1 Skaggs School of Pharmacy, University of California, San Diego, 9500 Gilman
Drive, La Jolla, CA 92093, USA
2 Department of Medicine, University of California, San Diego, 9500 Gilman
Drive, La Jolla, CA 92093, USA
3 Department of Medicine and Howard Hughes Medical Institute, University of
California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Author for correspondence (e-mail:
syevans{at}ucsd.edu)
Accepted 22 March 2005
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SUMMARY |
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Key words: T-box, Tbx20, Heart development, Proliferation, Nmyc1 (N-myc)
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Introduction |
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Tbx20 is a T-box transcription factor that is expressed throughout the
early cardiac crescent, and later in both myocardium and endocardium
(Iio et al., 2001;
Kraus et al., 2001
).
Expression of Tbx20 or its homologues in cardiac structures has been conserved
from Drosophila to mammals
(Plageman and Yutzey, 2005
).
In zebrafish and Xenopus, loss- or gain-of-function of Tbx20 causes
abnormal cardiogenesis (Brown et al.,
2005
; Stennard et al.,
2003
; Szeto et al.,
2002
). Injection of antisense morpholinos to Tbx20 in zebrafish
prevent heart looping, and result in defects in chamber morphology, aberrant
expression of ventricular-specific myosin heavy chain in atrium and
upregulation of Tbx5 (Szeto et al.,
2002
). In Xenopus, the cardiac mass of Tbx20 morphants is
reduced, although no downstream targets of Tbx20 have been identified, and
Tbx5 expression is normal (Brown et al.,
2005
). These results demonstrate a crucial role for Tbx20 in
cardiac morphogenesis, but do not provide mechanistic insight into how Tbx20
is required for normal heart formation.
To investigate the role of Tbx20 in mammalian heart, we have generated mice
that are homozygous null for Tbx20. Null mutants arrest development
in utero with arrested cardiac morphogenesis and hypoplastic hearts. Analysis
of these mutants has revealed that Tbx20 is a key component in a
genetic network controlling regional differences in proliferation and
regulating morphogenesis within the developing heart. We have identified Tbx2
as a crucial direct target for repression by Tbx20 and have discovered that
Tbx2 itself directly represses Nmyc1 (also known as N-myc) activity. Nmyc1 is
required for early myocardial proliferation, as demonstrated by severe cardiac
hypoplasia in mice that are homozygous null for Nmyc1
(Davis and Bradley, 1993).
Regional differences in proliferation rates within early looping heart have
been determined and found to be consistent in chick, rat and mouse
(Sedmera et al., 2003).
Relatively low proliferation is observed in the sinoatrial region, the
atrioventricular region, the outflow tract and within forming trabeculae of
the ventricular myocardium. Differential proliferation has both morphogenetic
and functional consequences. In forming ventricles, trabecular myocardium is
relatively more differentiated, providing contractile force and allowing for
proliferation of less-differentiated compact zone the future
thick-walled working myocardium
(Rumyantsev, 1991
). Other
regions of low proliferative activity in early looping heart correlate with
slow conducting myocardium, which acts as sphincters prior to valve
development (de Jong et al.,
1992
).
Aberrant proliferation may underlie some adverse phenotypic consequences of
T-box gene mutations in some human disorders and in cancer. TBX1 is
required for proliferation of cardiogenic progenitors that will contribute to
the outflow tract, a region which does not form normally in individuals with
di George syndrome (Xu et al.,
2004). Tbx5 has been shown to suppress proliferation of
cardiomyocytes (Hatcher et al.,
2001
). A missense mutation of TBX5, which is causative
for Holt Oram syndrome, lacks antiproliferative activity a
characteristic that can be blocked by treatment with wild-type TBX5.
Mechanisms by which either Tbx1 or Tbx5 affect proliferation are unknown. Tbx2
and Tbx3 have each been identified in senescence bypass screens, and either
Tbx2 or Tbx3 can immortalize mouse embryo fibroblasts and cooperate with the
oncogenes Myc or Ras to result in transformation
(Brummelkamp et al., 2002
;
Carlson et al., 2001
;
Carlson et al., 2002
;
Jacobs et al., 2000
). Tbx2 or
Tbx3 can promote immortalization by the direct repression of the tumor
suppressor cyclin-dependent kinase 2a, Cdkn2a
(Brummelkamp et al., 2002
;
Jacobs et al., 2000
;
Lingbeek et al., 2002
). Cells
that lack human Cdkn2a fail to senesce in culture and can be
propagated indefinitely (Kamijo et al.,
1997
). Cdkn2a promotes stabilization of the tumor
suppressor p53 (Sherr and Weber,
2000
). Tbx2 may also regulate proliferation/survival through
direct repression of p21, a cyclin dependent kinase inhibitor implicated in
senescence (Prince et al.,
2004
). Thus, Tbx2 and Tbx3 play crucial roles in cell cycle
control via suppression of senescence genes.
In addition to its role in regulating regional proliferation, we have found that Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development.
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Materials and methods |
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Chromatin immunoprecipitation (ChIP) assay
For in vivo ChIP experiments, extracts were prepared from 20 E8.75-9.5
wild-type mouse hearts. Embryos were dissected in ice-cold PBS. Following
gentle pipetting, tissue was crosslinked with 2% formaldehyde for 2 hours at
room temperature. Chromatin extraction and immunoprecipitations were performed
using a ChIP assay kit (Upstate, 17-295) according to manufacturer's protocol.
Protein-DNA crosslinking was reversed by overnight incubation at 65°C. A
PCR purification kit (QIAGEN, 28106) was used to recover DNA in 50 µl. The
following PCR primers against the 5' Tbx2 promoter region were used:
P-813 (5'-CTCCCTCTGAAGTGCATGGAC-3') and P-573
(5'-AGCGCAGAGGACCGATCTGAC-3'). As control, primers against an
unrelated region of Tbx2 promoter region were used: primer E
(5'-CTCTGGTTCCTAGGCAGGACTCGG-3') and primer F
(5'-TCCTCTGCAGTCTGCCTGTCTGTG-3').
The following PCR primers against the Nmyc1 intron 1 promoter region were used: P-4030 (5'-CCAGGCAGAGAAATAGCTTTAGCG-3') and P-4330 (5'-CCTTTCCATTCCCCTTCCTTCAGA-3'); P4630 (5'-CCAGGCAGTGCCTTGTGTGAAT-3') and P-4930 (5'-GCCAACCTCCAACTCTACAACC-3'). As control, primers against an unrelated region of Nmyc1 promoter region were used: primer G (5'-GAGGCTATGTGGCTTCTAGGAGAG-3') and primer H (5'-GGATGTTAGATGTCCAGTCTCACC-3').
The following PCR primers against the Isl1 5' promoter region were used: P-842 (5'-CGGGAGGAAAGGAACCAACCT-3') and P-581 (5'-CCGGAGTAGGACGGTTAGACC-3'). As controls, primers against an unrelated region of Isl1 promoter region were used: primer I (5'-CTCTGGTTCCTAGGCAGGACTCGG-3') and primer J (5'-GCGGTCTGCTGCTCGGCTCTCAGC-3').
Tbx20 polyclonal antibody was obtained from Orbigen (PAB-11248) and Tbx2 polyclonal antibody was obtained from Upstate (07-318).
Promoter cloning and luciferase transfection assay
A 1 kb genomic DNA fragment upstream of Tbx2 ATG was amplified with high
fidelity DNA polymerase (Novagen, 71086-3) and cloned into pGL3-basic vector
(Promega, E1751). Primers were: 5' primer
5'-CATCAGGGTTCTGCCATGGCTC-3', 3' primer
5'-GGCTCTCTCATCGGGACATCC-3'. A full-length 1.3 kb Nmyc1 intron 1
promoter fragment was cloned into pGL3-basic vector following PCR. Primers
were: 5' primer 5'-AGCGGTACTTGCGAAGCTTCGA-3', 3'
primer 5'-CGCCTCTCTTTTAATATCTCCGCT-3'.
A 1.5 kb genomic DNA fragment upstream of Isl1 ATG was amplified and cloned into pGL3-basic vector. Primers were: 5' primer 5'-GAATTCTGTGTGTCCCCTAATAAC-3', 3' primer 5'-AGAGGGAGTAATGTCCACAGTGAA-3'.
Transfections were carried out in HEK 293 cells according to standard techniques by FUGENE6 (Roche). Cells were lysed 48 hours after transfection, and luciferase and ß-galactosidase activities were measured on a Luminoskan Ascent luminometer (Thermo Labsystems). For luciferase reporters, CMV-ß-galactosidase was used to control for transfection efficiency. Normalized luciferase activities were compared with a pGL3 control to calculate relative repression. Results are from one representative experiment carried out in triplicates and expressed as mean±s.d. At least three independent transfection experiments were performed.
Site-directed mutagenesis
The QuickChange sited-directed mutagenesis kit (Stratagene, 200518) was
used to make point mutations in T-box binding sites in the promoter region
according to the manufacture's protocol.
Whole-mount cell death (TUNEL) assay
Whole-mount TUNEL staining was performed with In Situ Cell Death Detection
Kit (Roche, 1 684 817) followed a modified protocol
(Chi et al., 2003;
Hensey and Gautier, 1998
;
Yamamoto and Henderson,
1999
).
Whole-mount proliferation assay
The whole-mount mouse embryo cell proliferation assay was performed as
described (Nagy et al., 2003).
Anti-phospho-Histone H3 (Ser10) (1:100 dilution) was obtained from Upstate
(06-570).
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Results |
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Myocardial differentiation and anteroposterior patterning occur in Tbx20 mutant hearts
Tbx20 is expressed in throughout early differentiating myocardium,
suggesting that it might play a role in differentiation. To investigate this,
we performed whole-mount in situ hybridization to examine RNAs encoding
myofibrillar proteins myosin light chain 2a (MLC2a) and ß myosin heavy
chain (ß-MHC), both expressed throughout myocardium, myosin light chain
2v (MLC2v), restricted to ventricle and atrioventricular canal, and
myosin heavy chain (
-MHC), restricted to the atrioventricular canal and
forming atrium at early stages. Expression of MLC2a, MLC2v and myosin heavy
chain was intact in Tbx20 mutants, demonstrating that myocardial
differentiation had occurred. Expression of
-MHC, however, was greatly
reduced relative to wild-type controls, suggesting that some aspects of
specification or differentiation may be perturbed in Tbx20-null mice
(Fig. 2A-H).
|
Tbx2 is upregulated in Tbx20 mutants and is directly repressed by Tbx20
Hearts in Tbx20-null mice closely resembled those described for
transgenic mice overexpressing Tbx2 in myocardium
(Christoffels et al., 2004).
Indeed, we found that Tbx2 is highly upregulated in Tbx20 null mice, beginning
at early cardiac crescent stages (Fig.
3A-F). These data suggested that upregulation of Tbx2 could
account for the heart phenotype in Tbx20 mutants. Transgenic mice
overexpressing Tbx2 in myocardium exhibit decreased expression of several
genes, including atrial natriuretic peptide (Nppa), the
muscle-specific gene chisel (Smpx Mouse Genome Informatics)
and the transcriptional co-activator Cited1. Expression of each of
these genes is greatly reduced or absent in Tbx20 mutants
(Fig. 3G-L).
|
Tbx2 is most closely related to Tbx3, and the two genes are expressed in an
overlapping pattern in developing heart
(Hoogaars et al., 2004),
suggesting that they may, in some instances, be regulated coordinately.
However, Tbx3 expression was unaltered in Tbx20 mutants
(Fig. 3O-P).
We investigated whether upregulation of Tbx2 in Tbx20 mutants reflected direct repression by Tbx20 in wild-type hearts. Analysis revealed two conserved T-box recognition sites between 677-688 bp upstream of a putative transcription start site of the Tbx2 gene. ChIP analysis was performed on extracts from E8.75-E9.5 hearts and demonstrated that Tbx20 protein was recruited to this region, but not to an unrelated 5' genomic region of Tbx2 (Fig. 3Q). Co-transfection assays with a luciferase reporter driven by a 1 kb Tbx2 promoter and a Tbx20 expression vector demonstrated a fourfold repression of the Tbx2 promoter by Tbx20. This repression was relieved by mutation of the conserved T-box sites (Fig. 3R). These data demonstrated that Tbx20 directly represses Tbx2 within developing heart.
Proliferation, but not apoptosis, is affected in Tbx20 mutants
Reduced heart size in Tbx20-null mutants suggested an increase in
cell death or decrease in cell proliferation, or both. TUNEL assays
demonstrated no differences in apoptosis between wild-type or mutant embryos
at E7.5 or E8.5 (Fig. 4A-D).
Whole-mount immunostaining with antibody to phosphorylated histone H3,
however, demonstrated decreased proliferation in Tbx20 mutant hearts
relative to hearts of wild-type littermates at E8.0 and E8.5
(Fig. 4E-L). Proliferation
rates were assessed by examination of sections. At E8.0, the number of
phosphorylated histone H3 positive nuclei within myocardium of Tbx20
mutants was reduced from 3.5% in wild type to 1.4% in mutants. At E8.5, the
number of positive nuclei was reduced from 3.7% in wild type to 1.0% in
mutants, indicating significant reduction in proliferation rate in myocardium
of Tbx20 mutants. To ensure that the proliferative decrease in heart
was specific, we assessed proliferation rates in neural folds, which do not
express Tbx20 at this stage, and found no significant difference between
wild-type and mutant embryos (5.9% and 5.7%, respectively).
|
|
Tbx2 directly represses Nmyc1
ChIP analysis with E8.75-E9.5 embryonic heart extracts revealed that Tbx2
was recruited to both clusters of T-box sites within intron1 of Nmyc1
(Fig. 5M-1,M-2). Control sites
further upstream were negative (Fig.
5M-3). Co-transfection of an Nmyc1 intron 1-luciferase reporter
with a Tbx2 expression vector in HEK293 cells demonstrated significant
downregulation of the Nmyc1 promoter fragment by Tbx2
(Fig. 5O). By contrast, similar
studies with Tbx20 demonstrated in vivo binding, and in vitro activation of
the Nmyc1 promoter fragment by Tbx20, demonstrating specificity of repression
by Tbx2 (Fig. 5N). Repression
by Tbx2 of the Nmyc1 promoter was dose dependent. These data demonstrate that
Tbx2 directly binds to T-box consensus sites within intron 1 of the
Nmyc1 gene to repress transcriptional activity of Nmyc1.
Tbx20 is required for expression of Bmp2 and Bmp5
Our data demonstrated downregulation of Nmyc1 in Tbx20
mutants. Cardiac hypoplasia in Tbx20 mutants, or
myocardial-Tbx2 transgenics
(Christoffels et al., 2004),
however, appears to be more severe than observed in Nmyc1-null mice
(Charron et al., 1992
;
Moens et al., 1993
;
Sawai et al., 1993
),
suggesting that perturbation of genes in addition to Nmyc1 might
account for the severity of the growth phenotype in Tbx20-null mice.
Accordingly, we examined expression of bone morphogenetic proteins, which have
been demonstrated to play a role in early myocardial growth, often in a
redundant fashion (Kim et al.,
2001
; Liu et al.,
2004
; Solloway and Robertson,
1999
; Zhang and Bradley,
1996
). Results demonstrated that expression of Bmp4 and Bmp7 was
not downregulated in Tbx20-null mice, whereas expression of Bmp2 and
Bmp5 was severely downregulated (Fig.
6).
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Discussion |
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Our studies suggest that T-box genes regulate the Nmyc1 promoter
with consequences for organ morphogenesis and implications for human
congenital disease and cancer. Tbx2, which is normally expressed in outflow
tract and atrioventricular canal, is upregulated throughout the heart in
Tbx20 mutants. The cardiac phenotype of Tbx20 mutants mimics
that of mice overexpressing Tbx2 in myocardium
(Christoffels et al., 2004),
suggesting that upregulation of Tbx2 could account for the observed cardiac
phenotype of Tbx20-null mice. We have provided evidence that
Tbx2 is a direct target of Tbx20 in developing heart. In vivo ChIP
analysis performed on embryonic heart extracts has demonstrated direct binding
of Tbx20 to a region of the Tbx2 promoter containing conserved T-box
consensus sites. Transfection studies demonstrated that activity of this
promoter was repressed by co-transfection with a Tbx2 expression
vector, in a manner dependent on presence of conserved T-box sites within the
promoter. It should be noted that Tbx2 and Tbx20 are co-expressed in outflow
tract and atrioventricular canal, suggesting that repression of Tbx2
by Tbx20 is context dependent.
To define targets of Tbx2 that could explain defects in proliferation, we
examined two cell cycle genes previously implicated in cardiomyocyte
proliferation, cyclin A2 and Nmyc1
(Chaudhry et al., 2004;
Davis and Bradley, 1993
;
Murphy et al., 1997
), and
found that both were downregulated in Tbx20 mutant hearts. During our
analysis, we observed similar regional differences in expression of both these
genes in wild-type heart. Regions of relatively low expression coincided with
regions expressing Tbx2, consistent with the idea that Tbx2 might be
suppressing proliferation by acting directly on either cyclin A2 or Nmyc1. No
consensus T-box sites were identified in putative promoter regions of cyclin
A2. However, a cluster of conserved T-box sites was identified within intron 1
of Nmyc1. This intron is within a human NMYC1 transgene that
recapitulates expression pattern of the endogenous Nmyc1 gene in newborn mice
(Zimmerman et al., 1990
).
Sequences within exon1 and/or intron 1 of Nmyc1 direct tissue-specific
expression in cancer cell lines, and contain both positive and negative
regulatory elements, some acting at a post-transcriptional level
(Strieder and Lutz, 2002
).
Nmyc1 is expressed in early myocardial cells, and is required for normal
proliferative growth of the heart (Charron
et al., 1992; Moens et al.,
1993
; Sawai et al.,
1993
). Mice that are homozygous null for Nmyc1 arrest
their development at
E9.5, and die between E10.5 and E11.5, with severely
hypoplastic hearts, undivided thin-walled ventricles and few trabeculae.
Factors that regulate Nmyc1 expression during embryogenesis have not
been defined previously. We have found that Tbx2 directly binds to Nmyc1
enhancer elements activity in transfection studies. Tbx20 is also able to bind
to this Nmyc1 enhancer, but does not repress its activity in our in vitro
assay system. This observation suggests the possibility that Tbx20 may block
Tbx2 repression of Nmyc1 by competitive binding. Although both Tbx20
and Nmyc1 mutants have hypoplastic hearts, the cardiac hypoplasia in
Tbx20 mutants is more severe than that of Nmyc1 mutant mice.
Close resemblance of the Tbx20 cardiac phenotype to that of ß-MHC-Tbx2
transgenic mice suggests that aberrant regulation of additional downstream
targets of Tbx2 may work in concert with Nmyc1 repression to contribute to the
severely hypoplastic phenotype. In this regard, we observed downregulation of
two BMP growth factor genes, Bmp2 and Bmp5, in
Tbx20 null mice. Previous studies have demonstrated that ablation of
these genes can affect early myocardial development
(Solloway and Robertson, 1999
;
Zhang and Bradley, 1996
).
Intriguingly, studies in chick embryos have demonstrated that Bmp2 can induce
expression of Tbx2 in heart (Yamada et
al., 2000
). Here, we observe downregulation of Bmp2 in a
situation where Tbx2 is overexpressed in the heart, suggesting that there may
be a negative feedback loop between Bmp2 and Tbx2. Interactions
between Tbx2, Tbx20 and BMPs will be a subject for future investigation.
Our results suggest a model in which regional expression of Tbx2 in outflow tract and atrioventricular canal suppresses Nmyc1 expression to result in relatively low rates of proliferation. By contrast, in chamber myocardium, Tbx20 represses Tbx2, preventing its repression of Nmyc1 and allowing for higher rates of proliferation. Regulation of Nmyc1 by Tbx2 was dose dependent, suggesting that differential proliferation rates can be regulated by the amount of Tbx2 present.
Our data have demonstrated a role for Tbx20 in control of regional
proliferation at the early heart tube stage. Mutations in other genetic
pathways have demonstrated a role in later growth of ventricular myocardium,
resulting in mid-gestational embryonic lethality. These include
neuregulin/erbB signaling from endocardium to myocardium
(Carraway, 1996;
Negro et al., 2004
), Fgf/Fgfr
signaling from endocardium and epicardium to myocardium
(Lavine et al., 2005
), and
Bmp10 signaling within myocardium, negatively regulated by Nkx2-5
(Chen et al., 2004
;
Pashmforoush et al., 2004
). As
Tbx20 is expressed in ventricular myocardium during midgestation, it may also
be working in concert with these pathways to control later cardiac growth.
Evidence suggests that Tbx2 may regulate Nmyc1 in other contexts.
Downregulation of Nmyc1 is an early response in retinoic acid-induced
differentiation of neuroblastoma cells; Tbx2 is expressed in neuroblastoma
cells, as demonstrated by microarray analysis
(Schulte et al., 2005;
Thiele et al., 1985
). It will
be of great interest to investigate interactions between Nmyc1 and
Tbx2 in neuroblastoma. In melanocytes and melanoma cells, Tbx2 is a
direct target of Mitf, a basic helix-loop-helix leucine zipper transcription
factor that is central to pathways controlling proliferation and
differentiation of melanoblasts and melanocytes, and is likely to be a
negative regulator of cell cycle progression
(Vance and Goding, 2004
). B16
melanoma cells differentiate in response to retinoic acid and Tbx2 is an
immediate-early target (Niles,
2003
). These data suggest that Tbx2 plays a key role in cell cycle
regulation in both melanocytes and melanoma cells, and that Nmyc1 may
be a target of Tbx2 in this context.
A role for Tbx2 in cancer is postulated from the observation that
overexpression of Tbx2 allowed bypass of senescence in cells predisposed to
senesce; Tbx2 is amplified in breast, ovarian and pancreatic cancer cells
(Rowley et al., 2004). Tbx2
can bypass senescence by direct repression of senescence genes. If Tbx2
represses Nmyc1 in cancer cells, consequences of this repression may
depend on relative expression levels of Tbx2 and Nmyc1. For example, high
levels of Nmyc1 can trigger senescence. Therefore moderate downregulation of
Nmyc1 by Tbx2 in this context could bypass senescence, promoting
immortalization. However, severe downregulation of Nmyc1 by Tbx2
could prevent proliferation, promoting differentiation and rendering
transformation less likely. Bypass of senescence is a property of stem cells
(Park et al., 2004
). The
ability of Tbx2 to bypass senescence, either by targeting of senescence genes
or downregulation of Nmyc1 suggests that Tbx2 might play a role in
stem cell maintenance.
The T-box genes Tbx2, Tbx3, Tbx4 and Tbx5 are closely related, and may have
arisen from an ancient duplication that gave rise to the precursor genes
Tbx2/3 and Tbx4/5 (Agulnik et al.,
1996). Tbx2 and Tbx3 are expressed in a partially overlapping
manner during development, particularly in the heart. Tbx2 knockout
mice are embryonic lethal between E11.5 and E14.5, and exhibit morphological
defects in outflow tract and in atrioventricular canal
(Harrelson et al., 2004
). No
proliferative differences were observed in the heart of Tbx2-null
mice and wild-type littermates, despite previous data demonstrating a role for
Tbx2 in cell cycle control. One potential reason for this is functional
redundancy between Tbx2 and Tbx3, as both have been shown to repress the same
senescence genes, are amplified in breast cancer and cooperate with oncogenes
to transform cells (Rowley et al.,
2004
). These observations suggest that Tbx3 may also target
Nmyc1. Tbx3 is highly expressed in the developing central conduction
system of the heart, a region characterized by low rates of proliferation
(Hoogaars et al., 2004
;
Sedmera et al., 2003
). Tbx3 is
mutated in ulnar mammary syndrome, where phenotypic defects may result from
proliferative abnormalities (Bamshad et
al., 1999
); it will be of interest to examine the role of Tbx3 and
Nmyc1 in this regard. Recent microarray analysis has demonstrated high levels
of Tbx3 expression in an Acth (adrenal corticotropic hormone; Pomc1
Mouse Genome Informatics) -producing small cell lung carcinoma
(Turney et al., 2004
). Small
cell lung carcinomas are associated with Nmyc1 amplification. Investigating
potential interactions between Tbx3 and Nmyc1 in this context is
warranted.
Tbx5 has been shown to exhibit antiproliferative activity in cardiomyocytes
(Hatcher et al., 2001). A
missense mutation that causes Holt-Oram syndrome can act in a
dominant-negative fashion to counteract the antiproliferative activity of the
wild-type gene. The close relationship of Tbx2 and Tbx5 suggest that perhaps
Tbx5 also can target the conserved T-box elements within the Nmyc1 promoter.
Tbx5 is highly expressed in the atrioventricular canal and in the developing
cardiac conduction system, regions of relatively low proliferative activity
(Hoogaars et al., 2004
).
In addition to their role in proliferation, Tbx2 and Tbx20 regulate genes
that specify regional identity within the heart. Loss-of-function studies have
demonstrated that Tbx2 is required in atrioventricular canal to repress
expression of chamber-specific genes Cited1, chisel and Nppa
(Harrelson et al., 2004).
These chamber-specific genes are downregulated in ß-MHC-Tbx2 transgenics
(Christoffels et al., 2004
)
and in Tbx20 mutants, in which Tbx2 is similarly upregulated throughout
myocardium. Tbx20, however, may also be required to activate expression of
chamber specific genes, as Tbx20 and Nkx2-5 can cooperatively activate the
Nppa promoter in some cell contexts
(Stennard et al., 2003
). We
have found two other regionally specific genes that appear to be regulated by
Tbx20 independently of Tbx2. Expression of
-MHC is downregulated in
Tbx20 mutants, but is not affected in ß-MHC-Tbx2 transgenics;
Hand1 is downregulated in Tbx20 mutants, but not affected in Tbx2
knockouts. Another chamber-specific gene, Irx4, which is expressed in
ventricular chamber myocardium, is downregulated in Tbx20-null mice
but was not examined in ß-MHC-Tbx2 transgenics. Several genes that are
downregulated in Tbx20 mutants are downregulated in Nkx2-5
(Nppa, chisel, Cited1, Hand1, Irx4) and/or Tbx5
mutants (Nppa, Irx4) (Harvey,
2002
), suggesting that Tbx20, Nkx2-5 and Tbx5 may cooperatively
regulate a subset of downstream targets. Mutations in Nkx2-5 and
Tbx5 cause congenital disease in humans
(Seidman and Seidman, 2002
),
opening the possibility that causative mutations may also be found in
Tbx20.
In addition to Nmyc1, two other downstream targets of Tbx20,
Hand1 and Irx4, give cardiac phenotypes in null mice,
although their cardiac phenotypes are distinct from those of Tbx20 mutants
(Bruneau et al., 2000;
Olson, 2004
;
Srivastava, 1999
). These
observations demonstrate that, in addition to the control of regional
proliferation detailed here, Tbx20 is required for other crucial aspects of
heart development. Although both Tbx20 and Nmyc1 mutants
have severely hypoplastic hearts, the cardiac phenotype in Tbx20-null
mice is slightly more severe than that of Nmyc1 mutant mice,
suggesting that factors in addition to Nmyc1 downregulation
contribute to the phenotype.
Phenotypes observed following Tbx20 morpholino injection into
zebrafish or Xenopus embryos
(Brown et al., 2005;
Stennard et al., 2003
;
Szeto et al., 2002
) support a
conserved role for Tbx20 in chamber morphogenesis and specification,
although downstream targets may be species specific. In zebrafish, morphant
hearts exhibit no distinction between chambers, and aberrantly express
ventricular myosin heavy chain in atria. In contrast to our results with
Tbx20-null mice, Tbx5 is strongly upregulated in zebrafish
tbx20 morphants. Expression of tbx5 in zebrafish differs
from that in mouse, becoming restricted to ventricle, not atrium, suggesting
species specific differences in the regulation of these genes. Tbx20
morphant frogs also exhibit severe reduction in heart size. No downstream
targets of Tbx20 were identified in the frog experiments, and
XNppa expression is unaffected in Tbx20 morphants. Although
Tbx5 was not a target of Tbx20 knockdown, combined injection
of morpholinos against Tbx5 and Tbx20 synergistically
affected heart development, suggesting concerted activity of these two
transcription factors in cardiogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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