1 Integrative Morphology Group, Department of Anatomy, University of Vienna,
Waehringerstrasse 13, A-1090 Vienna, Austria
2 Developmental Biology Program, The Victor Chang Cardiac Research Institute,
384 Victoria Street, Darlinghurst, NSW 2010, Australia
3 Developmental Biology Division, National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, UK
4 The Neural Development Unit, The Institute of Child Health, 30 Guilford
Street, London WC1N 1EH, UK
5 St Vincent's Clinical School, and School of Biotechnology and Biomolecular
Sciences University of New South Wales, Kensington, NSW, Australia
Authors for correspondence (e-mail:
wolfgang.weninger{at}meduniwien.ac.at;
s.dunwoodie{at}victorchang.unsw.edu.au)
Accepted 4 January 2005
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SUMMARY |
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Although abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos, laterality defects are only observed in a proportion of these embryos. We have therefore used a combination of high-resolution imaging and three-dimensional (3D) modeling to systematically document the full spectrum of Cited2-associated cardiac defects. Previous studies have focused on the role of Cited2 in cardiac neural crest cell development, as Cited2 can bind the transcription factor Tfap2, and thus affect the expression of Erbb3 in neural crest cells. However, we have identified Cited2-associated cardiac defects that cannot be explained by laterality or neural crest abnormalities. In particular, muscular ventricular septal defects and reduced cell density in the atrioventricular (AV) endocardial cushions are evident in Cited2-null embryos. As we found that Cited2 expression tightly correlated with these sites, we believe that Cited2 plays a direct role in development of the AV canal and cardiac septa. We therefore propose that, in addition to the previously described reduction of cardiac neural crest cells, two other distinct mechanisms contribute to the spectrum of complex cardiac defects in Cited2-null mice; disruption of normal left-right patterning and direct loss of Cited2 expression in cardiac tissues.
Key words: Cited2, Left-right axis, Heart development, Mouse
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Introduction |
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The Cited2 gene affects cardiac morphogenesis and was identified
from studies of differential gene expression in the early embryo
(Dunwoodie et al., 1998),
cytokine function (Sun et al.,
1998
) and transcriptional regulation during hypoxia
(Bhattacharya et al., 1999
).
Cited2 expression is widespread during early mouse and chicken
development (Dunwoodie et al.,
1998
; Schlange et al.,
2000
). Furthermore, expression of Cited2 can be activated
by factors, including low oxygen tension, cytokines, lipopolysacharide and
shear stress (Bhattacharya et al.,
1999
; Sun et al.,
1998
; Yokota et al.,
2003
). This may account for the complexity of expression in
developing embryos.
Cited2 interacts with CBP and p300 - homologous proteins with intrinsic
acetyltransferase and E4 ubiquitin ligase activities
(Chan and La Thangue, 2001;
Grossman et al., 2003
). It
inhibits the transcriptional activity of Hif1
, through competition for
overlapping binding sites on CBP/p300 (De
Guzman et al., 2004
; Freedman
et al., 2003
). This may represent a paradigm for Cited2 function,
as it also appears to displace Ets1 from CBP/p300
(Yokota et al., 2003
). In
addition, Cited2 interacts directly with the transcription factors Lhx2 and
Tfap2 (Bamforth et al., 2001
;
Glenn and Maurer, 1999
).
Cited2-null embryos display numerous developmental defects
(Bamforth et al., 2001;
Martinez Barbera et al., 2002
;
Yin et al., 2002
). Cardiac
defects previously reported in Cited2-null embryos include: double
outlet right ventricle (DORV); atrial septal defect (ASD); ventricular septal
defect (VSD); overriding aorta; persistent truncus arteriosus (PTA); and
pulmonary artery stenosis. Previously, these defects had only been considered
in light of Tfap2 and Hif1
, and were only described at late stages of
gestation (13.5 dpc onwards) when the heart has essentially reached its final
morphology (Bamforth et al.,
2001
; Martinez Barbera et al.,
2002
; Yin et al.,
2002
). We determined the developmental origins of the cardiac
defects by examining cardiac morphogenesis in detail at a number of embryonic
stages, and determined the expression pattern of Cited2 in relation
to heart development.
We observed that the heart loops abnormally in a proportion of Cited2-null embryos, and that these embryos have right atrial isomerism (RAI). In these embryos, Nodal, Lefty2 and Pitx2c are not expressed in lateral mesoderm, and Lefty1 expression in the prospective floor plate (PFP) is abnormal. This shows that abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos. In addition, we performed a detailed analysis of the endpoint cardiac phenotypes using episcopic fluorescence image capturing (EFIC) and three-dimensional (3D) modeling. A number of abnormalities were identified in Cited2-null embryos that lacked a laterality defect, and some of these were consistent with a reduction in cardiac neural crest (CNC) cells. There were, however, two defects that did not seem to relate to abnormalities in laterality or CNC cells: reduced cell density in the AV endocardial cushions and muscular ventricular septal defects (VSD). These defects did, however, directly correlate with Cited2 expression and this suggests that Cited2 also plays a direct role in cardiac tissue.
In summary, we demonstrate that Cited2-null embryos display a spectrum of cardiovascular defects: some consistent with a laterality defect, others with a reduction in neural crest cells and yet others concordant with Cited2 acting directly within cardiac tissue to affect morphogenesis.
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Materials and methods |
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Morphology, histology, RNA in situ hybridization and X-gal staining
EFIC was used for high resolution morphological studies
(Weninger and Mohun, 2002).
Histology, RNA in situ hybridization and X-gal staining were performed as
described (Kaufman, 1992
;
Hogan et al., 1994
;
Harrison et al., 1995
). cDNA
probe details are as follows: Cited2
(Dunwoodie et al., 1998
);
Nodal, Lefty1/2 and Pitx2c probes were a gift from Christine
Biben and Richard Harvey.
Determination of cell density in endocardial cushions
Cell density of the AV cushions was measured in three Cited2
wild-type and three Cited2-null embryos at 10.5 dpc. Wax sections of
each embryo were cut at a 45° angle (midway between frontal and
transverse) and every section collected. The number of cells in the central
section plus two before and two after the central section were counted.
Cushion area was measured using Image J software v1.24o (NIH), and the results
expressed as density of cells per µm2 cushion. Cell density was
compared using analysis of variance.
Scanning electron microscopy (SEM)
Following incubation in Karnovsky's fixative (2% paraformaldehyde, 2.5%
glutaraldehyde and 0.1 M sodium phosphate buffer at pH 7.2), SEM was performed
(Webb et al., 1998).
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Results |
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Pitx2c is initially expressed in the left LPM, and then at later
stages expression persists on the left side of various LPM-derived tissues
such as the sinus venosus, common atrial chamber, cardinal vein, vitelline
vein, septum transversum and foregut
(Yoshioka et al., 1998). In
all Cited2 wild-type (n=6) and heterozygous (n=37)
embryos examined, the normal pattern of Pitx2c expression was
observed in the left LPM and later the left horn of the sinus venosus
(Fig. 3J,K). However, in four
out of 12 Cited2-null embryos, Pitx2c expression was
entirely absent from this left lateral mesoderm
(Fig. 3L). This indicates that
Cited2 acts upstream of Pitx2c expression in lateral
mesoderm and is consistent with the fact that Pitx2c is not expressed
because of an absence of Nodal expression in the lateral
mesoderm.
In summary, one-third of Cited2-null embryos showed loss of Nodal, Lefty2 and Pitx2c expression in left LPM, and loss of Lefty1 expression in the anterior PFP; this is consistent with the proportions of null embryos, which had RAI (Table 2).
|
Cited2-null embryos develop a spectrum of severe cardiovascular defects
Some cardiovascular defects that occur in Cited2-null embryos have
previously been reported (Bamforth et al.,
2001; Yin et al.,
2002
). Yet, the full spectrum of defects, their incidence and
association to one another has remained unexplored. As Cited2-null
embryos die late in gestation (Table
1) we examined a total of 28 Cited2-null embryos on a
mixed C57Bl6;129 genetic background between 14.5 and 15.5 dpc using
high-resolution images obtained from EFIC and 3D models
(Weninger and Mohun, 2002
).
This revealed that Cited2-null embryos showed a broad range of
cardiac, vascular and other defects, and were classified into four groups
based on the predominant cardiac defect
(Table 2). The major defect of
group 1 embryos is right isomerism, the other abnormalities being largely
attributable to the patterning defects that isomerism would impart. The
incidence of isomerism in 14.5-15.5 dpc hearts is consistent with our finding
that
40% of embryos examined between 8.5 and 12.5 dpc have a laterality
defect. As embryos in groups 2 to 4 do not show isomerism, the spectrum of
cardiovascular defects they exhibit are likely to result from other factors,
such as the reduction of CNC cells, or the lack of Cited2 expression
in cardiac tissues.
Cardiovascular defects associated with right isomerism
Nine out of 28 embryos (group 1; Table
2) demonstrated RPI and RAI as a component of a complex cardiac
phenotype (compare Fig. 4A-C
with 4D-F, Fig.
5A,C with
5B,D). Three-dimensional
reconstruction of these Cited2-null hearts shows symmetrical
arrangement of the atria, superior venae cavae and pulmonary veins
(Fig. 6;
Fig. 7A,B). A single common
atrium is apparent with rudimentary ridges protruding into the lumen from the
dorsal side. Both atrial appendages resemble the appearance of the normal
right atrium (Figs 6,
7,
8). The superior venae cavae
and inferior vena cava, along with multiple small mediastinal veins and a
common pulmonary vein, drain into the central atrial area, which is bordered
by the rudimentary ridges (Fig.
4B,G,H; Fig. 6C).
In three embryos, an extra vessel (possibly a persisting right umbilical vein)
enters the central atrial compartment from the caudal aspect
(Fig. 6C').
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|
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In summary, nine out of 28 Cited2-null embryos examined have a
right isomeric pulmonary and cardiac phenotype in combination with defects of
the AV junction, ventricular septum, outflow tract and aortic arches. Each
lesion (except for the muscular VSD) has previously been reported in
association with aberrant left-right embryonic patterning (see
Maclean and Dunwoodie, 2004;
Schneider and Brueckner,
2000
).
Cardiovascular defects in the absence of isomerism
Isomerism was not evident in the 15/28 Cited2-null embryos
(Table 2). Group 2 embryos
exhibited similar defects as those in group 1 but lack right isomerism. Group
3 defects were restricted to outflow tract malformations (DORV, TGA and
overriding aorta) frequently in combination with muscular VSDs. Three
Cited2-null embryos had multiple VSDs only (group 4), and a further
four had a normal cardiac phenotype.
Endocardial cushions have reduced cell density in Cited2-null hearts
Cited2 is expressed in myocardium adjacent to endocardial cushions
of the outflow tract and AV canal, and in some endocardial and cushion cells
(Fig. 1I,J,N,Q). In addition,
defects in endocardial cushions were detected at 14.5 dpc in
Cited2-null embryos (Fig.
4G; Fig.
8A,B,D,D'). Therefore, we examined formation of endocardial
cushions of the AV canal at 10.5 dpc (Fig.
9). The average cell density was significantly reduced
(P<0.0001) in Cited2-null hearts
(1.83x10-3
cells/µm2±0.18x10-3) compared with that
in wild-type hearts (2.68x10-3
cells/µm2± 0.15x10-3) (compare Fig.
9E with
9F-H). Furthermore, the area of
the endocardial cushions appeared to be reduced in the Cited2-null
hearts, this is despite the fact that the overall sizes of the hearts compared
were comparable.
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Discussion |
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A range of cardiac defects is observed in Cited2-null embryos. This variability in expressivity/penetrance of the phenotype is probably due to the effect(s) that polymorphism of a modifier(s) has on Cited2 function. For example, the incidence of right isomerism occurs in about 33% of Cited2-null embryos with the 129/Olac;C57BL/6 genetic background compared with about 50% when the mixed SJL;C57BL/6 genetic background of the MLC3F-nlacZ-2E transgene is introduced.
Cited2 and the establishment of laterality
Establishment of left-right patterning can be divided into three phases: an
initial break in embryonic symmetry through cilia-dependent processes
originating in the node; establishment of asymmetric gene expression in
lateral mesoderm; and transfer of positional information to developing organs.
The Nodal signaling pathway, which is central to this process in the lateral
mesoderm, is an important predictor of the phenotype
(Collignon et al., 1996;
Lowe et al., 1996
). For
example, left-sided expression results in situs solitus, right-sided
expression in situs inversus, bilateral expression in left isomerism and
absent expression in right isomerism.
The laterality defect in Cited2-null embryos is characterized by
right atrial and RPI (100% concordant), and 50% of these individuals have
altered abdominal situs. In addition, right atrial isomerism always occurs
where the heart is abnormally looped. As Cited2 is expressed in and
around the ventral node, it is possible that Cited2 is required for
cilia-dependent processes or for the relay of the asymmetric signal to the
lateral mesoderm. Mutation in Dnah5, Dnah11/Lrd, Hfh4/Foxj1 results
in immotile cilia of the node, random expression of Nodal and the
complete spectrum of laterality phenotypes: situs solitus, situs inversus,
left and right isomerism (Brody et al.,
2000; Chen et al.,
1998
; Collignon et al.,
1996
; Lowe et al.,
1996
; Meno et al.,
1996
; Oh and Li,
2002
; Olbrich et al.,
2002
; Supp et al.,
1999
; Supp et al.,
1997
). Moreover, mutations in genes that abrogate all nodal cilia
(Kif3a, Kif3b, polaris, Wim) or just the sensory cilia
(polycistin 2) result in absent, or bilateral Nodal expression that
would result in situs inversus and left isomerism
(Huangfu et al., 2003
;
Marszalek et al., 1999
;
Morgan et al., 1998
;
Moyer et al., 1994
;
Murcia et al., 2000
;
Nonaka et al., 1998
;
Pennekamp et al., 2002
;
Takeda et al., 1999
;
Wu et al., 2000
). As
Cited2-null embryos exhibit only situs solitus or right isomerism, it
is unlikely that this gene is required only for cilia-dependent processes of
the node and the breaking of embryonic symmetry.
Consistent with Nodal acting as a left-sided determinant, we have shown that complete absence of Nodal expression in lateral mesoderm is predictive of the right isomeric phenotype in Cited2-null embryos. This lack of Nodal suggests that Cited2 is required for either the initiation or maintenance of Nodal expression, and thus requires Cited2 to be expressed in lateral mesoderm. Appropriately, we have demonstrated low-level bilateral expression of Cited2 in mouse LPM and we suggest that it acts in this tissue to initiate or maintain Nodal expression.
It is not clear how loss of Cited2 might affect Nodal
expression. Cited2 binds CBP/p300, Tfap2 and Lhx2/3, and through its
interaction with CBP/p300 affects the transcriptional activity of Hif1 and
Ets1 (Bamforth et al., 2001;
Braganca et al., 2002
;
De Guzman et al., 2004
;
Freedman et al., 2003
;
Glenn and Maurer, 1999
;
Yahata et al., 2000
;
Yokota et al., 2003
).
Investigation of the known Cited2-binding proteins sheds no light on its
function, as mutations in these genes are not associated with laterality
defects (Barton et al., 1998
;
Goodman and Smolik, 2000
;
Iyer et al., 1998
;
Ryan et al., 1998
;
Schorle et al., 1996
;
Shikama et al., 2003
;
Zhang et al., 1996
).
The expression of Lefty1 and Lefty2 is abnormal in a
proportion of Cited2-null embryos. Consistent with the loss of
Nodal expression in the left LPM, Lefty2 expression was not
detected. In addition, Lefty1 expression was largely not detected in
the left PFP as the loss of Nodal expression in the left LPM might
predict. Intriguingly, however, Lefty1 expression was readily
detected in the posterior PFP. Regulatory elements directing anterior and
posterior expression of Lefty1 and Lefty2 have been
identified (Saijoh et al.,
1999), and so it is possible that Cited2, directly or
indirectly, acts through an enhancer that controls the anterior, but not
posterior, expression of Lefty1.
A proportion of Cited2-null embryos do not express Pitx2c
in left lateral mesoderm, or in right isomeric hearts at later stages of
development. This may simply reflect the fact that Pitx2c expression
requires Nodal signaling, and that Nodal is not expressed in
Cited2-null embryos with laterality defects. This assumes that
Pitx2c expression in the heart requires prior expression of
Pitx2c in the lateral mesoderm. No evidence contradictory to this
exists, and the fact that expression of Pitx2c in the anterior
lateral mesoderm at 8.5 dpc is consistent with the sites of Pitx2c
expression in the heart at later stages (13.5-14.5 dpc) supports this
hypothesis (Campione et al.,
2001). In addition, there are no cases in which Pitx2c
expression is observed in the heart in the absence of prior expression in the
anterior lateral mesoderm.
It is possible that Pitx2c expression in the heart has little to
do with prior gene activation by Nodal, and that Cited2 might have a more
direct role in its expression. This hypothesis is supported by the overlapping
expression patterns of Cited2 and Pitx2c; both are expressed
in the primary atrial septum, pulmonary veins and superior vena cava. In
addition, they are expressed in the atrial myocardium, AV canals and outflow
tract, although Pitx2c is only expressed on the left side of these
structures (Campione et al.,
2001; Franco and Campione,
2003
; Franco et al.,
2000
). Furthermore, embryos null for either Cited2 or
Pitx2c develop similar cardiac defects
(Franco and Campione, 2003
;
Liu et al., 2001
).
A very recent report proposes that Cited2 acts directly upstream
of Pitx2c, and that the severe and complex heart defects apparent in
Cited2-null embryos, that lack a laterality defect, occur because
Cited2 is required to directly activate Pitx2c expression in the
heart (Bamforth et al., 2004).
This hypothesis seems unlikely for the following reasons. First, Bamforth et
al. (Bamforth et al., 2004
)
show that the activity of the Pitx2c P1 promoter can be increased by
about 1.5-fold following co-transfection of Cited2 and the A and C
isoforms of transcription factor Tfap2 (in the hepatoma cell line
Hep3B cells). However, the degree of transactivation is slight in comparison
with a 25-fold increase in activation of the same promoter fragment through
the Nodal-responsive Fast1/Foxh1 binding sites
(Shiratori et al., 2001
). This
suggests that if Cited2-Tfap2a/c activates Pitx2c expression in the
heart, it does so at relatively low levels and thus is not a major determinant
of Pitx2c expression.
In addition, if the Cited2-Tfap2a/c complex activates Pitx2c in
the heart, one would expect in Cited2-null embryos where laterality
is normally established (and Pitx2c is expressed in left lateral
mesoderm) that Pitx2c expression would be greatly reduced or absent
in the heart. Evidence supporting this was not provided by Bamforth et al.
(Bamforth et al., 2004): three
Cited2-null hearts of a mixed genetic background (C57BL6/J;129Sv)
were examined for Pitx2c expression, one showed reduced expression;
however, the laterality status of these hearts was not determined. By
contrast, we find that Pitx2c expression in the heart is coupled with
its expression in lateral mesoderm: Cited2-null embryos with right
isomerism lack Pitx2c expression in the left lateral mesoderm and in
the heart; those without a laterality defect express Pitx2c in both
left lateral mesoderm and the heart. Our data thus argues against Cited2
playing a direct and major role in the expression of Pitx2c in the
heart, and instead supports the hypothesis that loss of Pitx2c in the
heart occurs only when this gene is not expressed in the left lateral mesoderm
at an earlier developmental stage. Importantly, the Cited2-null
embryos, which express Pitx2c in the heart, still develop a complex
array of cardiac defects, indicating that Cited2 expression is
required in the heart for normal development and that it has
Pitx2c-independent functions.
Cited2, heterotaxia and congenital heart disease
Our studies of the Cited2-null phenotype in the mouse demonstrate
that mutation of a single gene can result in a remarkably wide spectrum of
cardiac malformations. To date, no mutations have been identified in
CITED2 in human disorders; however, the right isomeric phenotype of
Cited2-null embryos resembles that seen in some individuals with
heterotaxia and, in particular, asplenia (Ivemark) syndrome (OMIM 208530)
(Ivemark, 1955). In humans,
heterotaxia is a clinically and genetically heterogeneous disorder that is
associated with a broad range of cardiac malformations. It occurs in
approximately one in 10,000 live born infants or 1% of children with
congenital heart disease, and common cardiac manifestations are complex,
including TGA, DORV or AV septal defects
(Hutchins et al., 1983
;
Rose et al., 1975
;
Ruttenberg et al., 1964
;
Van Mierop and Wiglesworth,
1962
). Thus, the Cited2-null embryo provides a useful
model with which to investigating the etiology of heterotaxia in humans.
Furthermore, as the Cited2 mutation results in a spectrum of
phenotypes that vary widely in their severity, it raises the possibility that
many less severe congenital heart defects share a common cause with each other
and with heterotaxia.
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ACKNOWLEDGMENTS |
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Footnotes |
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