1 Laboratory of Developmental Biology, National Heart Lung and Blood Institute,
National Institutes of Health, Bethesda, MD 20892-8019, USA
2 Department of Pediatrics, National Naval Medical Center, Uniformed Services
University of the Health Sciences, Bethesda, MD 20814-5000, USA
3 Pediatric Cardiology, Children's National Medical Center, Washington, DC
20010, USA
4 Department of Anatomy and Cell Biology, Medical University of South Carolina,
Charleston, SC 29425, USA
5 Pediatric Cardiology, Medical University of South Carolina, Charleston, SC
29425, USA
6 Department of Pediatrics, Duke University Medical Center, Durham, NC 27710,
USA
7 The Jackson Laboratory, Bar Harbor, ME 04609, USA
* Author for correspondence (e-mail: loc{at}nhlbi.nih.gov)
Accepted 20 October 2004
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SUMMARY |
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Key words: Congenital heart defects, Cardiovascular anomalies, ENU mutagenesis, Mouse mutants, Ultrasound imaging
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Introduction |
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Besides mutations in transcription factors, mutations in PTPN11,
encoding protein tyrosine phosphatase SHP-2, has been linked to Noonan and
LEOPARDS syndromes (Taraglia et al., 2001), which are characterized by
pulmonary stenosis, atrial septal defects, and early onset hypertrophic
cardiomyopathy (Noonan) or conduction anomalies (LEOPARD). Linkage has also
been established for mutations in 7-dehydrocholesterol reductase in
Smith-Lemli-Opitz syndrome (for a review, see
Jira et al., 2003), which is
characterized by AV canal defects, together with microcephaly, cleft palate,
postaxial polydactyly or syndactyly. Finally, mutations in Jagged1, a
ligand in the Notch signaling pathway, has been shown to cause Alagille
syndrome, which is characterized by pulmonary outflow and other cardiac
defects, bile duct paucity and other anomalies
(Li et al., 1997a
;
Oda et al., 1997
). Except for
Smith-Lemli-Opitz syndrome, these mutations are autosomal dominant. It is
important to note that not all individuals exhibiting the various syndromic
congenital cardiovascular malformations have mutations in the identified
genes. Rather, evidence suggests these congenital heart diseases are
genetically heterogeneous.
Given the many difficulties involved in tracking the genetic basis for
congenital heart anomalies in human populations, the pursuit of mouse models
has become an invaluable avenue of investigation. Using embryonic stem cell
gene-targeting approaches, gene function can be investigated by generating
mice in which the endogenous allele has been knocked out or replaced. Indeed
some of the gene-targeted mouse lines generated exhibit phenotypes resembling
those seen in human congenital heart disease. The involvement of Tbx1
in DiGeorge syndrome was in fact first indicated by the analysis of
Tbx1 knockout mice (Lindsay et
al., 2001; Merscher et al.,
2001
; Jerome and Papaioannou,
2001
), and only later was confirmation obtained for TBX1
mutations in patients with DiGeorge syndrome
(Yagi et al., 2003
). Overall,
studies with transgenic and knockout mouse models show that mice can be used
effectively to model human congenital heart disease, although in many
instances, the identification of genes or mutations causing cardiovascular
defects has been serendipitous.
In this study, we explored the use of N-ethyl-N-nitrosourea (ENU)
mutagenesis as an alternative approach for identifying genes that may
contribute to congenital heart defects. Chemical mutagenesis allows a genome
wide scan for genes that may be important in heart development and disease.
Typically, such mutagenesis is conducted in an inbred mouse strain such as
C57BL6/J, and when mutations of interest are found, the mutant mice are
interbred with a different inbred mouse strain, allowing the rapid mapping of
mutations by tracking linkage of the mutant phenotype with polymorphic
microsatellite DNA markers. ENU mutagenesis has been successfully employed for
a variety of focused phenotypic screens in mice, such as for mutations that
cause cataracts, behavioral or circadian rhythm perturbations,
atherosclerosis, hypertension and obesity
(Favor, 1986;
Vitaterna et al., 1994
;
Pickard et al.,1995
;
Svenson et al., 2003
). Of
crucial importance to the success of such screens is access to an appropriate
high-throughput phenotyping tool in our case, one suitable for the
detection of congenital cardiovascular anomalies in mouse fetuses. In this
study, we showed the efficacy of an advanced clinical ultrasound system for
non-invasive cardiovascular phenotyping of mouse fetuses.
Ultrasound is routinely used clinically for assessing human cardiovascular
structure and function, and has been used to examine mouse fetuses
(Gui et al., 1996;
Huang et al., 1998b
;
Srinivasan et al., 1998; Zhou et al.,
2002
; Maki et al.,
2002
; Leatherbury et al.,
2003
). There are several advantages with ultrasound phenotyping.
First, it is non-invasive, and thus fetuses can be examined in utero, allowing
for horizontal studies over many days. Second, it provides assessments of both
cardiovascular structure and function. Third, it is a high-throughput
procedure, as a hundred or more fetuses can be screened in a day. For this
prenatal fetal screen, we adopted a recessive breeding scheme, with the
expectation that this would yield the most deleterious phenotypes. Overall,
our studies showed that mouse mutations causing congenital heart disease can
be efficiently recovered by high-throughput fetal ultrasound phenotyping. Such
studies hold promise in yielding new insights into the genetic basis for human
congenital heart disease.
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Materials and methods |
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Ultrasound imaging and Doppler echocardiography
An Acuson Sequoia C256 ultrasound system with a 15 MHz L8 linear phased
array transducer was used to scan pregnant female mice placed on a heating pad
and anesthesized with isofluorane anesthesia (1.5% in medical air). ECG
electrodes and a rectal probe continuously monitored the mother's heart rate
(450-550 bpm) and body temperature (36-37°C), respectively. Hair was
removed from the abdomen, and pre-warmed ultrasound gel was applied. Color
flow and spectral Doppler imaging were obtained from embryonic day (E)12.5 to
E19.5. From E14.5, M-mode and 2D images were obtained from the fetus using
short axis views of the ventricles and great vessels, apical three/four/five
chamber views, and long axis views of the left ventricle, aorta and pulmonary
arteries.
Necropsy and histology
Stillborn pups or pups that died within a day of birth were retrieved and
fixed in 10% buffered formalin. Necropsy was performed to examine the heart,
great vessels and aortic arch arteries. For histology, the heart and
surrounding vessels were paraffin-wax embedded, sectioned, and stained with
Hematoxylin and Eosin. Some were processed and examined by episcopic
fluorescence image capture (Weninger and
Mohun, 2002). Pups with apparent skeletal anomalies were further
processed for Alizaren Red and Alcian Blue staining.
Genome scan analysis
To examine heritability and facilitate mapping of the mutation, G2 carrier
females were intercrossed with C3H mice to generate C3H/C57BL6 hybrid
offspring. These were then further intercrossed for cardiovascular phenotyping
by echocardiography, followed by analysis via necropsy and histopathology. DNA
collected from the affected fetuses was PCR amplified using primers for 48
C3H/C57BL6 polymorphic microsatellite markers. The resultant PCR products were
pooled and separated by capillary electrophoresis on the Avant 3100 Genetic
Analyzer (Applied Biosystems), and the data generated were analyzed using the
method of Neuhaus and Beier for recombinant interval haplotype analysis
(Neuhaus and Beier, 1998).
Thus, DNA markers located near the ends of each mouse chromosome are used to
demarcate intervals that are treated as haplotypes for the purpose of linkage
analysis. For the larger chromosomes (1, 2, 3, 4, 6), one or two additional
markers are included to provide one or two additional intervals (proximal,
middle, distal; see Table 2).
The frequency with which recombinant haplotypes are found across the entire
genome in the affected fetuses is tracked. The ENU-induced mutation is
expected to lie in a chromosome interval that is consistently homozygous B6 in
most or all of the affected fetuses, i.e. non-recombinant for C3H markers
(corresponding to a 0 or 1 in Table
2).
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Results |
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Cardiovascular anomalies detected by ultrasound
The cardiovascular anomalies that could be detected by prenatal fetal
ultrasound included arrhythmias, outflow regurgitations, increased outflow
velocity, heart failure, hypertrophy and ectopia cordis
(Table 1; see below).
Arrhythmia was easily observed by spectral Doppler analysis and included
premature systole, pause, bradycardia and tachycardia. However, bradycardia
accounted for nearly half (42%) of the arrhythmias, which frequently is a
manifestation of dying fetuses. Consistent with this, we note that 18% of
fetuses with heart failure also exhibited bradycardia. Heart failure was
discerned by dynamic 2D imaging, and is characterized by poor contractile
function, pericardial effusion and hydrops
(Fig. 1B). Contractile motion
of the beating heart was assessed qualitatively using 2D video sequences, and
quantitatively with measurements of ejection fraction, fractional area change
and shortening fraction. In many instances, heart failure was confirmed with
the examination of dead pups, which typically showed congested heart, lung and
liver (Table 1).
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Hypertrophy was detected using M-mode imaging and is defined as those fetuses with a wall thickness more than two standard deviations from the mean. Owing to difficulty in achieving the optimal imaging planes, M-mode data was obtained for less than 25% of the fetuses screened, and thus hypertrophy is underrepresented in the screen. Nevertheless, 23% of fetuses identified with heart defects exhibited hypertrophy (Table 1). This was observed in 77 fetuses from 44 families. The most infrequently observed defect was ectopia cordis, a condition where the heart develops outside the chest cavity. This is readily observed by 2D imaging, and was found in only 4 fetuses (Table 1).
Approximately 50% of the fetuses with outflow regurgitation and 25% of fetuses with other abnormal ultrasound presentations were ultrasound scanned two or three times on different days (Table 1). In almost all instances, the rescanning confirmed the original ultrasound presentations, sometimes even showing progression of the fetus into heart failure or death. Overall, excluding families exhibiting arrhythmias only, 61 (23%) families had serious cardiovascular defects. All affected fetuses died prenatally or at birth, except for some fetuses with isolated increased outflow velocity. By contrast, fetuses diagnosed with hypertrophy as the predominant defect all survived to term, but then expired at birth.
To obtain more specific diagnosis of the cardiovascular defects, whenever possible stillborn or dead pups were retrieved for necropsy and histological analysis (Table 1). Such studies revealed a wide range of cardiovascular malformations, including persistent truncus arteriosus (PTA), transposition of the great arteries (TGA), double outlet right ventricle (DORV), Tetralogy of Fallot, pulmonary atresia, right-sided aortic arch, interrupted aortic arch (IAA), ventricular (VSD) and atrial (ASD) septal defects, common atrioventricular canal (AVC), pulmonary atresia, aortic stenosis, coronary artery defects, hypertrophy and hypoplastic left ventricle. Some of the mutant families exhibited cardiovascular defects in conjunction with craniofacial or skeletal anomalies, or limb defects. Below we present, by way of example, the detailed analyses of 5 families. To assist in the histological examination of the hearts from the mutant animals, we show in Fig. 2, histological sections of hearts from normal newborn mice.
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Connexin43 mutation causing conotruncal malformation and coronary anomalies
Family 193 showed outflow regurgitation together with an abnormal spectral
Doppler signal that suggested hypertensive pulmonary outflow. Necropsy of dead
pups revealed an unusual conotruncal malformation consisting of bulges or
pouches at the base of the heart (Fig.
7A). In addition, defects involving the coronary arteries were
evident. Most striking were coronary aneurysms found in the walls and at the
base of the pulmonary and aortic outflows
(Fig. 7B,C). In addition,
typically a large peritruncal coronary vein was seen at the base of the
outflows (Fig. 7B,C).
Histological analysis showed that the conotruncal malformation consisted of
sinusoidal trabeculations (Fig.
7E-G). In these regions and also in the RV chamber, there was
marked thinning of the compact layer. Large subepicardial coronary vessels
were observed, which were particularly abundant in the peritruncal region of
the heart (Fig. 7D-G). Some of
these gave rise to large sinuses. In some instances, the coronary artery was
observed to insert below the level of the valves
(Fig. 7I). In addition, the
semilunar valves were thickened (Fig.
7K), and a VSD can be seen
(Fig. 7J). Among this
constellation of phenotypes, the conotruncal malformation stands out as being
reminiscent of the connexin43 knockout mouse
(Reaume et al., 1995). Indeed,
sequencing analysis confirmed a mutation in connexin43, a G to A substitution
that generated a premature stop codon at amino acid position 45
(Fig. 7L; Table 3). The 44-amino acid
polypeptide generated by this mutant Gja1W45X allele is
expected to terminate after the first transmembrane domain of connexin43, and
thus would be incapable of forming a gap junction channel. It is interesting
to note that the phenotype in the Gja1W45X mutant is much
more severe than that of the connexin43 knockout mouse. Although the mutation
in this family was never formally mapped by genome scanning, the phenotype has
been shown to segregate exclusively with the Gja1W45X
mutation. Thus in the fifth generation of breeding, we have found the same
cardiovascular/pouch phenotype in 18 homozygote Gja1W45X
mutant animals.
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Discussion |
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In family 26, PTA was observed in conjunction with thymus hypoplasia,
micrognathia and other craniofacial defects phenotypes that are
reminiscent of DiGeorge and velocardiofacial syndromes. Previous knockout
mouse studies showed that Tbx1 and CRKL, two genes situated
in the syntenic region of chromosome 22, which is deleted in DiGeorge
patients, have cardiovascular and craniofacial defects similar to those in
DiGeorge patients (Lindsay et al.,
2001; Jerome and Papaioannou,
2001
; Merscher et al.,
2001
; Guris et al.,
2001
). In addition, antisense attenuation of UFD1L,
another gene in this region, has been associated with outflow septation
defects in chick embryos (Yamagishi et
al., 2003
). However, some DiGeorge patients have a 10p rather than
22q11 chromosomal deletion, suggesting there is at least one additional
DiGeroge locus (Monaco et al.,
1991
; Schuffenhauer et al.,
1998
; Van Esch et al.,
1999
). Yet other DiGeorge patients show no detectable chromosome
deletions (Lichtner et al.,
2002
). DNA sequencing analysis of many such patients showed most
have no detectable mutation in the Tbx1 coding sequence
(Gong et al., 2001
;
Conti et al., 2003
;
Yagi et al., 2003
). The
mutation in family 26 is situated on mouse chromosome 2, in a 5-Mb region
containing 124 known or predicted genes. Although mouse chromosome 2 contains
regions of synteny with human 10p, this interval appears largely syntenic to
human chromosome 15.
In family 166, VSDs were linked with forelimb anomalies that included
oligodactyly and syndactyly, and possibly other radial ray defects (indicated
by the abnormal flexure of the forearm). These phenotypes are reminiscent of
those in Holt-Oram syndrome, which has been linked with TBX5
mutations (Basson et al., 1997;
Li et al., 1997b
). We note
that studies using a Tbx5 knockout mouse model showed ASD and VSD,
and subtle defects of the front paw and wrist in some of the heterozygous
knockout animals (Bruneau et al.,
2001
). The mutation in family 166 was mapped to mouse chromosome
4, which does not contain Tbx5 nor Sall4, the two genes
linked with Holt-Oram or the closely related Okihiro syndromes, respectively
(Basson et al., 1997
;
Li et al., 1997b
;
Kohlhase et al., 2002
).
Clinical studies have shown that 30-70% of patients with Holt-Oram syndrome
have mutations in Tbx5 (Cross et
al., 2000
; Mori and Bruneau,
2004
), leaving open the possibility that the mutation in family
166 may have a relevance for Holt-Oram syndrome.
The mutation in family 182 exhibiting TGA was mapped to chromosome 4, which
has a strong candidate gene, Hspg2, encoding the heparan sulfate
proteoglycan perlecan. Previous studies showed perlecan knockout mouse
surviving past E10.5-E11 can exhibit TGA
(Costell et al., 1999; Hirasawa
et al., 1999; Costell et al.,
2002
). However, fine mapping studies have narrowed the interval to
a 5.7 Mb segment on chromosome 4 that does not include Hspg2. We note
that unlike the perlecan knockout mouse, mutants in family 182 exhibit heart
situs anomalies never seen in the perlecan knockout mouse
(Costell et al., 2002
). Besides
Hspg2, there are only two other genes known to be associated with
TGA, cryptic, a member of the EGF-CFC family of membrane receptors
(Gaio et al., 1999
), and
activin receptor type IIB (Oh and Li,
1997
). Neither of these genes are situated on chromosome 4,
suggesting that the mutation in family 182 may correspond to a novel gene not
previously known to be associated with TGA and the specification of
laterality.
Semaphorin 3C mutation
The PTA and IAA defects in family 53 were shown to arise from a point
mutation in Sema3C. The Sema3CL605P mutation
causes a non-conservative amino acid substitution in the immunoglobulin
domain, a protein region previously shown to be dispensable for biological
activity (Koppel et al.,
1997). Nevertheless, the high degree of sequence conservation in
this protein domain would suggest an essential function. As semaphorins are
known to function as dimers, perhaps this immunoglobulin domain may have a
role in semaphorin dimerization (Koppel
and Raper, 1998
). Consistent with this possibility, previous
studies showed that replacement of the immunoglobulin domain in Sema3A with
the Fc domain of IgG allowed the retention of semaphorin activity
(Eickholt et al., 1999
). If
indeed this mutation interferes with protein dimerization, homozygous mutant
animals would expected to be functional nulls, which would explain why they
have phenotypes similar to the Sema3C knockout animals. At the same time, it
would predict that Sema3C activity may be reduced in heterozygote animals
because of dominant interference with protein dimerization. The latter could
account for the finding of cardiovascular defects in heterozygous
Sema3CL605P mutants.
The semaphorin family of proteins, including Sema3C, provide guidance cues
for axon pathfinding, and also has been suggested to play an important role in
modulating neural crest cell migration
(Kolodkin et al., 1993;
Luo et al., 1993
;
Eickholt et al., 1999
;
Brown et al., 2001
) (for a
review, see Tamagnone and Comoglio,
2004
). Sema3C is found along the migratory paths of cardiac neural
crest cells, and cardiac neural crest cells express plexinA2, a co-receptor
mediating semaphorin signaling (Brown et
al., 2001
). As deployment of neural crest cells to the heart plays
an essential role in aortic arch remodeling and outflow tract septation
(Hutson and Kirby, 2003
), it
is significant that cardiac neural crest abundance appears to be reduced in
the Sema3C knockout mouse embryo
(Brown et al., 2001
). In
conjunction with the cardiac phenotype, we also observed pigmentation defects
in the Sema3CL605P mutant ectopic pigmentation in
the chest cavity in conjunction with skin hypopigmentation. As Sema3C
transcripts are expressed in the ectoderm
(Chilton and Guthrie, 2003
),
this suggests a previously unknown role for Sema3C in targeting
and/or maintaining crest-derived melanocyte precursors in the skin.
ENU-induced connexin43 mutation
Another ENU-induced mutation identified is the connexin43 allele
Cx43W45X. This mutation generated a premature stop codon at amino
acid 45, resulting in a hypothetical 44-amino acid polypeptide that spans the
first transmembrane domain, ending shortly after the first extracellular loop.
This N-terminal truncated connexin43 polypeptide cannot form a gap junction
channel, which probably requires all four transmembrane domains, as well as
the two extracellular loops that mediate docking of the gap junction
hemichannels (Foote et al.,
1998). Although this mutation might be predicted to be a
functional null, the cardiovascular phenotype is more severe than that of the
connexin43 knockout mouse. Thus the coronary aneurysms, VSD and semilunar
valve abnormalities have never been seen in the connexin43 knockout mouse.
This difference is unlikely to be caused by strain background effects, as we
have maintained the connexin43 knockout mouse line in a C57BL6/J background.
These findings suggest that the N-terminal polypeptide encoded by
Cx43W45X may exert dominant-negative effects, such as via
heteromeric interactions with other connexin polypeptides. The N-terminal
polypeptide also may interact with other membrane or cytoplasmic proteins, an
intriguing possibility given the evidence for protein-protein interactions
involving the C terminus of connexin43 and a variety of other proteins, such
as ZO1, ß-catenin, and others (Toyufuku et al., 1998;
Ai et al., 2000
;
Giepmans et al., 2001a
;
Giepmans et al., 2001b
). Our
previous studies have indicated that the conotruncal heart defects in the
connexin43 knockout mouse arises from perturbations in the migratory behavior
of two extracardiac cell populations, the cardiac neural crest and
proepicardially derived cells (Huang et
al., 1998a
; Huang et al.,
1998b
; Li et al.,
2002
). Using this new connexin43 mutant mouse model, we hope to
further elucidate connexin43 structure-function relationships and the
mechanism through which connexin43 regulates neural crest and proepicardial
cell motility.
Dominant and recessive inheritance
Our mutagenesis screen was designed to recover recessive mutations, with
the expectation that this would increase the sensitivity of the screen in
uncovering mutations causing severe congenital heart defects. This expectation
was in fact realized. However, mutations causing human congenital heart
disease are largely autosomal dominant. This disparity could partly reflect
the bias inherent in a recessive screen, or perhaps gene dosage regulation in
mouse is different than in human. We note that mice with homozygous
Tbx1 deficiency have DiGeorge-like outflow anomalies, but no outflow
anomalies were found in heterozygous mutants; these mice showed mostly mild
defects involving derivatives of the fourth pharyngeal arch (Jerone and
Papaioannou, 2001; Lindsay et al.,
2001; Merscher et al.,
2001
). However, it is also probably the case that recessive
mutations causing human congenital heart disease are difficult to recover, as
the incidence of pairing required to generate homozygote offspring in a
randomly breeding population is likely to be very low. In addition, as most
ENU-induced mouse mutations causing serious heart defects caused prenatal or
neonatal lethality, this might predict early miscarriages that would be missed
in clinical studies of live births (given a term mouse fetus is
developmentally equivalent to a 8-9 weeks gestation human embryo). Finally, we
note that in fact heterozygous offspring in a number of our ENU families have
cardiovascular defects. This was observed for the heart defects in family 166,
and the outflow septation and arch anomalies elicited by the
Sema3CL605P mutation in family 53. These observations
suggest that ENU-induced mutations will be invaluable in providing novel
insights into gene function, and in some cases, may provide mutant alleles
that may more closely model the genetics of human congenital heart
disease.
Future prospects for ENU mutagenesis
Our ongoing screen has recovered mutants with a variety of other
cardiovascular phenotypes. These new mouse models undoubtedly will help to
define developmental pathways that play an essential role in congenital heart
disease. We anticipate recovering not only novel alleles of known genes, but
also novel genes that play a role in a wide variety of congenital heart
defects. In contrast to analysis in human pedigrees, mouse models provide the
opportunity to examine phenotype-genotype correlations in well-defined genetic
backgrounds. This may make it possible to test for multigenic contributions,
as well as the role of gene-environment interactions in congenital heart
disease. Such studies may provide some insights into the often-observed
variable penetrance and genetic heterogeneity associated with human congenital
heart disease.
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ACKNOWLEDGMENTS |
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