines compared with
embryos with slightly more remaining Tbx20, perhaps because the embryos died
at a stage at which RV development is rudimentary. Alternatively, the more
restricted expression of the Tbx20a splice variant in the outflow
tract suggests that this isoform may have specific functions that are more
important than the other more widely expressed isoforms. In embryos derived
from line a6, which survived slightly longer than those from the severe b12
line, clear defects in RV formation were observed, indicating that Tbx20 is
also important for RV formation. Furthermore, marker analysis revealed defects
in cardiac chamber differentiation. Therefore, loss of Tbx20 affects
morphogenesis and differentiation of individual segments of the developing
heart. The phenotypes of Tbx20 RNAi embryos are reminiscent of those
observed in embryos lacking Smarcd3, which encodes Baf60c, a
muscle-restricted subunit of the BAF chromatin remodeling complex
(Lickert et al., 2004).
Indeed, significant downregulation of Tbx20 was observed in
Smarcd3 knockdown embryos.
Atrioventricular and outflow tract cushion formation was abrogated in
Tbx20 knockdown embryos. Tbx20 is expressed both in the
myocardium and in the endocardium, both of which are crucial cell types in
cardiac cushion formation (Barnett and
Desgrosellier, 2003; Chang et
al., 2004
). At the present time we cannot distinguish whether the
defects in cushion formation are due to a primary defect in endocardial
function, or are secondary to the loss of Tbx20 in the adjacent myocardium.
However, the severe defects in development of valves in embryos with a mild
knockdown of Tbx20 (line a4) in which myocardial differentiation is
not greatly affected, indicates that this may be a primary defect due to loss
of Tbx20 in the endocardium. In support of this hypothesis, Tbx20 has been
shown to interact with Gata5 (Stennard et
al., 2003
), which is required for endocardial cell differentiation
(Nemer and Nemer, 2002
).
Transcription factor networks in heart development
Tbx20 is important for the expression of several key regulators of cardiac
morphogenesis, and thus we propose that the loss of Tbx20 affects heart
development via a breakdown of transcription factor networks. The complexity
of these interactions is further enhanced by the observation that Tbx20 can
interact with other transcription factors to regulate high-level
tissue-specific expression of essential cardiac transcription factors. Indeed,
Tbx20 regulates expression of both Nkx2-5 and Mef2c, and
Tbx20 can interact with Isl1 and Gata4 to activate the AHF enhancers of both
genes. As the AHF enhancers of both Nkx2-5 and Mef2c rely on
Isl1- and Gata-binding sites and on Tbx20, this provides a common mechanism
for the integration of transcription factor inputs for the AHF. The persistent
expression of Isl1 in cardiac progenitors
(Laugwitz et al., 2005), or
the establishment of active chromatin at Isl1-dependent enhancers, may explain
the widespread effect of loss of the Isl1-binding sites in the Nkx2-5
enhancer. Tbx20 has been shown to interact with other important cardiac
transcription factors, including Gata4, Nkx2-5 and Tbx5
(Plageman and Yutzey, 2004
;
Stennard et al., 2003
;
Takeuchi et al., 2003
), and
thus the roles of Tbx20 in chamber differentiation may similarly rely on these
interactions. However, some degrees of specificity of interactions must exist,
as not all enhancers tested for activation by Tbx20 could respond, while they
were responsive to Tbx5. This specificity extends to T-box transcription
factors expressed in overlapping domains: Tbx1 is also important for AHF
formation, but appears to do so via direct activation of fibroblast growth
factor and forkhead transcription factor genes, instead of cardiac
transcription factors such as those affected by loss of Tbx20
(Hu et al., 2004
;
Xu et al., 2004
). A
combinatorial interaction in myocardial development is also observed for
Nkx2-5 and Tbx5, which regulate and interact with several other cardiac
transcription factors, including Gata4
(Bruneau, 2002
;
Bruneau et al., 2001
;
Garg et al., 2003
;
Tanaka et al., 1999
). Thus,
self-reinforcing transcription factor networks are central to cardiac gene
expression and morphogenesis. Tbx20 appears to be a crucial co-activator in
this process, as its nodes of interaction are widespread and positioned at key
transition points in heart development, including positive interactions with
Nkx2-5, Gata4 and Isl1 in AHF and chamber differentiation.
Tbx20 dose and congenital heart defects
An intermediate (60%) reduction in Tbx20 levels resulted in grossly normal
heart morphology, but with impaired outflow tract septation, RV hypoplasia and
defective valve formation. These defects resemble several human congenital
heart defects, such as persistent truncus arteriosus, hypoplastic RV and
Ebstein's anomaly of the tricuspid valve. This suggests that as for several
other cardiac transcription factors
(Bruneau, 2003;
Lickert et al., 2004
), partial
loss of function of Tbx20 may be an important etiology of human
congenital heart defects. Dominant mutations in NKX2-5, GATA4, TBX5
and TBX1 have been shown to cause congenital heart defects in humans
(Basson et al., 1997
;
Garg et al., 2003
;
Li et al., 1997
;
Schott et al., 1998
;
Yagi et al., 2003
), and Tbx20
can interact with several of these transcription factors. Thus, the combined
interactions between the cardiac transcription factors implicated in human
disease create an interacting network that depends on precise stoichiometry of
protein-protein interactions. Interestingly, several individuals with
NKX2-5 mutations have congenital heart defects that affect the
tricuspid valve and the outflow tract
(Benson et al., 1999
;
Goldmuntz et al., 2001
;
McElhinney et al., 2003
),
structures that are the most affected in the mild Tbx20 knockdown
embryos. It is possible that impaired interaction of the mutant NKX2-5 protein
with Tbx20 would be a contributory factor to the tricuspid and conotruncal
defects in these individuals. Thus, it is very likely that Tbx20 can play
important roles in dose-sensitive transcriptional regulatory complexes, and
that dose-dependent tissue-specific transcription is disrupted in human
congenital heart malformations because of TBX20 mutations or
mutations in genes required for interactions with Tbx20. TBX20 is
therefore a strong candidate gene for human congenital heart defects.
Tbx20 and motoneuron development
Motoneuron development relies on patterning cues and cell-type specific
transcriptional programs, to yield the complexity of regulatory neurons that
innervate somatic and visceral muscles. In particular, dorsoventral patterning
by Shh signaling established domains of transcription factors that are crucial
for the initiation of motoneuron differentiation
(Briscoe et al., 2000;
Litingtung and Chiang, 2000
).
This is subsequently accomplished by the localized expression of specific
transcription factors that together initiate the terminal differentiation of
specific motoneuron subtypes (Price and
Briscoe, 2004
; Shirasaki and
Pfaff, 2002
; Thaler et al.,
2004
). We have demonstrated that Tbx20 plays an important role in
motoneuron development. This appears to be primarily in regulating the
expression of Isl2 and Hb9, which are essential regulators
of motoneuron differentiation downstream of Isl1
(Arber et al., 1999
;
Thaler et al., 1999
;
Thaler et al., 2004
). As Isl1
expression was not affected in Tbx20 knockdown embryos, it is likely that
Tbx20 is an important parallel or interacting modulator of the activity
conferred upon the Isl2 and Hb9 enhancers by Isl1. As with
most markers of differentiating motoneurons, Tbx20 is regulated by
Shh patterning. Therefore, Tbx20 lies at the interface between patterning and
differentiation, and probably has a role as a reinforcing factor in the
transcriptional regulation of motoneuron differentiation.
Conclusions
In conclusion, we have demonstrated that Tbx20 dose is a crucial
determinant of heart morphogenesis, and particularly of the AHF derivatives,
the RV and outflow tract. Tbx20 interacts with and regulates important cardiac
transcription factors, leading to the conclusion that it plays a central role
in coordinating a crucial transcription factor network in heart formation.
Future studies will identify the extent of these interactions and how altered
Tbx20 dose modifies these networks.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/2463/DC1
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahn, D., Ruvinsky, I., Oates, A. C., Silver, L. M. and Ho, R. K. (2000). tbx20, a new vertebrate T-box gene expressed in the cranial motor neurons and developing cardiovascular structures in zebrafish. Mech. Dev. 95,253 -258.[CrossRef][Medline]
Ahn, D., Kourakis, M. J., Rohde, L. A., Silver, L. M. and Ho, R. K. (2002). T-box gene tbx5 is essential for formation of the pectoral limb bud. Nature 417,754 -758.[CrossRef][Medline]
Arber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T. M. and Sockanathan, S. (1999). Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity. Neuron 23,659 -674.[CrossRef][Medline]
Barnett, J. V. and Desgrosellier, J. S. (2003). Early events in valvulogenesis: a signaling perspective. Birth Defects Res. C Embryo Today 69,58 -72.[CrossRef][Medline]
Basson, C. T., Bachinsky, D. R., Lin, R. C., Levi, T., Elkins, J. A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T. A., Leblanc-Straceski, J. et al. (1997). Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 15,30 -35.[CrossRef][Medline]
Benson, D. W., Silberbach, G. M., Kavanaugh-McHugh, A.,
Cottrill, C., Zhang, Y., Riggs, S., Smalls, O., Johnson, M. C., Watson, M. S.,
Seidman, J. G. et al. (1999). Mutations in the cardiac
transcription factor NKX2.5 affect diverse cardiac developmental pathways.
J. Clin. Invest. 104,1567
-1573.
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101,435 -445.[CrossRef][Medline]
Brown, C. O., 3rd, Chi, X., Garcia-Gras, E., Shirai, M., Feng,
X. H. and Schwartz, R. J. (2004). The cardiac determination
factor, Nkx2-5, is activated by mutual cofactors GATA-4 and Smad1/4 via a
novel upstream enhancer. J. Biol. Chem.
279,10659
-10669.
Brown, D. D., Binder, O., Pagratis, M., Parr, B. A. and Conlon, F. L. (2003). Developmental expression of the Xenopus laevis Tbx20 orthologue. Dev. Genes Evol. 212,604 -607.[Medline]
Brown, D. D., Martz, S. N., Binder, O., Goetz, S. C., Price, B.
M., Smith, J. C. and Conlon, F. L. (2005). Tbx5 and Tbx20 act
synergistically to control vertebrate heart morphogenesis.
Development 132,553
-563.
Bruneau, B. G. (2002). Transcriptional
regulation of vertebrate cardiac morphogenesis. Circ.
Res. 90,509
-519.
Bruneau, B. G. (2003). The developing heart and congenital heart defects: a make or break situation. Clin. Genet. 63,252 -261.[CrossRef][Medline]
Bruneau, B. G., Logan, M., Davis, N., Levi, T., Tabin, C. J., Seidman, J. G. and Seidman, C. E. (1999). Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev. Biol. 211,100 -108.[CrossRef][Medline]
Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D., Gessler, M., Nemer, M., Seidman, C. E. et al. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106,709 -721.[CrossRef][Medline]
Cai, C. L., Liang, X., Shi, Y., Chu, P. H., Pfaff, S. L., Chen, J. and Evans, S. (2003). Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877-889.[CrossRef][Medline]
Carson, C. T., Kinzler, E. R. and Parr, B. A. (2000). Tbx12, a novel T-box gene, is expressed during early stages of heart and retinal development. Mech. Dev. 96,137 -140.[CrossRef][Medline]
Chang, C. P., Neilson, J. R., Bayle, J. H., Gestwicki, J. E., Kuo, A., Stankunas, K., Graef, I. A. and Crabtree, G. R. (2004). A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis. Cell 118,649 -663.[CrossRef][Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Christoffels, V. M., Habets, P. E., Franco, D., Campione, M., de Jong, F., Lamers, W. H., Bao, Z. Z., Palmer, S., Biben, C., Harvey, R. P. et al. (2000). Chamber formation and morphogenesis in the developing mammalian heart. Dev. Biol. 223,266 -278.[CrossRef][Medline]
Christoffels, V. M., Hoogaars, W. M., Tessari, A., Clout, D. E., Moorman, A. F. and Campione, M. (2004). T-box transcription factor Tbx2 represses differentiation and formation of the cardiac chambers. Dev. Dyn. 229,763 -770.[CrossRef][Medline]
Delot, E. C., Bahamonde, M. E., Zhao, M. and Lyons, K. M.
(2003). BMP signaling is required for septation of the outflow
tract of the mammalian heart. Development
130,209
-220.
Dodou, E., Verzi, M. P., Anderson, J. P., Xu, S. M. and Black,
B. L. (2004). Mef2c is a direct transcriptional target of
ISL1 and GATA factors in the anterior heart field during mouse embryonic
development. Development
131,3931
-3942.
Durocher, D., Charron, F., Warren, R., Schwartz, R. J. and
Nemer, M. (1997). The cardiac transcription factors Nkx2-5
and GATA-4 are mutual cofactors. EMBO J.
16,5687
-5696.
Garg, V., Kathiriya, I. S., Barnes, R., Schluterman, M. K., King, I. N., Butler, C. A., Rothrock, C. R., Eapen, R. S., Hirayama-Yamada, K., Joo, K. et al. (2003). GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424,443 -447.[CrossRef][Medline]
Garrity, D. M., Childs, S. and Fishman, M. C.
(2002). The heartstrings mutation in zebrafish causes heart/fin
Tbx5 deficiency syndrome. Development
129,4635
-4645.
Goldmuntz, E., Geiger, E. and Benson, D. W.
(2001). NKX2.5 mutations in patients with tetralogy of fallot.
Circulation 104,2565
-2568.
Habets, P. E., Moorman, A. F., Clout, D. E., van Roon, M. A.,
Lingbeek, M., van Lohuizen, M., Campione, M. and Christoffels, V. M.
(2002). Cooperative action of Tbx2 and Nkx2.5 inhibits ANF
expression in the atrioventricular canal: implications for cardiac chamber
formation. Genes Dev.
16,1234
-1246.
Hadjantonakis, A. K., Gertsenstein, M., Ikawa, M., Okabe, M. and Nagy, A. (1998). Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76,79 -90.[CrossRef][Medline]
Harrelson, Z., Kelly, R. G., Goldin, S. N., Gibson-Brown, J. J.,
Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (2004).
Tbx2 is essential for patterning the atrioventricular canal and for
morphogenesis of the outflow tract during heart development.
Development 131,5041
-5052.
Hemann, M. T., Fridman, J. S., Zilfou, J. T., Hernando, E., Paddison, P. J., Cordon-Cardo, C., Hannon, G. J. and Lowe, S. W. (2003). An epiallelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat. Genet. 33,396 -400.[CrossRef][Medline]
Hiroi, Y., Kudoh, S., Monzen, K., Ikeda, Y., Yazaki, Y., Nagai, R. and Komuro, I. (2001). Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat. Genet. 28,276 -280.[CrossRef][Medline]
Hoogaars, W. M., Tessari, A., Moorman, A. F., de Boer, P. A., Hagoort, J., Soufan, A. T., Campione, M. and Christoffels, V. M. (2004). The transcriptional repressor Tbx3 delineates the developing central conduction system of the heart. Cardiovasc. Res. 62,489 -499.[CrossRef][Medline]
Horb, M. E. and Thomsen, G. H. (1999). Tbx5 is
essential for heart development. Development
126,1739
-1751.
Hu, T., Yamagishi, H., Maeda, J., McAnally, J., Yamagishi, C.
and Srivastava, D. (2004). Tbx1 regulates fibroblast growth
factors in the anterior heart field through a reinforcing autoregulatory loop
involving forkhead transcription factors. Development
131,5491
-5502.
Iio, A., Koide, M., Hidaka, K. and Morisaki, T. (2001). Expression pattern of novel chick T-box gene, Tbx20. Dev. Genes Evol. 211,559 -562.[CrossRef][Medline]
Jerome, L. A. and Papaioannou, V. E. (2001). Di George syndrome phenotype in mice mutant for the T-box gene, Tbx1.Nat. Genet. 27,286 -291.[CrossRef][Medline]
Kelly, R. G., Brown, N. A. and Buckingham, M. E. (2001). The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev. Cell 1,435 -440.[CrossRef][Medline]
Kioussi, C., Briata, P., Baek, S. H., Rose, D. W., Hamblet, N.
S., Herman, T., Ohgi, K. A., Lin, C., Gleiberman, A., Wang, J. et al.
(2002). Identification of a Wnt/Dvl/beta-Catenin Pitx2
pathway mediating cell-type-specific proliferation during development.
Cell 111,673
-685.[CrossRef][Medline]
Kothary, R., Clapoff, S., Darling, S., Perry, M. D., Moran, L. A. and Rossant, J. (1989). Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. Development 105,707 -714.[Abstract]
Kraus, F., Haenig, B. and Kispert, A. (2001). Cloning and expression analysis of the mouse T-box gene Tbx20. Mech. Dev. 100,87 -91.[CrossRef][Medline]
Kunath, T., Gish, G., Lickert, H., Jones, N., Pawson, T. and Rossant, J. (2003). Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat. Biotechnol. 21,559 -561.[CrossRef][Medline]
Laugwitz, K. L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard, S., Lin, L. Z., Cai, C. L., Lu, M. M., Reth, M. et al. (2005). Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433,647 -653.[CrossRef][Medline]
Lawson, N. D., Scheer, N., Pham, V. N., Kim, C. H., Chitnis, A.
B., Campos-Ortega, J. A. and Weinstein, B. M. (2001). Notch
signaling is required for arterial-venous differentiation during embryonic
vascular development. Development
128,3675
-3683.
Lawson, N. D., Vogel, A. M. and Weinstein, B. M. (2002). sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev. Cell 3,127 -136.[CrossRef][Medline]
Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I., Curtis, A. R., Yi, C. H., Gebuhr, T., Bullen, P. J., Robson, S. C., Strachan, T. et al. (1997). Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15,21 -29.[CrossRef][Medline]
Lickert, H., Takeuchi, J. K., von Both, I., Walls, J. R., McAuliffe, F., Adamson, S. L., Henkelman, R. M., Wrana, J. L., Rossant, J. and Bruneau, B. G. (2004). Baf60c is essential for function of BAF chromatin remodelling complexes in heart development. Nature 432,107 -112.[CrossRef][Medline]
Lien, C. L., Wu, C., Mercer, B., Webb, R., Richardson, J. A. and
Olson, E. N. (1999). Control of early cardiac-specific
transcription of Nkx2-5 by a GATA-dependent enhancer.
Development 126,75
-84.
Lien, C. L., McAnally, J., Richardson, J. A. and Olson, E. N. (2002). Cardiac-specific activity of an Nkx2-5 enhancer requires an evolutionarily conserved Smad binding site. Dev. Biol. 244,257 -266.[CrossRef][Medline]
Lin, Q., Schwarz, J., Bucana, C. and Olson, E. N.
(1997). Control of mouse cardiac morphogenesis and myogenesis by
transcription factor MEF2C. Science
276,1404
-1407.
Lincoln, J., Alfieri, C. M. and Yutzey, K. E. (2004). Development of heart valve leaflets and supporting apparatus in chicken and mouse embryos. Dev. Dyn. 230,239 -250.[CrossRef][Medline]
Lindsay, E. A., Vitelli, F., Su, H., Morishima, M., Huynh, T., Pramparo, T., Jurecic, V., Ogunrinu, G., Sutherland, H. F., Scambler, P. J. et al. (2001). Tbx1 haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410,97 -101.[CrossRef][Medline]
Litingtung, Y. and Chiang, C. (2000). Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3. Nat. Neurosci. 3, 979-985.[CrossRef][Medline]
Liu, C., Liu, W., Palie, J., Lu, M. F., Brown, N. A. and Martin,
J. F. (2002). Pitx2c patterns anterior myocardium and aortic
arch vessels and is required for local cell movement into atrioventricular
cushions. Development
129,5081
-5091.
Liu, W., Selever, J., Wang, D., Lu, M. F., Moses, K. A.,
Schwartz, R. J. and Martin, J. F. (2004). Bmp4 signaling is
required for outflow-tract septation and branchial-arch artery remodeling.
Proc. Natl. Acad. Sci. USA
101,4489
-4494.
Lyons, I., Parsons, L. M., Hartley, L., Li, R., Andrews, J. E., Robb, L. and Harvey, R. P. (1995). Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev. 9,1654 -1666.[Abstract]
McElhinney, D. B., Geiger, E., Blinder, J., Benson, D. W. and Goldmuntz, E. (2003). NKX2.5 mutations in patients with congenital heart disease. J. Am. Coll. Cardiol. 42,1650 -1655.[CrossRef][Medline]
Meilhac, S. M., Esner, M., Kelly, R. G., Nicolas, J. F. and Buckingham, M. E. (2004). The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell 6,685 -698.[CrossRef][Medline]
Meins, M., Henderson, D. J., Bhattacharya, S. S. and Sowden, J. C. (2000). Characterization of the human TBX20 gene, a new member of the T-Box gene family closely related to the drosophila H15 gene. Genomics 67,317 -332.[CrossRef][Medline]
Merscher, S., Funke, B., Epstein, J. A., Heyer, J., Puech, A., Lu, M. M., Xavier, R. J., Demay, M. B., Russell, R. G., Factor, S. et al. (2001). TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104,619 -629.[CrossRef][Medline]
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder,
J. C. (1993). Derivation of completely cell culture-derived
mice from early-passage embryonic stem cells. Proc. Natl. Acad.
Sci. USA 90,8424
-8428.
Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R. (2003). Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Nemer, G. and Nemer, M. (2002). Cooperative
interaction between GATA5 and NF-ATc regulates endothelial-endocardial
differentiation of cardiogenic cells. Development
129,4045
-4055.
Packham, E. A. and Brook, J. D. (2003). T-box
genes in human disorders. Hum. Mol. Genet.
12,R37
-R44.
Papaioannou, V. E. (2001). T-box genes in development: from hydra to humans. Int. Rev. Cytol. 207, 1-70.[Medline]
Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T. and Jessell, T. M. (1996). Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84,309 -320.[CrossRef][Medline]
Plageman, T. F., Jr and Yutzey, K. E. (2004).
Differential expression and function of tbx5 and tbx20 in cardiac development.
J. Biol. Chem. 279,19026
-19034.
Price, S. R. and Briscoe, J. (2004). The generation and diversification of spinal motor neurons: signals and responses. Mech. Dev. 121,1103 -1115.[CrossRef][Medline]
Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W.,
Silberbach, G. M., Moak, J. P., Maron, B., Seidman, C. E. and Seidman, J.
G. (1998). Congenital heart disease caused by mutations in
the transcription factor NKX2-5. Science
281,108
-111.
Searcy, R. D., Vincent, E. B., Liberatore, C. M. and Yutzey, K.
E. (1998). A GATA-dependent nkx-2.5 regulatory element
activates early cardiac gene expression in transgenic mice.
Development 125,4461
-4470.
Sharpe, J. (2004). Optical projection tomography. Annu. Rev. Biomed. Eng. 6, 209-228.[CrossRef][Medline]
Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A.,
Hecksher-Sorensen, J., Baldock, R. and Davidson, D. (2002).
Optical projection tomography as a tool for 3D microscopy and gene expression
studies. Science 296,541
-545.
Shirasaki, R. and Pfaff, S. L. (2002). Transcriptional codes and the control of neuronal identity. Annu. Rev. Neurosci. 25,251 -281.[CrossRef][Medline]
Stennard, F. A., Costa, M. W., Elliott, D. A., Rankin, S., Haast, S. J., Lai, D., McDonald, L. P., Niederreither, K., Dolle, P., Bruneau, B. G. et al. (2003). Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev. Biol. 262,206 -224.[CrossRef][Medline]
Szeto, D. P., Griffin, K. J. and Kimelman, D.
(2002). hrT is required for cardiovascular development in
zebrafish. Development
129,5093
-5101.
Takeuchi, J. K., Ohgi, M., Koshiba-Takeuchi, K., Shiratori, H.,
Sakaki, I., Ogura, K., Saijoh, Y. and Ogura, T. (2003). Tbx5
specifies the left/right ventricles and ventricular septum position during
cardiogenesis. Development
130,5953
-5964.
Tanaka, M., Chen, Z., Bartunkova, M., Yamazaki, N. and Izumo,
S. (1999). The cardiac homeobox gene Csx/Nkx2.5 lies
genetically upstream of multiple genes essential for heart development.
Development 126,1269
-1280.
Thaler, J., Harrison, K., Sharma, K., Lettieri, K., Kehrl, J. and Pfaff, S. L. (1999). Active suppression of interneuron programs within developing motor neurons revealed by analysis of homeodomain factor HB9. Neuron 23,675 -687.[CrossRef][Medline]
Thaler, J. P., Koo, S. J., Kania, A., Lettieri, K., Andrews, S., Cox, C., Jessell, T. M. and Pfaff, S. L. (2004). A postmitotic role for Isl-class LIM homeodomain proteins in the assignment of visceral spinal motor neuron identity. Neuron 41,337 -350.[CrossRef][Medline]
Vintersten, K., Monetti, C., Gertsenstein, M., Zhang, P., Laszlo, L., Biechele, S. and Nagy, A. (2004). Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals. Genesis 40,241 -246.[CrossRef][Medline]
von Both, I., Silvestri, C., Erdemir, T., Lickert, H., Walls, J., Henkelman, R. M., Rossant, J., Harvey, R. P., Attisano, L. and Wrana, J. L. (2004). Foxh1 is essential for development of the anterior heart field. Dev. Cell 7, 331-345.[CrossRef][Medline]
Xu, H., Morishima, M., Wylie, J. N., Schwartz, R. J., Bruneau,
B. G., Lindsay, E. A. and Baldini, A. (2004). Tbx1 has a dual
role in the morphogenesis of the cardiac outflow tract.
Development 131,3217
-3227.
Yagi, H., Furutani, Y., Hamada, H., Sasaki, T., Asakawa, S., Minoshima, S., Ichida, F., Joo, K., Kimura, M., Imamura, S. et al. (2003). Role of TBX1 in human del22q11.2 syndrome. Lancet 362,1366 -1373.[CrossRef][Medline]
Related articles in Development: