1 Howard Hughes Medical Institute and Developmental Genetics Program, Skirball
Institute of Biomolecular Medicine, New York University School of Medicine,
New York, NY 10016, USA
2 Departments of Pediatrics and Cell and Developmental Biology, Vanderbilt
University Medical Center, Nashville, TN 37232, USA
3 Cardiovascular Division, University of Pennsylvania, Philadelphia, PA 19104,
USA
Author for correspondence (e-mail:
kbriegel{at}med.miami.edu)
Accepted 3 May 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Lbh (Limb-bud and heart), Gene regulation, Heart development, Mouse, Congenital heart disease, Nkx2.5, Tbx5
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously identified a novel mouse protein, encoded by the
Lbh (Limb-bud and heart) gene
(Briegel and Joyner, 2001). Lbh
is a member of a highly conserved family of small acidic proteins of
12
kDa in vertebrates that do not exhibit any known structural motifs. A
Xenopus orthologue of Lbh, termed XlCl2, was
originally cloned as a maternal RNA of unknown function that becomes activated
in fertilized oocytes by polyadenylation
(Paris et al., 1988
;
Paris and Philippe, 1990
). Lbh
proteins share a glutamate-rich putative transcriptional activation domain at
the carboxyl terminus that is preceded by a putative nuclear localization
signal, but do not have any apparent DNA-binding domain
(Briegel and Joyner, 2001
). In
keeping with the protein structure, Lbh localizes to the nucleus and can
activate transcription in a reporter assay in mammalian tissue culture cells
(Briegel and Joyner, 2001
).
Thus, Lbh could act as a tissue-specific transcription cofactor.
During mouse embryogenesis, the dynamic spatiotemporal expression pattern
of Lbh reflects the onset of formation and patterning events in the
limb buds and in the heart (Briegel and
Joyner, 2001). Lbh expression is also detected in
primitive gut endoderm, branchial arches, ventral tail ectoderm, urogenital
ridge, otic vesicles, oral epithelium and in neural crest-derived sensory
neurons (Briegel and Joyner,
2001
). At the initial stages of limb outgrowth, Lbh is
expressed in ventral limb ectoderm and the apical ectodermal ridge (AER).
These limb ectodermal compartments provide the cues for both ventral limb
specification and proximodistal limb outgrowth
(Chen and Johnson, 1999
;
Tickle, 1999
). In the heart,
Lbh expression initiates in the crescent-shaped precardiac mesoderm
as early as expression of the homeodomain transcription factor Nkx2.5
(Briegel and Joyner, 2001
;
Lints et al., 1993
). Notably,
the cardiac crescent expresses Lbh in an anterior-to-posterior
gradient with highest levels of expression in anterior pro-cardiomyocytes
(Briegel and Joyner, 2001
). At
the completion of cardiac looping, Lbh expression is highest in the
right ventricle (RV), the atrio-ventricular canal (AVC) and the sinus venosus
(SV), but is excluded from atrial myocardium and endocardial structures. Once
chamber formation has occurred, the right-sided Lbh expression in
ventricular myocardium is lost and Lbh transcripts are distributed
more uniformly in the outer compact zone of RV and left ventricular (LV)
myocardium, but remain absent from atrial myocardium
(Briegel and Joyner, 2001
).
Interestingly, the Xenopus orthologue, XlCL2, is also
specifically expressed in the embryonic heart, suggesting a functional
conservation of Lbh proteins in vertebrate cardiogenesis
(Gawantka et al., 1998
). Both
Lbh and XlCl2 continue to be expressed at high levels in the
adult heart (Briegel and Joyner,
2001
; Paris and Philippe,
1990
). Although these findings suggest important roles of Lbh
during limb and heart development, the in vivo function of Lbh has
remained unknown.
We mapped the murine Lbh locus to mouse chromosome 17E2, and the
human LBH gene to human chromosome 2p23.3. To specifically study the
function of Lbh in heart development, we engineered mice that express an
Lbh transgene uniformly throughout the developing myocardium from the
3-somite stage onwards using a heart-specific promoter of the cardiac ankyrin
repeat protein (Carp; Ankrd1 Mouse Genome
Informatics) (Kuo et al.,
1999; Zou et al.,
1997
). We demonstrate that normal anteroposterior and later
chamber-specific localization, as well as gene-dosage of Lbh in the
primitive heart tube is important for normal heart morphogenesis because
enforced expression of Lbh in mice leads to a spectrum of
cardiovascular defects. Most strikingly, the cardiac phenotypes of
Carp-Lbh transgenic mice mimic CHD reported in humans trisomic for
chromosomal region 2p23, where LBH maps. Mice hemizygous for the
Carp-Lbh transgene develop OFT anomalies, including PS or PA due to
subvalvular obstruction of the pulmonary infundibulum with excessive valve
tissue, as well as DORV, D-TGA and TOF. In addition, characteristic defects in
IFT morphogenesis, cardiac septation, heart position and in ventricular
development were observed. Finally, we show that Lbh expressed in tissue
culture cells inhibits Nkx2.5- and Tbx5-mediated activation of cardiac target
genes and that Anf (Nppa Mouse Genome Informatics),
a common target gene, is downregulated in Carp-Lbh transgenic mice.
Taken together, our studies provide strong evidence that LBH is a
candidate gene for CHD associated with partial trisomy 2p syndrome and that
Lbh deregulation interferes with normal cardiac development, in part through
the attenuation of Nkx2.5 and Tbx5 transcription factor function.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of Carp-Lbh transgenic mice
A HindIII-BamHI fragment comprising an amino-terminally
Flag-tagged Lbh coding region from pcDNA/NFlag-Lbh
(Briegel and Joyner, 2001) was
blunt-end ligated into the SnaBI site of the transgenic vector PEVII
(Kimmel et al., 2000
). A 2.5
kb Carp cardiac-specific promoter contained on a
BamHI-XhoI fragment (Kuo
et al., 1999
; Zou et al.,
1997
) (generous gift from Dr Kenneth Chien), was blunt-end ligated
into the ClaI site of PEVII, creating the final transgenic construct
Carp-Lbh. The 3.5 kb transgene was released from vector sequences by
cleaving with SalI and microinjected into the pronuclei of one-cell
FVB/N embryos (Hogan et al.,
1994
). Transgenic progeny were identified by PCR and confirmed by
Southern blotting analysis as previously described
(Kimmel et al., 2000
).
RNA in situ hybridization
Whole-mount and section in situ hybridization was performed as described
previously (Chen et al., 2002;
Schaeren-Wiemers and Gerfin-Moser,
1993
; Wilkinson,
1992
). The gene-specific antisense probes used were to
Lbh (P1) (Briegel and Joyner,
2001
), lacZ (P2)
(Kimmel et al., 2000
),
Gata4 (Molkentin et al.,
1997
), Nkx2.5 (Lints
et al., 1993
), Tbx5
(Bruneau et al., 1999
) and
Anf (Zeller et al.,
1987
).
Histological analysis and immunohistochemistry
Whole mouse embryos and adult hearts were fixed for 18-24 hours in 4%
paraformaldehyde at +4°C, dehydrated in increasing concentrations of
ethanol and embedded in paraffin wax. Transverse, frontal and coronal sections
were cut at 4-6 µm and stained with Haematoxylin and Eosin.
Anti-phospho-histone H3 staining on paraffin sections was performed as
described by Shin et al. (Shin et al.,
2002), except that after immunostaining, sections were mounted in
SlowFade Antifade with DAPI (Molecular Probes). Phospho-histone H3-positive
cells were quantified by histomorphometry using Metamorph software (Universal
Imaging Corporation). This data was statistically analyzed with a paired
Student's t-test.
Polymeric dye injections
After CO2 euthanasia of mice, blue Batson's no. 17 casting
solution (Polysciences) was infused into the RV followed by injection of red
Batson's no. 17 polymeric dye solution into the LV of sick adult transgenic
mice and normal wild-type mice. After dye polymerization overnight at room
temperature, the tissue was digested with maceration solution according to the
manufacturer's protocol.
Transfections
NIH3T3 cells (2.5 x105 cells/well of a 12-well plate on
the day prior to transfection) were transfected using Lipofectamine 2000
reagent (Invitrogen) with 200 ng of Anf-human growth hormone (Anf-hGH)
reporter (Chen et al., 2002),
200 ng each of pCGN-Nkx2.5 and Gata4
(Durocher et al., 1997
) and
500 ng of pcDNA3-N-FLAG-TBX5
(Hiroi et al., 2001
)
expression plasmids. For synergy studies 600 ng of a pcDNA/NFLAG-Lbh
expression plasmid (Briegel and Joyner,
2001
) were co-transfected. 50 ng of pRL-CMV (Promega) were used to
normalize for transfection efficiencies and pBluescript was added to equalize
the amount of DNA to 1.6 µg per transfection. Representative results of at
least three independent experiments performed in duplicates were statistically
analyzed using a paired Student's t-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transgenic mis-expression of Lbh throughout the developing myocardium
The cardiac expression pattern of Lbh during mouse embryogenesis
and the linkage of LBH to human chromosome 2p23 suggest that
LBH gain of function could play a role in CHD commonly associated
with partial trisomy 2p syndrome. To test this hypothesis and also to examine
the function of Lbh in cardiovascular development, we designed an experiment
to perturb the normal Lbh gene dosage and expression pattern during
heart morphogenesis in mice. Transgenic mice were generated that express an
Lbh transgene uniformly throughout the developing myocardium from the
3-somite stage onwards under the control of a cardiomyocyte-specific promoter
of the Cardiac ankyrin repeat protein gene (Carp)
(Kuo et al., 1999;
Zou et al., 1997
)
(Fig. 2A). Transgene expression
was determined by RNA in situ analysis with a lacZ-specific antisense
probe (P2) that detects a short lacZ tag contained in the transgene
(Fig. 2A,D,F,H). Of the initial
10 transgenic lines, six showed robust cardiac-specific transgene expression
and germline transmission, and, hence, were used for further analyses (see
Table 1). In contrast to
endogenous Lbh expression in ventricular myocardium of wild-type
embryos with highest levels of expression in RV, AVC and SV
(Fig. 2C,E,G)
(Briegel and Joyner, 2001
), the
Carp-Lbh transgene was uniformly expressed in both ventricular and
atrial cardiomyocytes in all of 38 stable transgenic embryos from the six
selected transgenic lines that were analyzed on embryonic days 9.0 to 12.5
(E9.0-12.5) (Fig. 2D,F,H). Five
lines expressed the Carp-Lbh transgene at comparably high levels
(lines no. 3, 9, 16, 23 and 25; Table
1). Only one line (line no. 17) showed a slightly weaker, albeit
equally broad transgene expression in the heart
(Table 1). Transgene expression
was also visible in somites (Fig.
2D,F), but alterations in somite formation were not evident, and
hence not analyzed further. As expected, neither endogenous Lbh, nor
the Lbh transgene were expressed in endocardial structures
(Fig. 2G,H). Thus, during
cardiogenesis the Carp promoter fragment drives expression of
Lbh ectopically in atrial cardiomyocytes and dramatically increases
the level of expression in myocardium of the OFT and LV.
|
|
|
OFT and septation defects in postnatal and adult Carp-Lbh transgenic mice
Gross morphological and histological analyses of hemizygous (F0
+ F1; n=39) and homozygous (F2; n=12)
postnatal and adult Carp-Lbh transgenic mice (total number=51)
revealed different classes of cardiovascular malformations, which were
consistent between individual lines (Table
1). Eleven of the 51 transgenic mice analyzed (22%) exhibited
anomalies of the OFT. Characteristically, six of these 11 transgenic animals
displayed either PS (n=4) or PA (n=2)
(Table 1;
Fig. 3B,D,H). Furthermore, a
D-TGA was apparent in 3/11 animals with OFT defects (see below). 2/11
transgenic mice had only minor OFT defects
(Table 1).
To investigate potential abnormalities in blood flow that frequently occur in common with these OFT anomalies, we injected a blue polymeric dye into the RV (venous blood), followed by injection of red polymeric dye into the LV (systemic blood) of adult wild-type (n=2) and Carp-Lbh transgenic (n=2) animals showing signs of sickness. Whereas in wild-type hearts the aorta was filled with red polymeric dye only, one of the Carp-Lbh transgenic hearts displayed abnormal filling of the aorta with both red and blue polymeric dyes, indicative of mixing of systemic with venous blood, consistent with an overriding aorta and a PDA (Fig. 3G,H). Moreover, PS and a VSD (identified by the filling of the LV with blue dye) were apparent in this transgenic animal (Fig. 3H,J; data not shown). This combination of RVOT obstruction, overriding aorta, VSD and secondary ventricular hypertrophy is typical of human TOF (Fig. 3J). One additional transgenic mouse displayed RVOT defects in conjunction with TOF, as confirmed by histological sectioning (Table 1). The second transgenic heart examined by intra-cardiac dye filling displayed normal blood flow, but had a D-TGA (Table 1; data not shown). In addition, minor ASD was apparent in 4/6 Carp-Lbh transgenic mice with pulmonary trunk obstruction (Table 1; Fig. 6F; data not shown). One transgenic animal (line 9; F2) had a small VSD in the absence of any other cardiac defects (Table 1; data not shown). Thus, ectopic myocardial Lbh expression during heart development affected both OFT morphogenesis and cardiac septation.
Abnormal IFT morphology and heart position in Carp-Lbh transgenic mice
Seven of 51 Carp-Lbh transgenic mice (14%) exhibited IFT
deformities, including abnormal pulmonary venous return, either in isolation
(n=6) or in association with OFT defects (n=1)
(Table 1;
Fig. 4B,D). In contrast to
control hearts, the pulmonary veins in these transgenic hearts formed abnormal
connections to the right atrium, in addition to the left, through an enlarged
common sinus, which presumably represented a rudimentary left superior vena
cava (Fig. 4A-D). This
phenotype suggests that both pulmonary vein development, and IFT remodeling
were perturbed in a subset of Carp-Lbh transgenic mice.
Moreover, six of 51 postnatal and adult Carp-Lbh transgenic mice (12%) displayed an abnormal heart position in the absence of visceral heterotaxy. The transgenic hearts were shifted either to the right (dextrocardia; 4/51), to the left (levocardia; 1/51) or to the middle of the body (mesocardia; 1/51; Table 1). An adult Carp-Lbh transgenic heart with dextrocardia is shown in Fig. 4F. Whereas in wild-type mice, the heart is positioned to the left of the midline with the apex inclined towards the left, the transgenic heart was rotated to the right with dextro-positioned apex, great arteries and IFT (Fig. 4E-H). This phenotype most probably was secondary to hypoplastic growth of LV cardiomyocytes (Fig. 4I,J), rather than caused by defects in cardiac laterality.
|
|
Histological analysis of neonatal and embryonic Carp-Lbh transgenic hearts
To examine disease progression and the primary morphological lesions that
cause the cardiac disease phenotype in Carp-Lbh transgenic mice, we
performed histopathology on wild-type and transgenic mice at different stages
in heart development. To assess the effect of Lbh overexpression at
birth, when the pulmonary blood circulation becomes established and vital
because of closures of the intra-atrial foramen ovale and the ductus
arteriosus, we analyzed the hearts of Carp-Lbh transgenic mice
(n=2) from two independent lines that had died shortly after birth.
As shown in Fig. 6, the
pulmonary valves of P0 wild-type hearts consist of three thin leaflets derived
from endocardial tissue, and the RVOT beneath the valves forms a cavity
(Fig. 6A,C). In the P0
Carp-Lbh transgenic mouse shown, the pulmonary valves appeared
normal, however, the RVOT below the valves was filled with excessive valve
tissue and myocardial cells (Fig.
6B,D), causing a subvalvular obstruction of the pulmonary
infundibulum. In addition, the transgenic heart displayed a PFO with a right
to left shunting most probably caused by increased pressures on the right side
of the heart as a result of the pulmonic obstruction
(Fig. 6F). The second
transgenic newborn that died exhibited isolated IFT anomalies (data not
shown).
To investigate cardiac valve and septae formation in more detail, we
analyzed transient transgenic embryos (n=8), as postnatal
Carp-Lbh founder transgenics showed the most severe phenotypes
(Table 1), between E12-14.
Fig. 6G-L shows histological
sections from a wild-type and a transient transgenic littermate at E13. In
addition to levocardia, ventricular hyperplasia and abnormal trabeculation,
severe OFT anomalies were observed in the Carp-Lbh transgenic embryo
(Fig. 6H,J,L). In wild-type
embryos, the ridge-like endocardial cushions of the OFT, which give rise to
the septae and the valves of the great arteries
(Fishman and Chien, 1997),
only line the inner conotruncal myocardium
(Fig. 6I). However, in the
transgenic embryo shown, the OFT ridges were not only drastically enlarged,
but also extended to ectopic sites in the cavity of the RV
(Fig. 6H,J). Moreover, a DORV
and a VSD were apparent (Fig.
6J,L). Thus, the cardiovascular phenotypes of neonatal and
embryonic Carp-Lbh transgenic mice are consistent with the spectrum
of cardiac malformations observed in late postnatal and adult animals.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Range of cardiovascular malformations in Carp-Lbh transgenic mice
The phenotypes of Carp-Lbh transgenic mice suggest that enforced
expression of Lbh in heart muscle cells from the 3-somite stage
onwards affects different steps in heart morphogenesis, including OFT
valvulogenesis, IFT morphogenesis, cardiac septation, as well as cardiomyocyte
growth and function, leading to secondary cardiovascular defects and
dysfunction in postnatal and adult transgenic animals. The spectrum of cardiac
malformations was consistent between the independent transgenic lines, but was
more severe in some lines and varied among different individuals of one line
(Table 1). Although this could
reflect different Lbh transgene levels, we detected only slight
variations in transgene levels in five out of six lines, as assessed by RNA in
situ hybridization. Furthermore, homozygous animals with clearly elevated
transgene expression levels did not show more severe phenotypes than their
hemizygous littermates (Table
1; data not shown). This suggests that the diversity of cardiac
defects in Carp-Lbh transgenic mice could have been evoked not only
by a gene dosage effect, but also by the abnormal distribution of Lbh in
transgenic hearts.
Lbh interferes with OFT valvulogenesis
Carp-Lbh transgenic mice displayed a spectrum of OFT defects (PS
and PA with or without TOF) that is characteristic for DiGeorge Syndrome, a
human neural crest (NC) ablation defect, caused by haploinsufficiency of genes
located on chromosome 22p11, in particular TBX1
(Goldmuntz et al., 1998;
Jerome and Papaioannou, 2001
;
Lindsay et al., 1999
;
Merscher et al., 2001
).
Cardiac NC colonizes the primordial endocardial cushions, which is a
prerequisite to cardiac valve and septae formation (reviewed by
Kirby, 1999
). However, in
Carp-Lbh transgenic mice these OFT anomalies appear to be of
myocardial origin, because the Carp-Lbh transgene was not expressed
in endocardium or cardiac NC (Fig.
2) (Kuo et al.,
1999
; Zou et al.,
1997
). Valve formation is controlled by reciprocal signaling
between the myocardium and endocardial cushions in valve-forming regions (OFT,
AVC), which induces epithelial-mesenchymal transformation (EMT) in the valve
cushions (reviewed by Barnett and
Desgrosellier, 2003
). In the OFT these tissue interactions also
control a process called `myocardialization', in which myocardial cells invade
the conotruncal mesenchymal cushions, contributing to muscular pulmonary
infundibulum and outlet septum (van den
Hoff et al., 1999
). Overgrowth of the sub-pulmonic region of
embryonic and P0 Carp-Lbh transgenic mice with both mesenchymal valve
tissue and myocardial cells, leading to pulmonary obstruction and eventually
to a regression of the pulmonary artery in adult transgenics, suggests that
overexpression of Lbh in OFT myocardium perturbed the myocardial
signaling that controls both of these processes. Notch signaling has been
shown to promote EMT in endocardial cushions
(Noseda et al., 2004
;
Timmerman et al., 2004
).
Notably, gene mutations in Jagged 1 (JAG1), a Notch ligand, in human
Alagille Syndrome (Li et al.,
1997
; Oda et al.,
1997
) or gene ablation of Hey2, encoding a
hairy/Enhancer-of-split-related basic helix-loop-helix transcription factor
acting downstream of Notch, in mice
(Donovan et al., 2002
), cause
OFT and cardiac septation defects similar to those observed in
Carp-Lbh transgenic mice. Another myocardial signal that regulates
EMT during cardiac valve formation, as well as OFT myocardialization, is
transforming growth factor ß2 (Tgfß2)
(Bartram et al., 2001
;
Boyer et al., 1999
;
Camenisch et al., 2002
).
Tgfß2-deficient mice, not only have hyperplastic valves but also DORV
with accompanying VSD (Bartram et al.,
2001
), an anomaly we also observed in Carp-Lbh transgenic
mice.
Mis-regulation of Lbh perturbs late sino-atrial morphogenesis
Ectopic expression of Lbh in atrial cardiomyocytes most probably
contributed to both abnormal pulmonary venous return and to defects in atrial
septation in Carp-Lbh transgenic mice. The primordial pulmonary veins
originate as an outgrowth of atrial muscle cells that anastomose with the
pulmonary venous plexus and subsequently colonize the pulmonary veins in a
caudocranial fashion (Larsen,
1997; Millino et al.,
2000
). Although the role of myocardium in pulmonary vein formation
is not known, mis-expression of Lbh in atrial muscle cells that
encase the pulmonary veins indicates that proper pulmonary vein muscle
development is a prerequisite for the correct positioning of the pulmonary
veins. Alternatively, ectopic Lbh might have altered atrial
myocardial function, which could impair atrial-venous differentiation and
cardiac morphology by changing the normal blood flow and hemodynamics
(Huang et al., 2003
;
le Noble et al., 2003
).
Altered hemodynamic forces may also have contributed to ASD, PFA and other
morphological defects in Carp-Lbh transgenic mice
(Larsen, 1997
;
le Noble et al., 2003
).
Furthermore, Lbh transgene expression might have directly interfered
with the molecular pathways that control atrial septation (see below).
Overexpression of Lbh impairs ventricular cardiomyocyte growth
During heart development, Lbh is predominantly expressed in
proliferative ventricular myocardium
(Briegel and Joyner, 2001),
suggesting a role of Lbh in ventricular cardiomyocyte growth. In keeping with
this idea, ventricular growth defects including hyperplasia and hypoplasia
were observed in a cohort of Carp-Lbh transgenic mice. Proliferation
of cardiomyocytes normally stops after birth and transits into hypertrophic
cell growth, which is accompanied by increased myofibril density and
cardiomyocyte cell fusion (Pasumarthi and
Field, 2002
). However, in hyperplastic transgenic hearts,
proliferation of ventricular cardiomyocytes was prolonged into postnatal
stages. In contrast, hypoplasia in transgenic mice was due to a failure of
ventricular cardiomyocytes to undergo hypertrophic growth, which was evident
by reduced cardiomyocyte size and fusion
(Fig. 5F). As the Carp
promoter is down-regulated upon birth (Kuo
et al., 1999
; Zou et al.,
1997
), these postnatal ventricular growth defects could be the
result of transgenic Lbh protein being more stable than its mRNA. Furthermore,
the abnormal position of some Carp-Lbh transgenic hearts appeared to
be the consequence of asymmetric ventricular hypoplasia
(Fig. 4J) or hyperplasia
(Fig. 6H), rather than of
aberrant left-right patterning, as expression of Pitx2, a
POU-homeodomain transcription factor and major determinant for organ asymmetry
(Capdevila et al., 2000
), and
early looping morphogenesis was unaltered in these mice (data not shown).
What is the molecular basis for Lbh function?
The biochemical properties of Lbh suggest a role of this protein in cardiac
gene regulation (Briegel and Joyner,
2001). Consistent with this notion, we found that expression of
Lbh in tissue culture cells predominantly represses the transcriptional
activities of Nkx2.5 and Tbx5 (Bruneau et
al., 2001
; Hiroi et al.,
2001
), two key regulators of cardiogenesis
(Biben et al., 2000
;
Bruneau et al., 2001
;
Liberatore et al., 2000
;
Lyons et al., 1995
;
Schott et al., 1998
;
Takeuchi et al., 2003
). In
accord with these data, expression of Anf, a common Nkx2.5/Tbx5
target gene (Bruneau et al.,
2001
), was markedly downregulated in early Carp-Lbh
transgenic embryos. Since Lbh lacks a DNA binding domain and therefore could
not compete for Nkx2.5/Tbx5 DNA binding sites in the promoters of cardiac
genes, we favor the idea that Lbh modulates Nkx2.5 and Tbx5 transcriptional
activities by directly interacting with these factors at the protein level. In
support of this idea, the cardiac disease phenotypes observed in
Carp-Lbh transgenic mice, as well as in individuals with partial
trisomy 2p syndrome are remarkably similar to CHD caused by haploinsufficiency
of NKX2.5 or TBX5: ASD, TOF and ventricular abnormalities
(Basson et al., 1997
;
Biben et al., 2000
;
Bruneau et al., 2001
;
Schott et al., 1998
). In
addition, Carp-Lbh transgenic mice and individuals with partial
trisomy 2p syndrome have in common with NKX2.5 hapoinsufficiency, PS, DORV and
PDA (Benson et al., 1999
;
Schott et al., 1998
), whereas
anomalous pulmonary return and left ventricular hyperplasia have also been
reported in TBX5-haploinsufficient individuals with Holt-Oram syndrom and in
Tbx5 mouse mutants (Basson et al.,
1997
; Bruneau et al.,
2001
). However, unlike NKX2.5 and TBX-deficiencies
(Bruneau et al., 2001
;
Schott et al., 1998
)
Carp-Lbh transgenic mice did not display cardiac rhythm disturbances,
as assessed by electrocardiograms (data not shown), which could be due to the
absence of Carp-promoter activity in the conduction system
(Kuo et al., 1999
;
Zou et al., 1997
). There is
also significant overlap between the cardiac phenotypes observed in
Carp-Lbh transgenic mice and Gata4 deficiencies: ASD, TOF, PS, PDA,
DORV, VSD membranous type, excessive pulmonary valve tissue, dextrocardia and
ventricular hypoplasia (Garg et al.,
2003
; Pu et al.,
2004
). Although we did measure only a modest inhibitory effect of
Lbh on Gata4 transcriptional activity in cell-based reporter assays, our
genetic data would suggest that the Lbh transgene also interfered
with Gata4-dependent pathways. Taken together, our data indicate that Lbh can
regulate cardiac gene expression by modulating the combinatorial activities of
key cardiac transcription factors, as well as their individual functions in
cardiogenesis.
Potential role of LBH in human congenital heart disease
Most importantly, Lbh gain of function during heart development of
transgenic mice virtually mimics CHD that has been reported in individuals
trisomic for the chromosomal region 2p23, to which the human LBH
maps. This provides the first evidence that CHD associated with partial
trisomy 2p syndrome is due in part to increased gene dosage, and most likely
also abnormal cardiac distribution of LBH in some individuals. A
deregulation of LBH gene expression could result if the translocation
breakpoint occurs in proximity of the LBH locus. Indeed, in over
one-third of these individuals the chromosomal breakpoint maps within 2p23
(Taylor Clelland et al.,
2000). Interestingly, triplication of chromosomal band 2p23 also
seems to be associated with postaxial limb defects, such as polydactyly,
clinodactyly, long tapering fan-like digits and bilateral simian creases
(Cassidy et al., 1977
;
Francke and Jones, 1976
;
Hahm et al., 1999
;
Lurie et al., 1995
). These
anomalies might in part be due to dysfunction of the AER, but also to
defective dorsoventral limb patterning. Since Lbh is expressed both
in the AER and in ventral limb ectoderm during mouse limb development
(Briegel and Joyner, 2001
), it
is tempting to speculate that increased LBH gene dosage is also
involved in the partial trisomy 2p limb phenotypes. In conclusion, our
findings suggest a pivotal role of Lbh in normal heart development, as well as
in human CHD, as a trans-acting modulator of key cardiac transcription
factors.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barnett, J. V. and Desgrosellier, J. S. (2003). Early events in valvulogenesis: a signaling perspective. Birth Defects 69,58 -72.[CrossRef]
Bartram, U., Molin, D. G. M., Wisse, L. J., Mohamad, A.,
Sanford, L. P., Doetschman, T., Speer, C. P., Poelmann, R. E. and
Gittenberger-de Groot, A. C. (2001). Double-outlet right
ventricle and overriding tricuspid valve reflect disturbances of looping,
myocardialization, endocardial cushion differentiation, and apopotosis in
TGF-ß2-knockout mice. Circulation
103,2745
-2752.
Basson, C. T., Bachinsky, D. R., Lin, R. C., Levi, T., Elkins, J. A., Soults, J., Grayzel, D., Kroumpouzou, E., Traill, T. A., Leblanc-Straceski, J. et al. (1997). Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 15,30 -35.[CrossRef][Medline]
Benson, D. W., Silberbach, G. M., Kavanaugh-McHugh, A., Cottrill, C., Zhang, Y., Riggs, S., Smalls, O., Johnson, M. C., Watson, M. S., Seidman, J. G. et al. (1999). Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. Nat. Genet. 15,30 -35.[CrossRef]
Biben, C., Weber, R., Kesteven, S., Stanley, E., McDonald, L.,
Elliott, D. A., Barnett, L., Koentgen, F., Robb, L., Feneley, M. et
al. (2000). Cardiac septal and valvular dysmorphogenesis in
mice heterozygous for mutations in the homeobox gene Nkx2-5. Circ.
Res. 87,888
-895.
Boyer, A. S., Ayerinskas, I. I., Vincent, E. B., McKinney, L. A., Weeks, D. L. and Runyan, R. B. (1999). TGFß2 and TGFß3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart. Dev. Biol. 208,530 -545.[CrossRef][Medline]
Briegel, K. J. and Joyner, A. L. (2001). Identification and characterization of Lbh, a novel conserved nuclear protein expressed during early limb and heart development. Dev. Biol. 233,291 -304.[CrossRef][Medline]
Bruneau, B. G., Logan, M., Davis, N., Levi, T., Tabin, C. J., Seidman, J. G. and Seidman, C. E. (1999). Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev. Biol. 211,100 -108.[CrossRef][Medline]
Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. 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]
Camenisch, T. D., Molin, D. G. M., Person, A., Runyan, R. B., Gittenberger-de Groot, A. C., McDonald, J. A. and Klewer, S. E. (2002). Temporal and distinct TGFß ligand requirements during mouse and avian endocardial cushion morphogenesis. Dev. Biol. 248,170 -181.[CrossRef][Medline]
Capdevila, J., Vogan, K. J., Tabin, C. J. and Izpisua Belmonte, J. C. (2000). Mechanisms of left-right deterimination in vertebrates. Cell 101,9 -21.[CrossRef][Medline]
Cassidy, S. B., Heller, R. M., Chazen, E. M. and Engel, E. (1977). The chromosome 2 distal short arm trisomy syndrome. J. Pediatr. 91,934 -938.[Medline]
Chen, F., Kook, H., Milewski, R., Gitler, A. D., Lu, M. M., Li, J., Nazarian, R., Schnepp, R., Jen, K., Biben, C. et al. (2002). Hop is an unusual homeobox gene that modulates cardiac development. Cell 110,713 -723.[CrossRef][Medline]
Chen, H. and Johnson, R. L. (1999). Dorsoventral patterning of the vertebrate limb: a process governed by multiple events. Cell Tissue Res. 296, 67-73.[CrossRef][Medline]
Donovan, J., Kordylewska, A., Jan, Y. N. and Utset, M. F. (2002). Tetralogy of fallot and other congenital heart defects in Hey2 mutant mice. Curr. Biol. 12,1605 -1610.[CrossRef][Medline]
Durocher, D., Charron, F., Warren, R., Schwartz, R. J. and
Nemer, M. (1997). The cardiac transcription factors Nkx2-5
and GATA-4 are mutual cofactors. EMBO J.
16,5687
-5696.
Fishman, M. C. and Chien, K. R. (1997).
Fashioning the vertebrate heart: earliest embryonic decisions.
Development 124,2099
-2117.
Francke, U. and Jones, K. L. (1976). The 2p partial trisomy syndrome. Duplication of region 2p23 leads to 2pter in two members of a t(2;7) translocation kindred. Am. J. Dis. Child. 130,1244 -1249.[Medline]
Garg, V., Kathiriya, I. S., Barnes, R., Schluterman, M. K., King, I. N., Butler, C. A., Rothrock, C. R., Eapen, R. S., Hirayama-Yamada, K., Joo, K. et al. (2003). GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424,443 -447.[CrossRef][Medline]
Gawantka, V., Pollet, N., Delius, H., Vingron, M., Pfister, R., Nitsch, R., Blumenstock, C. and Niehrs, C. (1998). Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning. Mech. Dev. 77,95 -141.[CrossRef][Medline]
Goldmuntz, E., Clark, B. J., Michell, L. E., Jawad, A. F.,
Cuneo, B. F., Reed, L., McDonald-McGinn, D., Chien, P., Feuer, J.,
Zackai, E. H. et al. (1998). Frequency of 22q11 deletions in
patients with conotruncal defects. J. Am. Coll.
Cardiol. 32,492
-498.
Hahm, G. K., Barth, R. F., Schauer, G. M., Reiss, R. and Opitz, J. M. (1999). Trisomy 2p syndrome: a fetus with anencephaly and postaxial polydactyly. Am. J. Med. Genet. 87, 45-48.[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]
Hogan, B. M. L., Beddington, R. S. P., Constantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hofmann, J. I. and Kaplan, S. (2002). The
incidence of congenital heart disease. J. Am. Coll.
Cardiol. 39,1890
-1900.
Huang, C., Sheikh, F., Hollander, M., Cai, C., Becker, D., Chu,
P.-H., Evans, S. and Chen, J. (2003). Embryonic atrial
function is essential for mouse embryogenesis, cardiac morphogenesis and
angiogenesis. Development
130,6111
-6119.
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]
Kimmel, R. A., Turnbull, D. H., Blanquet, V., Wurst, W., Loomis,
C. A. and Joyner, A. L. (2000). Two lineage boundaries
coordinate vertebrate apical ectodermal ridge formation. Genes
Dev. 14,1377
-1389.
Kirby, M. L. (1999). Contribution of neural crest to heart and vessel morphology. In Heart Development (ed. R. Harvey and N. Rosenthal), pp.179 -193. San Diego, CA: Academic Press.
Kuo, H., Chen, J., Ruiz-Lozano, P., Zou, Y., Nemer, M. and
Chien, K. R. (1999). Control of segmental expression of the
cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways
in murine cardiogenesis. Development
126,4223
-4234.
Larsen, W. J. (1997). Human Embryology. Philadelphia: Churchill Livingston.
le Noble, F., Moyon, D., Pardanaud, L., Yuan, L., Djonov, V., Matthijsen, R., Breant, C., Fleury, V. and Eichmann, A. (2003). Flow regulates arterial-venous differentiation in the chick embryo yolk sac. Development 131,361 -375.[CrossRef][Medline]
Li, L., Krantz, I. D., Deng, Y., Genin, A., Banta, A. B., Collins, C. C., Qi, M., Trask, B. J., Kuo, W. L., Cochran, J. et al. (1997). Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat. Genet. 16,243 -251.[CrossRef][Medline]
Liberatore, C. M., Searcy-Schrick, R. D. and Yutzey, K. E. (2000). Ventricular expression of tbx5 inhibits normal heart chamber development. Dev. Biol. 223,169 -180.[CrossRef][Medline]
Lindsay, E. A., Botta, A., Jurecic, V., Carattini-Rivera, S., Cheah, Y. C., Rosenblatt, H. M., Bradley, A. and Baldini, A. (1999). Congenital heart disease in mice deficient for the DiGeorge syndrome region. Nature 401,379 -383.[CrossRef][Medline]
Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I. and Harvey, R. P. (1993). Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119,969 .[Medline]
Lurie, I. W., Ilyina, H. G., Gurevich, D. B., Rumyantseva, N. V., Naumchik, I. V., Castellan, C., Hoeller, A. and Schinzel, A. (1995). Trisomy 2p: analysis of unusual phenotypic findings. Am. J. Med. Genet. 55,229 -236.[CrossRef][Medline]
Lyons, I., Parsons, L. M., Hartley, L., Li, R., Andrews, J. E., Robb, L. and Harvey, R. P. (1995). Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev. 9,1654 -1666.[Abstract]
Merscher, S., Funke, B. A., Epstein, J. A., Heyer, J., Puech, A., Lu, M. M., Xavier, R. J., Demay, M. B., Russell, R. G., Factor, S. et al. (2001). TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104,619 -629.[CrossRef][Medline]
Millino, C., Sarinella, F., Tiveron, C., Villa, A., Sartore, S. and Ausoni, S. (2000). Cardiac and smooth muscle cell contribution to the formation of the murine pulmonary veins. Dev. Dyn. 218,414 -425.[CrossRef][Medline]
Molkentin, J. D., Lin, Q., Duncan, S. A. and Olson, E. N. (1997). Requirements of the transcription factor GATA-4 for heart tube formation and ventral morphogenesis. Genes Dev. 11,1061 -1072.[Abstract]
Neu, R. L., Dennis, N. R. and Fisher, J. E. (1979). Partial 2p trisomy in a 46,XY,der(5),t(2;5)(p23;p15)pat infant; autopsy findings. Ann. Genet. 22, 33-34.
Noseda, M., McLean, G., Niessen, K., Chang, L., Pollet, I.,
Montpetit, R., Shahidi, R., Dorovini-Zis, K., Li, L., Beckstead, B. et
al. (2004). Notch activation results in phenotypic and
functional changes consistent with endothelial-to-mesenchymal transformation.
Circ. Res. 94,910
-917.
Oda, T., Elkahloun, A. G., Pike, B. L., Okajima, K., Krantz, I. D., Genin, A., Piccoli, D. A., Meltzer, P. S., Spinner, N. B., Collins, F. S. et al. (1997). Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nat. Genet. 16,235 -242.[CrossRef][Medline]
Paris, J. and Philippe, M. (1990). Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development. Dev. Biol. 140,221 -224.[CrossRef][Medline]
Paris, J., Osborne, H. B., Couturier, A., Le Guellec, R. and Philippe, M. (1988). Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis. Gene 72,169 -176.[CrossRef][Medline]
Pasumarthi, K. B. S. and Field, L. J. (2002).
Cardiomyocyte cell cycle regulation. Circ. Res.
90,1044
-1054.
Pu, W. T., Ishiwata, T., Juraszek, A. L., Ma, Q. and Izumo, S. (2004). GATA4 is a dosage-sensitive regulator of cardiac morphogenesis. Dev. Biol. 275,235 -244.[CrossRef][Medline]
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using dioxigenin-labeled cRNA probes. Histochemistry 100,431 -440.[CrossRef][Medline]
Schott, J. J., Benson, D. W., Basson, C. T., Pease, W.,
Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and
Seidman, J. G. (1998). Congenital heart disease caused by
mutations in the transcription factor NKX2-5. Science
281,108
-111.
Shin, C. H., Liu, Z. P., Passier, R., Zhang, C. L., Wang, D. Z., Harris, T. M., Yamagishi, H., Richardson, J. A., Childs, G. and Olson, E. N. (2002). Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell 110,725 -735.[CrossRef][Medline]
Takeuchi, J. K., Ohgi, M., Koshiba-Takeuchi, K., Shiratori, H.,
Sakaki, I., Ogura, K., Saijoh, Y. and Ogura, T.
(2003). Tbx5 specifies the left/right ventricles and ventricular
septum position during cardiogenesis. Development
130,5953
-5964.
Taylor Clelland, C. L., Levy, B., McKie, J. M., Duncan, A. M., Hirschhorn, K. and Bancroft, C. (2000). Cloning and characterization of human PREB; a gene that maps to a genomic region associated with trisomy 2p syndrome. Mamm. Genome 11,675 -681.[CrossRef][Medline]
Therkelsen, A. J., Hultén, M., Jonasson, J., Lindsten, J., Christensen, N. C. and Iversen, T. (1973). Presumptive direct insertion within chromosome 2 in man. Ann. Hum. Genet. 36,367 -373.[Medline]
Tickle, C. (1999). Morphogen gradients in vertebrate limb development. Semin. Cell Dev. Biol. 10,345 -351.[CrossRef][Medline]
Timmerman, L. A., Grego-Bessa, J., Raya, A., Bertran, E.,
Perez-Pomares, J. M., Diez, J., Aranda, S., Palomo, S., McCormick, F.,
Izpisua Belmonte, J. C. et al. (2004). Notch promotes
epithelial-mesenchymal transition during cardiac development and oncogenic
transformation. Genes Dev.
18, 99-115.
van den Hoff, M. J. B., Moorman, A. F. M., Ruijter, J. M., Lamers, W. H., Bennington, R. W., Markwald, R. R. and Wessels, A. (1999). Myocardialization of the cardiac outflow tract. Dev. Biol. 212,477 -490.[CrossRef][Medline]
Wilkinson, D. G. (1992). Whole mount in situ hybridization of vertebrate embryos. In In Situ Hybridization (ed. D. G. Wilkinson), pp.939 -947. Oxford: IRL Press.
Zeller, R., Bloch, K. D., Williams, B. S., Arceci, R. J. and Seidman, C. E. (1987). Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev. 1,693 -698.[Abstract]
Zou, Y., Evans, S., Chen, J., Kuo, H. C., Harvey, R. P. and
Chien, K. R. (1997). Carp, a cardiac ankyrin repeat protein,
is downstream in the Nkx2-5 homeobox gene pathway.
Development 124,793
-804.
|