1 Department of Genetics and Development, College of Physicians and Surgeons of
Columbia University, New York, NY 10032, USA
2 Department of Biology, Washington University, St Louis, MO 63130, USA
3 Department of Molecular Biology, Lewis Thomas Laboratory, Princeton
University, Princeton, NJ 08544, USA
4 Institute of Molecular Genetics and Development, Medical College of Georgia,
Augusta, GA 30912, USA
* Author for correspondence (e-mail: vep1{at}columbia.edu)
Accepted 29 July 2004
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SUMMARY |
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Key words: Tbx2, T-box, Heart development, Atrioventricular canal, Outflow tract, Cell cycle
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Introduction |
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Several T-box genes have been shown to play essential roles in heart
development. At 7.5 days post coitus (dpc), the embryonic mouse heart exists
as a crescent-shaped field of cells located in anterior splanchnic mesoderm.
As the embryo undergoes turning, the cardiac crescent fuses into an
anteroposteriorly oriented linear heart tube. The heart tube grows and
expands, and around 8.5 dpc begins a complex morphogenetic looping process
that eventually brings the posterior, venous aspect of the tube to a rostral
position dorsal to the outflow tract. As looping progresses through 9.5 dpc,
chamber formation and septation begins such that, by 10.5 dpc, the heart has
transformed from a linear tube into a four-chambered structure with
prospective right and left atria and ventricles
(Kaufman and Bard, 1999).
Chamber formation involves at least two important processes. First, two
myocardial domains on the outer curvature of the heart tube differentiate as
either an atrial or ventricular chamber. Importantly, part of the heart tube,
including the outflow tract (OFT), inner curvature, atrioventricular canal
(AVC) and inflow tract (IFT), escapes this developmental chamber program
(Moorman and Christoffels,
2003). Simultaneously, an epithelial-to-mesenchymal transformation
occurs on the inner walls of the AVC and OFT. These mesenchymal cells and
their cardiac jelly matrix form two sets of opposing structures called
endocardial cushions. The endocardial cushions grow, fuse, and serve as the
precursors of the valves and contribute to septation. Subsequently, cardiac
septation leads to division of the outflow tract into two separate outlets,
and division of the atrium and ventricle into right and left chambers
(Moorman and Christoffels,
2003
).
Of the six T-box genes known to be expressed in specific patterns during
cardiogenesis in mouse, Tbx1, Tbx2, Tbx3, Tbx5, Tbx18 and
Tbx20, targeted mutagenesis has demonstrated that Tbx1 and
Tbx5 have essential roles during cardiac development
(Braybrook et al., 2001;
Bruneau et al., 2001
;
Chapman et al., 1996
;
Christoffels et al., 2004
;
Habets et al., 2002
;
Hoogaars et al., 2004
;
Jerome and Papaioannou, 2001
;
Kraus et al., 2001a
;
Kraus et al., 2001b
;
Lindsay et al., 2001
).
Heterozygous Tbx1 mutants display abnormal aortic arch artery
remodelling, and homozygous mutants fail to septate the outflow tract
(Jerome and Papaioannou, 2001
;
Lindsay et al., 2001
).
Heterozygous Tbx5 mutants have conduction and septation defects,
accompanied by reduced embryonic expression of the cardiac factor genes
connexin40 (Cx40; Gja5 Mouse Genome Informatics) and
natriuretic precursor peptide type A (Nppa, formerly known as atrial
natriuretic factor or Anf). Homozygous Tbx5 mutants develop
hypoplastic left ventricles and atria, with altered embryonic expression of
several additional cardiac factors (Bruneau
et al., 2001
).
Previous work has shown that Tbx2 expression is first detected in
the mouse embryo at 8.5 dpc in the allantois
(Mahlapuu et al., 2001), and
at 8.75 dpc in OFT, AVC and IFT myocardium
(Christoffels et al., 2004
;
Habets et al., 2002
). At 9.5
dpc, Tbx2 is expressed in the myocardium of the OFT, inner curvature,
AVC and IFT (Christoffels et al.,
2004
; Habets et al.,
2002
). A similar pattern of expression is observed in the
developing chick heart (Gibson-Brown et
al., 1998b
; Yamada et al.,
2000
). Additionally, Tbx2 is expressed at 9.5 dpc in the
optic and otic vesicles, and in the naso-facial mesenchyme, and later in the
developing limbs and other internal organ primordia such as the lungs and
genitalia (Chapman et al.,
1996
; Gibson-Brown et al.,
1996
; Gibson-Brown et al.,
1998a
; Gibson-Brown et al.,
1998b
).
Based on the cardiac expression profile of Tbx2, and on evidence
that Tbx2 can act as a transcriptional repressor
(Chen et al., 2004;
Sinha et al., 2000
), a model
has been proposed whereby Tbx2 regionalizes chamber differentiation
to the prospective ventricle and atrium by repressing these programs in the
OFT, inner curvature, AVC and IFT at 9.5 dpc
(Christoffels et al., 2004
;
Habets et al., 2002
). In vitro
reporter assays and transgenic analyses in mice have shown that Tbx2 can
repress the transcription of Nppa, Cx40 and connexin43
(Cx43; Gja1 Mouse Genome Informatics), cardiac genes
whose expression is specifically restricted to the developing chambers
(Chen et al., 2004
;
Christoffels et al., 2004
;
Habets et al., 2002
).
Additionally, transgenic embryos in which Tbx2 is ubiquitously
expressed throughout the heart tube, under the control of a ßMHC
promoter fragment, exhibit arrested cardiac development at looping, and a
failure of chamber-specific myocardial gene expression, including
Nppa (Christoffels et al.,
2004
).
An alternative, yet compatible, hypothesis suggests that Tbx2
regulates cellular proliferation and/or survival via transcriptional
repression of downstream targets, such as p19ARF and
p16INK4a from the cyclin-dependent kinase inhibitor (Cdkn)
2a locus, p15INK4b from Cdkn2b, and p21 from
Cdkn1a (Jacobs et al.,
2000; Lingbeek et al.,
2002
; Prince et al.,
2004
). A senescence bypass screen using prematurely senescing
Bmi1/ murine embryonic fibroblasts
identified TBX2, with further analysis showing that senescence bypass
was likely achieved by downregulation of p19ARF expression and p53
protein levels. TBX2-expressing fibroblasts also exhibited reduced
p16INK4a and p15INK4b transcription
(Jacobs et al., 2000
).
Subsequent work showed that the p19ARF promoter contains a
functional T-box-binding element (Lingbeek
et al., 2002
). Others have shown that Tbx2 can specifically
regulate transcription of p21 (WAF)
(Prince et al., 2004
). These
results, in combination with the observation that TBX2 is amplified,
and sometimes overexpressed, in a subset of primary breast tumors, breast
tumor cell lines and pancreatic cancer cell lines
(Barlund et al., 2000
;
Jacobs et al., 2000
;
Mahlamaki et al., 2002
), has
led to the hypothesis that Tbx2 regulates cell proliferation or
apoptosis through p21, p15INK4b, p16INK4a, and/or
p19ARF and p53.
We have used targeted mutagenesis of Tbx2 in mice to gain a
greater insight into the function of this gene during normal embryonic
development. We engineered an 2.2 kb deletion of the endogenous
Tbx2 locus, including part of the T-box, to generate a null allele.
Mice heterozygous for the targeted locus appear normal and fertile, whereas
homozygous mutant embryos exhibit lethal cardiovascular defects, revealing a
crucial role for Tbx2 during cardiac development. Abnormal expression
of cardiac chamber markers Nppa, Cx40 and chisel (Csl;
Smpx Mouse Genome Informatics) is observed in the AVC of
homozygous mutants at 9.5 dpc. At 11.5-12.5 dpc, surviving homozygous mutants
exhibit abnormal OFT septation and other cardiac remodeling defects, although
at 9.5 dpc, markers of neural crest (NC) cells and cardiac progenitor
populations contributing to the arterial pole of the heart are normally
expressed. All homozygous mutants are dead by 14.5 dpc. Further studies
addressing the role of Tbx2 as a regulator of the cell cycle reveal
that loss of Tbx2 function in mouse embryos is not sufficient to
deregulate cell proliferation through p21, p15INK4b,
p16INK4a, p19ARF or p53.
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Materials and methods |
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This PCR generates a 180 bp product from the wild-type allele and an 88 bp product from the mutant allele (Fig. 2C).
Collection of embryos
The Tbx2tm1Pa allele has been maintained both on the
129 inbred background and on a mixed (129/C57/ICR) genetic background.
Heterozygous mice were intercrossed to generate homozygous mutant embryos.
Embryos were dissected in phosphate-buffered saline (PBS) containing 0.2%
bovine albumin (fraction V) (Sigma, St Louis, MO). Embryos for in situ
hybridization and immunocytochemistry were fixed in 4% paraformaldehyde
overnight, dehydrated in methanol and stored at 20°C. Embryos for
histology were fixed in Bouin's fixative, dehydrated in ethanol and stored at
4°C. Yolk sacs were used for genotyping by PCR.
Histology, in situ hybridization, immunocytochemistry, and Alcian Blue staining
Embryos were collected at 10.5, 11.5 and 12.5 dpc for histology.
Paraffin-embedded embryos were sectioned at 8 µm and stained with
Hematoxylin and Eosin Y. In situ hybridization was performed according to
previously described protocols (Wilkinson,
1992), using the following probes: mouse Tbx2, Tbx3, Tbx5,
Csl, Cx40, Cited1, MLC2v, ßMHC, eHAND and Crabp1;
and rat Nppa and Islet1. Stained whole-mount embryos were
post-fixed in 4% paraformaldehyde for vibratome sectioning. Embryos were
infiltrated with 4% sucrose in PBS, 30% sucrose in PBS, and transferred to
embedding mix (0.44% gelatin, 14% bovine serum albumen, 18% sucrose in PBS).
Embryos were embedded in fresh embedding mix with the addition of
glutaraldehyde (0.25% final concentration). Sections (50 µm) were cut on a
Vibratome 1000 Plus Sectioning System (The Vibratome Company, St Louis, MO).
Immunocytochemistry with rabbit anti-phospho-histone H3 primary IgG (Upstate
Biotechnology, Lake Placid, NY) was performed according to standard protocols
(Davis, 1993
). Secondary
antibody was peroxidase-conjugated goat anti-rat IgG (Jackson Immunoresearch
Laboratories, West Grove, PA). Stained whole-mount embryos were post-fixed in
4% paraformaldehyde and embedded in paraffin wax for sectioning. Sections were
counterstained with Nuclear Fast Red. Mitotic cells were counted in the heart
tube. Alcian Blue cartilage staining was performed as previously described
(Jegalian and De Robertis,
1992
).
Breeding the 1v-nlacZ-24 transgene and ß-galactosidase staining
Previously characterized 1v-nlacZ-24 transgenic mice
(Kelly et al., 2001) were bred
with Tbx2tm1Pa heterozygous males of a mixed 129/C57/ICR
background. Tbx2tm1Pa heterozygous mice carrying the
1v-nlacZ-24 transgene were crossed with Tbx2tm1Pa
heterozygous mice to collect embryos at 9.5 and 12.5 dpc. ß-Galactosidase
staining was performed according to a previously described protocol
(Kelly et al., 2001
).
RT-PCR expression analysis
At 9.5 and 10.5 dpc, both whole embryos and a dissected trunk region
including the heart were collected and stored in RNAlater RNA stabilization
reagent at 4°C or 20°C (QIAGEN, Valencia, CA). RNA was
extracted using RNeasy Protect Mini kit (QIAGEN, Valencia, CA), and cDNA was
reverse-transcribed using SuperScript III First-Strand Synthesis System
(Invitrogen Life Technologies, Carlsbad, CA). p19ARF,
p16INK4a, p15INK4b and p21 expression were assayed with
semi-quantitative real-time RT-PCR performed on a DNA Engine Opticon 2
Continuous Fluorescence Detection System (MJ Research, Waltham, MA). cDNA from
adult testis was used as a positive control for p19ARF+
p16INK4a and p15INK4b expression. Mandible from a 14.5
dpc embryo was used as a positive control for p21 expression. The following
primers were used for the analysis:
Expression units were calculated according to the following equation:
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Results |
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Heterozygotes containing the Tbx2tm1Pa allele on a
mixed 129/C57/ICR background are viable, fertile, and display no obvious
phenotypic abnormalities. No
Tbx2tm1Pa/Tbx2tm1Pa mutants were recovered
postnatally, indicating that the homozygous state is embryonic lethal
(Table 1). Homozygous embryos
collected between 8.5 and 18.5 dpc from heterozygous intercross matings were
present at Mendelian ratios, suggesting that no homozygous mutants are lost
due to preimplantation defects (Table
1, 2=4.85, P>0.05). However, all
homozygous mutants are dead by 14.5 dpc apparently due to cardiovascular
insufficiency (Table 2). The
mutant phenotype is first discernable at 9.5 dpc in 35% (n=25/72) of
homozygous mutants, by the absence of a constriction at the AVC and/or an
enlarged and dilated ventricle (Fig.
3A-F). At 10.5 dpc, 26% (n=14/53) of homozygous mutants
exhibit abnormal AVC morphology. Inflated pericardial sacs and generalized
edema indicate that these embryos are suffering from circulatory distress
(Fig. 3H). The hearts have
undergone looping, but show the absence or reduction of an atrioventricular
constriction (Fig. 3I).
Transverse sections reveal that endocardial cushion development is compromised
in both the AVC and OFT (Fig.
3J-O). Small endocardial cushions are observed in the OFT, which
appears shortened (Fig. 3L),
but cushion formation is more severely affected at the AVC, where only minor
cushion-like structures from the inner curvature can be identified
(Fig. 3O). Other homozygous
mutants display normal cushion and atrioventricular development
(Fig. 3K,N). Forty-two percent
(n=11/26) of homozygous mutants are dead at 11.5 dpc
(Table 2). Other
Tbx2-expressing tissues are also affected: the facial region is
dysmorphic with hypoplastic pharyngeal arches and the eyes have morphology
equivalent to that of 9.5 dpc embryos (Fig.
3H). These phenotypic features are currently under study (Z.H. and
V.E.P., unpublished).
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Outflow tract septation defects in Tbx2tm1Pa/Tbx2tm1Pa mutants, 11.5-12.5 dpc
Most homozygous mutants that survive to 12.5 dpc show signs of circulatory
distress, including pericardial effusion and generalized edema
(Fig. 4A), and all homozygous
mutants are dead by 14.5 dpc (Table
2). Gross morphological analysis of dissected 12.5 dpc hearts
reveals that many homozygous mutants display abnormal OFT development, such
that the base of the aorta is positioned to the right of the pulmonary trunk
(Fig. 4B). In wild-type
embryos, the OFT becomes divided by the fusion of endocardial cushions,
resulting in the formation of two separate outflow tracts exiting from
specific chambers: the aorta from the left ventricle, the pulmonary trunk from
the right ventricle. Histological analysis revealed that OFT septation is
delayed by 0.5 dpc in homozygous mutant embryos
(Fig. 4D-I), and that the
aortic outlet is not aligned with the left ventricle relative to normal 12.5
dpc littermates (Fig. 4J,K).
Histology showed that homozygous mutants surviving to 12.5 dpc have normal
atrioventricular cushion morphology (data not shown). Defects in aortic arch
artery remodeling were also observed. The right 6th arch artery, which
normally degenerates by 12.5 dpc, persists in half the homozygous mutants
analyzed at this age (n=3/6) (Fig.
4L). A rightward positioned aorta and failure of the OFT to
septate properly will lead to double-outlet right ventricle or other OFT
anomalies that could contribute to lethality at 13.5-14.5 dpc.
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Further expression analysis addressed possible explanations for the OFT
defects observed in Tbx2tm1Pa/Tbx2tm1Pa mutants
at 12.5 dpc. Cellular retinoic acid binding protein 1 (Crabp1) is a
NC marker gene normally expressed in dorsal-ventral stripes between the neural
tube and the pharyngeal arches at 9.5 dpc
(Fig. 5Q) (Giguere et al., 1990).
Crabp1 expression is normal in
Tbx2tm1Pa/Tbx2tm1Pa mutants (n=4/4;
Fig. 5R). A population of
splanchnic mesoderm that will contribute to myocardium at both poles of the
heart, including the OFT and right ventricle (RV), is marked by expression of
the LIM homeobox gene Islet1 at 9.5 dpc
(Fig. 5S)
(Cai et al., 2003
). Homozygous
mutant embryos have normal Islet1 expression (n=4/4;
Fig. 5T). The Fgf10
enhancer-trap transgene 1v-nlacZ-24 was bred onto the mutant
background to assess the integrity of the anterior heart field (AHF) in
Tbx2tm1Pa/Tbx2tm1Pa embryos
(Kelly et al., 2001
). The
transgene was normally expressed in RV and OFT myocardium, and pharyngeal arch
mesoderm, in 9.5 dpc homozygous mutant (n=7/7) and heterozygous
control embryos (Fig. 5U,V).
Transgenic homozygous mutant embryos at 12.5 dpc confirm a normal contribution
of the AHF to the RV and OFT (n=3/3)
(Fig. 5W,X).
Myocardial cell proliferation in Tbx2tm1Pa/Tbx2tm1Pa mutants, 9.5 dpc
Immunocytochemistry with an anti-phospho-histone H3 antibody was used to
assay cell proliferation in 9.5 dpc embryos. Whole-mount staining showed no
differences in the global pattern of phospho-histone H3-positive cells between
wild-type (n=2) and homozygous mutant (n=3) embryos. Cell
counts in the entire myocardium showed no difference in the percentage of
phospho-histone H3-positive cells between wild-type (1.97%, n=17,738
cells) and homozygous mutant populations (1.89%, n=28,155 cells;
2=0.87, P>0.05), demonstrating normal cell
proliferation levels in Tbx2tm1Pa/Tbx2tm1Pa
hearts at 9.5 dpc.
Digit duplication in Tbx2tm1Pa/Tbx2tm1Pa mutants at 14.5 dpc
In the most developmentally advanced homozygous mutant embryos recovered, a
bilateral, hindlimb-specific, digit IV duplication was observed
(n=3/3 embryos; Fig.
6A,B). Alcian Blue staining showed two cartilage condensations
within the first phalangeal segment of digit IV (n=4/4 hindlimbs;
Fig. 6C). Tbx2
expression has been reported in the interdigital mesenchyme between digits IV
and V at 13.5 dpc (Gibson-Brown et al.,
1996).
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Discussion |
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The cardiac phenotype of Tbx5 homozygous null mutant mice
(Bruneau et al., 2001), in
vitro reporter assays, and transgenic analysis of the regulation of the
chamber-specific gene Nppa
(Christoffels et al., 2004
;
Habets et al., 2002
) have
culminated in the following hypothesis regarding the development of chamber
myocardium in the 9.5 dpc mouse heart: Tbx5 activates a chamber
differentiation program and Tbx2 spatially restricts this program by
repressing a set of downstream target genes in non-chamber myocardium of the
AVC and OFT. Tbx5 homozygous mutants exhibit reduced expression of at
least two chamber-specific genes, Nppa and Cx40
(Bruneau et al., 2001
).
Biochemical evidence has shown that Tbx5 and Nkx2.5 can specifically and
cooperatively bind the promoter of Nppa, synergistically activating
reporter expression (Hiroi et al.,
2001
). Nkx2.5 is a homeodomain transcription factor that is one of
the earliest markers of the mouse cardiac lineage and interacts with a number
of factors in the cooperative regulation of downstream targets
(Bruneau et al., 2000
).
Nkx2.5 homozygous mutants display reduced expression of a number of
cardiac genes, including Nppa
(Tanaka et al., 1999
).
Biochemical experiments have shown that Tbx2 also has the capacity to
specifically regulate Nppa in cooperation with Nkx2.5 and that this
interaction is preferred in competitive binding assays between Tbx2, Tbx5 and
Nkx2.5 (Habets et al., 2002
).
Tbx2 can also specifically regulate the expression of other chamber-specific
genes, including Cx40
(Christoffels et al.,
2004
).
The embryonic expression profile of Tbx2, and both the
morphological and molecular aspects of the Tbx2 homozygous mutant
phenotype, support a model in which Tbx2-mediated repression
localizes chamber differentiation to the prospective ventricle and atrium at
9.5 dpc. Whole-mount in situ hybridization data confirm previously reported
results that Tbx2 is normally expressed in myocardium of the OFT,
inner curvature, AVC and IFT of the 9.5 dpc mouse heart
(Christoffels et al., 2004;
Habets et al., 2002
). While
AVC morphology is normal in a subset of
Tbx2tm1Pa/Tbx2tm1Pa mutants, all homozygous
mutants exhibit ectopic expression of the chamber-specific markers analyzed:
Nppa, Csl, Cx40 and Cited1. Importantly, there is no
evidence that Tbx2 directly affects anteroposterior patterning of the
heart tube, as the posterior expression boundaries of MLC2v and
eHAND are unaffected in homozygous mutants. Loss of Tbx2,
however, can affect the expression pattern of some genes, such as
ßMHC, which fails to be downregulated in
Tbx2tm1Pa/Tbx2tm1Pa atria.
The abnormal development of the AVC provides a plausible explanation for the lethality observed amongst Tbx2tm1Pa/Tbx2tm1Pa mutants at 10.5-11.5 dpc. Insulation of the ventricular and atrial chambers by a distinct myocardial zone is crucial for the mechanical and electrical isolation of chambers that are functionally separate in the mature heart. The molecular characterization described above suggests that Tbx2tm1Pa/Tbx2tm1Pa hearts are developing atrial and ventricular chambers whose conduction and contraction are coupled. The lack of functional separation between the developing chambers could explain the lethal cardiac distress that kills many homozygous mutants.
Tbx2 and remodeling of the outflow tract
Many Tbx2tm1Pa/Tbx2tm1Pa embryos escape the
cardiac distress imposed by ectopic chamber differentiation in the AVC at 9.5
dpc. Of those that survive, however, many experience similar distress by 12.5
dpc. The homozygous mutant population displays a range of defects in septation
and remodeling of the OFT and aortic arch arteries. Histological analysis
indicates misalignment of the aorta and pulmonary trunk with the appropriate
ventricles. Although interventricular septum formation is still incomplete by
12.5 dpc, persistence of such misalignment will eventually result in
double-outlet right ventricle, where both the aorta and pulmonary trunk emerge
from the right ventricle. There are several possible explanations for the
defects observed in 12.5 dpc
Tbx2tm1Pa/Tbx2tm1Pa mutants. First, the defects
may be due to misalignment of the OFT and the ventricles, as a secondary
consequence of torsional strains on looping imposed by abnormal AVC
development. Another possibility is that homozygous mutants surviving to 12.5
dpc experience primary defects in the elongation, septation and rotation of
the OFT, such that the ventricular outlets never achieve their final
alignment. The fact that Tbx2 is expressed in the OFT from 8.75 dpc
until late fetal stages supports the latter hypothesis
(Fig. 1G,I)
(Christoffels et al., 2004).
However, normal expression of Islet1 and the Fgf10
enhancer-trap 1v-nlacZ-24 transgene suggests that the population of
cells contributing the majority of myocardium to the OFT and RV are present
and normal in Tbx2tm1Pa/Tbx2tm1Pa mutants. A
third possibility is that Tbx2 is required in the cardiac NC for OFT
septation, or that Tbx2 targets regulate NC deployment during
septation and aortic arch artery remodelling
(Kirby and Waldo, 1995
),
although normal Crabp1 expression in homozygous mutant embryos at 9.5
dpc provides preliminary evidence against this hypothesis. Conditional
mutagenesis experiments will be required to resolve this issue.
Tbx2 and the developing limbs
Few Tbx2tm1Pa/Tbx2tm1Pa embryos survive past
13.5 dpc, but those that do display bilateral, hindlimb-specific, distal digit
IV duplications, revealing a late role for the gene during patterning of the
hindlimb autopod. Tbx2 is expressed in the interdigital mesenchyme
between developing digits IV and V of 13.5 dpc mouse embryos
(Gibson-Brown et al., 1996),
and our observations could be compatible with the involvement of Tbx2
in regulating apoptosis in this region. Recent work has implicated
Tbx2 as a posteriorizing influence during digit identity
specification in chick and mouse (Suzuki
et al., 2004
). Our results, however, do not address this possible
role for Tbx2. Further work will be required before the exact role of
Tbx2 during limb patterning and digit specification is
understood.
Tbx2 and the cell cycle
Despite overwhelming evidence connecting Tbx2 to the cell cycle
(Barlund et al., 2000;
Jacobs et al., 2000
;
Lingbeek et al., 2002
;
Mahlamaki et al., 2002
;
Prince et al., 2004
),
Tbx2tm1Pa/Tbx2tm1Pa embryos offer no evidence
that any of the implicated pathways are dysregulated during embryogenesis.
There is no difference in p19ARF, p16INK4a,
p15INK4b or p21 expression levels between wild-type embryos and
homozygous mutants at 9.5 or 10.5 dpc, as assessed by semi-quantitative
RT-PCR. Additionally, introduction of the Trp53tm1Tyj
mutation into the Tbx2tm1Pa line failed to rescue the
morphological range or presentation of the
Tbx2tm1Pa/Tbx2tm1Pa phenotype, eliminating the
possibility of a specific genetic interaction. Loss of Tbx2 function
is therefore not sufficient to upregulate expression of p19ARF,
p16INK4a, p15INK4b or p21 in 9.5-10.5 dpc mouse embryos,
nor can the Tbx2tm1Pa/Tbx2tm1Pa phenotype be
attributed to an excess of p53.
Although Tbx2tm1Pa/Tbx2tm1Pa embryos do not
exhibit precocious p19ARF, p16INK4a, p15INK4b
or p21 expression, or abnormal p53 function, these findings do not rule out a
role for Tbx2 in regulating the cell cycle. The knowledge that
TBX2 is capable of regulating p19ARF, p16INK4a,
p15INK4b and p21 expression in vitro
(Lingbeek et al., 2002;
Prince et al., 2004
), and that
TBX2-mediated regulation of p19ARF has an observable
biological effect on cellular senescence
(Jacobs et al., 2000
),
combined with the lack of a cell cycle phenotype in 9.5-10.5 dpc
Tbx2tm1Pa/Tbx2tm1Pa mutants, suggests that
compensating factors may participate in the regulation of this pathway.
Tbx3 is the most obvious candidate for several reasons. Within the
T-box family, Tbx2 and Tbx3 are more closely related to each
other than to any other family member, and both can function as
transcriptional repressors (Agulnik et al.,
1996
; Carlson et al.,
2001
; Sinha et al.,
2000
). Tbx2 and Tbx3 exhibit extensive
expression overlap in many tissues during mouse development, including the AVC
and the limbs (Chapman et al.,
1996
; Christoffels et al.,
2004
; Gibson-Brown et al.,
1996
; Hoogaars et al.,
2004
). Tbx3 has also been implicated in
p19ARF-mediated regulation of the cell cycle and cell death
(Carlson et al., 2002
;
Lingbeek et al., 2002
).
Therefore, the likely functional overlap between Tbx2 and
Tbx3 may account for our observations that p19ARF,
p16INK4a, p15INK4b and p21 expression, and p53 function,
are normal in Tbx2tm1Pa/Tbx2tm1Pa mutants at
9.5-10.5 dpc. We have initiated an analysis of Tbx2, Tbx3 double
mutants to investigate potential functional overlap, particularly with respect
to cardiac development and cell cycle regulation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Adell, T., Grebenjuk, V. A., Wiens, M. and Muller, W. E. G. (2003). Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon. Dev. Genes Evol. 213,421 -434.[CrossRef][Medline]
Agulnik, S. I., Garvey, N., Hancock, S., Ruvinsky, I., Chapman,
D. L., Agulnik, I., Bollag, R., Papaioannou, V. and Silver, L. M.
(1996). Evolution of mouse T-box genes by tandem
duplication and cluster dispersion. Genetics
144,249
-254.
Bamshad, M., Lin, R. C., Law, D. J., Watkins, W. S., Krakowiak, P. A., Moore, M. E., Franceschini, P., Lala, R., Holmes, L. B., Gebuhr, T. C. et al. (1997). Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 16,311 -315.[Medline]
Barlund, M., Monni, O., Kononen, J., Cornelison, R., Torhorst,
J., Sauter, G., Kallioniemi, O.-P. and Kallioniemi, A.
(2000). Multiple genes at 17q23 undergo amplification and
overexpression in breast cancer. Cancer Res.
60,5340
-5344.
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 cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 15,30 -35.[Medline]
Bollag, R. J., Siegfried, Z., Cebra-Thomas, J. A., Garvey, N., Davison, E. M. and Silver, L. M. (1994). An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus. Nat. Genet. 7, 383-389.[Medline]
Braybrook, C., Doudney, K., Marcano, A. C. B., Arnason, A., Bjornsson, A., Patton, M. A., Goodfellow, P. J., Moore, G. E. and Stanier, P. (2001). The T-box transcription factor gene TBX22 is mutated in X-linked cleft palate and ankyloglossia. Nat. Genet. 29,179 -183.[CrossRef][Medline]
Bruneau, B. G., Bao, Z.-Z., Tanaka, M., Schott, J.-J., Izumo, S., Cepko, C. L., Seidman, J. G. and Seidman, C. E. (2000). Cardiac expression of the ventricle-specific homeobox gene Irx4 is modulated by Nkx2-5 and dHand. Dev. Biol. 217,266 -277.[CrossRef][Medline]
Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. A., 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]
Carlson, H., Ota, S., Campbell, C. E. and Hurlin, P. J.
(2001). A dominant repression domain in Tbx3 mediates
transcriptional repression and cell immortalization: relevance to mutations in
Tbx3 that cause ulnar-mammry syndrome. Hum. Mol.
Genet. 10,2403
-2413.
Carlson, H., Ota, S., Song, Y., Chen, Y. and Hurlin, P. J. (2002). Tbx3 impinges on the p53 pathway to suppress apoptosis, facilitate cell transformation and block myogenic differentiation. Oncogene 21,3827 -3835.[CrossRef][Medline]
Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6.Nature 391,695 -697.[CrossRef][Medline]
Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (1996). Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206,379 -390.[CrossRef][Medline]
Chen, J. R., Chatterjee, B., Meyer, R., Yu, J. C., Borke, J. L., Isales, C. M., Kirby, M. L., Lo, C. W. and Bollag, R. J. (2004). Tbx2 represses expression of Connexin43 in osteoblastic-like cells. Calcif. Tissue Int. 74,561 -573.[CrossRef][Medline]
Christoffels, V. M., Hoogaars, W. M. H., Tessari, A., Clout, D. E. W., Moorman, A. F. M. and Campione, M. (2004). T-Box transcription factor Tbx2 represses differentiation and formation of the cardiac chambers. Dev. Dyn. 229,763 -770.[CrossRef][Medline]
Davenport, T. G., Jerome-Majewska, L. A. and Papaioannou, V.
E. (2003). Mammary gland, limb and yolk sac defects in mice
lacking Tbx3, the gene mutated in human ulnar mammary syndrome.
Development 130,2263
-2273.
Davis, C. A. (1993). Whole-mount immunohistochemistry. Methods Enzymol. 225,502 -516.[Medline]
Delorme, B., Dahl, E., Jarry-Guichard, T., Briand, J.-P.,
Willecke, K., Gros, D. and Theveniau-Ruissy, M. (1997).
Expression pattern of connexin gene products at the early developmental stages
of the mouse cardiovascular system. Circ. Res.
81,423
-437.
Dunwoodie, S. L., Rodriguez, T. A. and Beddington, R. S. P. (1998). Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene expression during mouse embryogenesis. Mech. Dev. 72, 27-40.[CrossRef][Medline]
Gibson-Brown, J. J., Agulnik, S. I., Chapman, D. L., Alexiou, M., Garvey, N., Silver, L. M. and Papaioannou, V. E. (1996). Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech. Dev. 56,93 -101.[CrossRef][Medline]
Gibson-Brown, J. J., Agulnik, S. I., Silver, L. M., Niswander,
L. and Papaioannou, V. E. (1998a). Involvement of T-box genes
Tbx2-Tbx5 in vertebrate limb specification and development.
Development 125,2499
-2509.
Gibson-Brown, J. J., Agulnik, S. I., Silver, L. M. and Papaioannou, V. E. (1998b). Expression of T-box genes Tbx2-Tbx5 during chick organogenesis. Mech. Dev. 74,165 -169.[CrossRef][Medline]
Giguere, V., Lyn, S., Yip, P., Siu, C.-H. and Amin, S. (1990). Molecular cloning of cDNA encoding a second cellular retinoic acid-binding protein. Proc. Natl. Acad. Sci. USA 87,6233 -6237.[Abstract]
Habets, P. E. M. H., Moorman, A. F. M., Clout, D. E. W., 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.
Herrmann, B. G., Labiet, S., Poustka, A., King, T. and Lehrach, H. (1990). Cloning of the T gene required in mesoderm formation in the mouse. Nature 343,617 -622.[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. H., Tessari, A., Moorman, A. F. M., de Boer, P. A. J., 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]
Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, R. T. and Weinberg, R. A. (1994). Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4,1 -7.[Medline]
Jacobs, J. J. L., Keblusek, P., Robanus-Maandag, E., Kristel, P., Lingbeek, M., Nederlof, P. M., van Welsem, T., van de Vijver, M. J., Koh, E. Y., Daley, G. Q. et al. (2000). Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19ARF) and is amplified in a subset of human breast cancers. Nat. Genet. 26,291 -299.[CrossRef][Medline]
Jegalian, B. G. and de Robertis, E. M. (1992). Homeotic transformations in the mouse induced by overexpression of a human Hox3. 3 transgene. Cell 71,901 -910.[Medline]
Jerome, L. A. and Papaioannou, V. E. (2001). DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1.Nat. Genet. 27,286 -291.[CrossRef][Medline]
Kaufman, M. H. and Bard, J. B. L. (1999). The Anatomical Basis of Mouse Development. San Diego, CA: Academic Press.
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.[Medline]
Kirby, M. L. and Waldo, K. L. (1995). Neural
crest and cardiovascular patterning. Circ. Res.
77,211
-215.
Kraus, F., Haenig, B. and Kispert, A. (2001a). Cloning and expression analysis of the mouse T-box gene Tbx18.Mech. Dev. 100,83 -86.[CrossRef][Medline]
Kraus, F., Haenig, B. and Kispert, A. (2001b). Cloning and expression analysis of the mouse T-box gene Tbx20.Mech. Dev. 100,87 -91.[CrossRef][Medline]
Lamolet, B., Pulichino, A.-M., Lamonerie, T., Gauthier, Y., Brue, T., Enjalbert, A. and Drouin, J. (2001). A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 104,849 -859.[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]
Lingbeek, M. E., Jacobs, J. J. L. and van Lohuizen, M.
(2002). The T-box repressors TBX2 and TBX3
specifically regulate the tumor suppressor gene p14ARF via
a variant T-site in the initiator. J. Biol. Chem.
277,26120
-26127.
Liu, W. and Saint, D. A. (2002). A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal. Biochem. 302,52 -59.[CrossRef][Medline]
Lyons, G. E., Schiaffino, S., Sassoon, D., Barton, P. and Buckingham, M. (1990). Developmental regulation of myosin gene expression in mouse cardiac muscle. J. Cell Biol. 111,2427 -2436.[Abstract]
Mahlamaki, E. H., Barlund, M., Tanner, M., Gorunova, L., Hoglund, M., Karhu, R. and Kallioniemi, A. (2002). Frequent amplification of 8q24, 11q, 17q, and 20q-specific genes in pancreatic cancer. Genes Chromosomes Cancer 35,353 -358.[CrossRef][Medline]
Mahlapuu, M., Ormestad, M., Enerback, S. and Carlsson, P.
(2001). The forkhead transcription factor Foxf1 is required for
differentiation of extra-embryonic and lateral plate mesoderm.
Development 128,155
-166.
Moorman, A. F. M. and Christoffels, V. M.
(2003). Cardiac chamber formation: development, genes, and
evolution. Physiol. Rev.
83,1223
-1267.
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.
Naiche, L. A. and Papaioannou, V. E. (2003).
Loss of Tbx4 blocks hindlimb development and affects vascularization
and fusion of the allantois. Development
130,2681
-2693.
O'Brien, T. X., Lee, K. J. and Chien, K. R. (1993). Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube. Proc. Natl. Acad. Sci. USA 90,5157 -5161.[Abstract]
Palmer, S., Groves, N., Schindeler, A., Yeoh, T., Biben, C.,
Wang, C.-C., Sparrow, D. B., Barnett, L., Jenkins, N. A., Copeland, N. G. et
al. (2001). The small muscle-specific protein Csl modifies
cell shape and promotes myocyte fusion in an Insulin-like Growth Factor
1-dependent manner. J. Cell Biol.
153,985
-997.
Papaioannou, V. E. (2001). T-Box genes in development: from hydra to humans. Int. Rev. Cytol. 207, 1-70.[Medline]
Prince, S., Carreira, S., Vance, K. W., Abrahams, A. and Goding,
C. R. (2004). Tbx2 directly represses the expression of the
p21WAF1 cyclin-dependent kinase inhibitor. Cancer
Res. 64,1669
-1674.
Ramakers, C., Ruijter, J. M., Lekanne Deprez, R. H. and Moorman, A. F. M. (2003). Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339,62 -66.[CrossRef][Medline]
Showell, C., Binder, O. and Conlon, F. L. (2004). T-box genes in early embryogenesis. Dev. Dyn. 229,201 -218.[CrossRef][Medline]
Sinha, S., Abraham, S., Gronostajski, R. M. and Campbell, C. E. (2000). Differential DNA binding and transcription modulation by three T-box proteins, T, TBX1 and TBX2. Gene 258,15 -29.[CrossRef][Medline]
Suzuki, T., Takeuchi, J., Koshiba-Takeuchi, K. and Ogura, T. (2004). Tbx genes specify posterior digit identity through Shh and BMP signaling. Dev. Cell 6, 43-53.[CrossRef][Medline]
Tanaka, M., Chen, Z., Bartunkova, S., Yamasaki, 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.
Taylor, W. R. and Stark, G. R. (2001). Regulation of the G2/M transition by p53. Oncogene 20,1803 -1815.[CrossRef][Medline]
Thomas, T., Yamagishi, H., Overbeek, P. A., Olson, E. N. and Srivastava, D. (1998). The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev. Biol. 196,228 -236.[CrossRef][Medline]
Walther, T., Schultheiss, H.-P., Tschope, C. and Stepan, H. (2002). Natriuretic peptide system in fetal heart and circulation. J. Hypertens. 20,785 -791.[CrossRef][Medline]
Wilkinson, D. G. (1992). Whole Mount In Situ Hybridization of Vertebrate Embryos. Oxford, UK: IRL Press.
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]
Yamada, M., Revelli, J.-P., Eichele, G., Barron, M. and Schwartz, R. J. (2000). Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2. Dev. Biol. 228,95 -105.[CrossRef][Medline]
Zindy, F., Quelle, D. E., Roussel, M. F. and Sherr, C. J. (1997). Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene 15,203 -211.[CrossRef][Medline]