1
Department of Neuroscience, University of Pennsylvania School of Medicine,
1115 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104, USA
2
Department of Medicine, Cardiovascular Division, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA
3
The Rockefeller University, New York, New York 10021, USA
*
Author for correspondence
(e-mail:raperj{at}mail.med.upen.edu
)
Accepted 31 May 2001
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SUMMARY |
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Key words: Semaphorin 3C, Mouse, Cardiac neural crest, Interrupted aortic arch, Persistent truncus arteriosus, PlexinA2, Chemoattractant, Sema3C
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INTRODUCTION |
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More recently a number of mutations have been engineered in mice that
produce similar cardiovascular defects (see Discussion). By analogy to the
avian model, these genes are likely to affect cardiac neural crest development
or function. In humans, outflow tract and other crest related anomalies are
present in patients with DiGeorge syndrome and are often associated with
deletions on chromosome 22q11 (Driscoll,
1994). Significant progress
has been made in producing mouse models of DiGeorge syndrome by deleting
homologous regions of mouse chromosome 16 (Lindsay,
1999
). More recent and ongoing
studies are identifying candidate genes within this region and characterizing
their functional roles in neural crest development (Epstein and Buck,
2000
; Merscher et al.,
2001
; Lindsay et al.,
2001
; Jerome and Papaioannou,
2001
; Guris et al.,
2001
).
The identification of essential genes is the first step in explaining how cardiac neural crest cells contribute to cardiovascular development. These genes will in turn define complex genetic and developmental pathways that control crest cell specification, migration, differentiation and survival. They will also identify signaling pathways by which crest cells interact with neighboring tissues within the pharyngeal arches and the outflow tract of the heart.
We present evidence that morphogenesis of the outflow tract and great
vessels is dependent upon semaphorin 3C (Sema3C), a signaling molecule
previously hypothesized to guide extending axons in the developing nervous
system. The semaphorins comprise a large family of phylogenetically conserved
secreted and transmembrane signaling proteins, some of which function as axon
guidance cues (Raper, 2000; Yu
and Kolodkin, 1999
). Class 3
semaphorins are secreted glycoproteins that contain an approximately 500 amino
acid N-terminal semaphorin domain, a C2 type immunoglobulin domain, and a
highly basic C-terminal tail. Four of them, including Sema3C, are
located on mouse chromosome 5 in a region syntenic to human chromosome
7q21-q31 (Tarantino et al.,
2000
). The first identified
vertebrate semaphorin, semaphorin 3A, is a secreted protein purified on the
basis of its ability to repel dorsal root ganglion axons in culture (Luo et
al., 1993
). semaphorin
3A knockout mice display a complex phenotype in which many different
tissues are affected (Behar et al.,
1996
; Taniguchi et al.,
1997
). Notably, the peripheral
nervous system of semaphorin 3A mutants is severely defasciculated
although most axons ultimately appear to connect with their appropriate
targets (Catalano et al.,
1998
). In knockout animals,
axons fail to avoid territories that would normally express semaphorin 3A
(Taniguchi et al., 1997
).
These mutant animals also experience postnatal hypertrophy of the right
ventricle (Behar et al., 1996
).
The cause of this hypertrophy has not yet been identified.
The functions of a related semaphorin, Sema3C, have been characterized in
vitro. Sema3C can act as either a repellent or an attractant for axons growing
in culture. For example, the growth cones of sympathetic neurons are repelled
by Sema3C (Koppel et al.,
1997), whereas growth cones of
rat cortical axons are attracted towards a source of Sema3C (Bagnard et al.,
1998
).
We have generated mice deficient in Sema3C. Mutants are cyanotic and die just after birth. Their death is attributable to aortic arch malformations and septation defects in the outflow tract of the heart. Our results suggest that Sema3C expression in the proximal cardiac outflow tract facilitates the entry of migrating neural crest cells that are essential for normal septation.
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MATERIALS AND METHODS |
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Gene targeting
The targeting vector was linearized with XhoI. Electroporation and
cell culture of E14 cells (Hooper et al.,
1987) were carried out as
previously described (Mombaerts et al.,
1996
). Genomic DNA from
G418-resistant ES cell colonies was digested with EcoRI and analyzed
by Southern blot hybridization with external probes 5' and 3' to
the targeting vector. Mice were backcrossed 4 generations into the CD1
background.
Cre recombination
The neo-selectable marker was removed from the targeted mutation by
crossing Sema3C heterozygotes (neoin) to CD1 mice
from a cre pedigree (bcre-23) that consisted of 6 kb of the 5'
flanking region of the Brn4/Pou3f4 promoter driving the expression of
the Cre recombinase gene (gift from K. Ahn and E. B. Crenshaw III).
PCR genotyping
Three to five mm of mouse tail was placed into 100 µl of buffer
containing 0.5 mg/ml proteinase K, 50 mM KCl, 10 mM Tris/HCl pH 8.3, 2 mM
MgCl2, 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20. After 2-10
hours digestion at 55°C, samples were heat inactivated for 10 minutes at
95°C. 1 µl of this sample was added to a standard 15 µl PCR reaction
with the following primer sets to amplify the wild-type and mutant alleles
respectively in Sema3C(neoin) mice;
5'-ttcccagtggggtagaacctagagc-3',
5'-gctatcaggacatagcgttggctac-3', and
5'-gagtctgtttctacagaaatgcatgggtt-3',
5'-ttcccagtggggtagaacctagagc-3'. In
Sema3C(neoout) mice the following primer sets
were used to amplify the wild-type and mutant alleles respectively
5'-ttcccagtggggtagaacctagagc-3',
5'-gcgattaccgttgatgttgagtggc-3', and
5'-gagtctgtttctacagaaatgcatgggtt-3',
5'-ttcccagtggggtagaacctagagc-3'. The conditions for the
thermocycler were: 94°C for 1 minute, 29 seconds followed by 94°C for
31 seconds/60.9°C for 1 minute 10 seconds/72°C for 1 minute 30 seconds
(35 times), 72°C for 5 minutes.
RT-PCR
RNA was isolated from E12.5 wild-type and Sema3C mutant embryos by
CsCl2 gradient centrifugation (Chirgwin et al.,
1979). The Superscript
Preamplification System Kit (GibcoBRL) was used to generate first strand cDNA
using 2 µg of total RNA from each mouse sample. PCR was performed using one
of 3 primer sets. Primer set #1 5'-aaatggctggcaaaggatcct-3',
5'-cggtccacagcaatctttgt-3', primer set #3
5'-cggggcaccgtgcaaaggtc-3',
5'-cagtggggtagaacctagagc-3', primer set #2
5'-ctgactgcgagctgatgattt-3',
5'-attggggctagtatagaca-3'. 30 cycles of PCR (same conditions as
above) were performed and samples were run on a 1.5% agarose gel.
Corrosion casting
To examine the structure of the vascular system, corrosion casting
(Polysciences # 07349) was performed. Briefly, P0 wild-type and
Sema3C mutant mice were anesthetized with ketamine and perfused with
phosphate-buffered saline through the left ventricle after a small hole was
introduced into the right atrium. A 1 ml tuberculin syringe was used to inject
polymer into the left ventricle under gentle pressure. After perfusion with
the polymer, the resin was left overnight to harden. After the mass had fully
cured, the tissue surrounding the cast was corroded away by using Maceration
Solution (Polysciences) at 50°C.
In situ hybridization
Digoxigenin-labeled antisense and sense riboprobes were synthesized by in
vitro run-off transcription of linearized plasmids, using SP6, T7 or T3 RNA
polymerase. Sense control probes gave no signal. Whole-mount in situ
hybridization was performed as described by Borycki et al. (Borycki et al.,
1999). Radioactive in situ
hybridization was performed as described previously (Wawersik and Epstein,
2000
).
Histology
Embryos and newborn mice were fixed in 4% paraformaldehyde in PBS for 24-48
hours. The skin was removed from newborn mice before fixation. After
dehydration and embedding in paraffin wax, 8 µm sections were placed on
glass slides. After dewaxing in xylenes and rehydration, sections were either
stained with Hematoxylin and Eosin, or processed for in situ
hybridization.
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RESULTS |
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A diagram of the targeting vector we used to mutate Sema3C is
shown in Fig. 1A. The majority
of the CCLARDPYCAWD-containing exon is replaced by a cassette that includes an
IRES followed by a Tau-lacZ fusion protein and a neomycin resistance
cassette flanked by LoxP sites (Mombaerts et al.,
1996). This targeting vector
was electroporated into E14 ES cells (Hooper et al.,
1987
). Ten of 96 clones that
developed resistance to G418 had undergone homologous recombination. Three of
these ES cell lines were expanded and injected into blastocysts to derive
chimeric mice. Chimeras from all 3 lines were capable of germline transmission
of the mutation. The targeting of ES cells and germline transmission were
confirmed by Southern blot hybridization
(Fig. 1B). The neomycin
resistance cassette was removed by crossing mice carrying the mutant
Sema3C locus to mice that express Cre recombinase in the germ cell
lineage (gift from K. Ahn and E. B. Crenshaw III). Proper recombination was
verified by PCR with a primer set flanking the excised cassette (data not
shown).
|
Sema3C mRNA is reduced and appropriately altered in
Sema3C mutant mice
Owing to the absence of specific antibodies for the mouse semaphorins, we
analyzed Sema3C mRNA in Sema3C mutant mice and their
wild-type littermates to determine the effectiveness of the Sema3C
mutation. Three primer sets were used in RT-PCR reactions to detect different
regions of the Sema3C mRNA (Fig.
1D). Primer set 1 (blue) amplifies a region of the mRNA 5'
of the targeted region, primer set 2 (red) a region 3' of the targeted
region, and primer set 3 (green) a region internal to the targeted region. All
three primer sets amplify appropriately sized PCR products in wild-type
animals (Fig. 1E). In
Sema3C mutant mice, only the primer sets to either side of the
deleted exon are capable of detecting Sema3C mRNA. Thus, while
Sema3C mRNA is produced in Sema3C mutant animals, this
message does not contain the exon modified by the targeting construct
(Fig. 1E, green lanes).
Sema3C mRNA is barely detectable by in situ hybridization in E12.5 mutant embryos (data not shown). Thus, the mutant form of Sema3C mRNA lacks a critical functional domain and is only expressed at a fraction of the wild-type level. The targeted Sema3C allele contains an IRES followed by a sequence encoding a Tau-lacZ fusion protein. The purpose of this addition is to harness the endogenous Sema3C promoter to drive the expression of tau-lacZ in cells that normally express Sema3C. Perhaps because of the low levels of Sema3C mRNA in mutant mice, no ß-galactosidase is detectable by X-gal histochemistry or immunocytochemistry in tissues that normally express Sema3C mRNA (data not shown).
Sema3C mutant mice die from persistent truncus arteriosus
and interruption of the aortic arch
Mice heterozygous for the Sema3C mutation are grossly
indistinguishable from their wild-type littermates. Some progeny of
heterozygous mice die within the first 24 hours after birth. These sick
neonates are cyanotic and when genotyped are found to be homozygous for the
Sema3C mutation. Interestingly, the penetrance of this phenotype is
highly dependent on the strain of mouse. Postnatal mortality of
Sema3C mutant mice is lower than 50% on both the 129 and C57BL/6
backgrounds. As the mutation was bred onto the CD1 background, however, the
penetrance increased with each successive backcross. Crossing heterozygotes
from the fourth backcross generation yields progeny in which the mortality
rate for Sema3C homozygote mutants is 96% (22 of the 23 mutants in
the first 8 litters examined). The powerful effect of strain upon the
penetrance of this phenotype may be useful for identifying modifier genes in
the future.
Analysis of 8 CD1 litters at birth demonstrates that wild-type, heterozygous and homozygous animals are present in the appropriate Mendelian ratios expected from a heterozygous cross (Table 1). There are occasional Sema3C mutant mice that do not die shortly after birth (e.g. 1 of 23 mutants from these 8 CD1 litters). These animals are viable, grossly indistinguishable from wild-type or heterozygous littermates, and fertile. The peripheral nervous system of mutant animals was examined for abnormalities. The cranial nerves of E10.5 and E11.5 Sema3C mutant mice were visualized with antibodies that stain neurofilaments and the sympathetic nervous system was examined with antibodies to tyrosine hydroxylase. No misprojections were detected, suggesting that Sema3C by itself is not required for grossly normal peripheral axon pathfinding. A detailed examination of the CNS of Sema3C mutant mice is now in progress.
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Pathological evaluation of homozygous mice that die shortly after birth reveals cardiac outflow tract and aortic arch abnormalities. The aortic arch is interrupted in 100% of mice that die postnatally. The interruptions occur either between the left common carotid and left subclavian arteries (type B interruption; Fig. 2B) or between the brachiocephalic and left common carotid arteries (type C interruption; Fig. 2C). Type B interruption of the aortic arch could result from inappropriate regression of the left fourth branchial arch artery. Duplication of the left common carotid is infrequently observed (Fig. 2D).
|
Sema3C mutant mice frequently exhibit defects in septation of the conotruncus known as persistent truncus arteriosus (PTA). Injection of methyl methacrylate resin into the left ventricle is a sensitive method for detecting communication between the aorta and the pulmonary artery (Fig. 3A,C). Seventy-five percent of P0 Sema3C mutant mice evaluated by this technique have incomplete septation of the conotruncus (Table 1). In animals with PTA, the common valve of the outflow tract has four cusps (Fig. 3C inset). A ventriculoseptal defect is present in these animals just below the common valve. Histological analysis of the heart of a Sema3C mutant mouse with PTA (Fig. 3D) identifies a defect in the membranous portion of the ventricular septum with an intact muscular septum (Fig. 3E). The histology of the ventricular wall (Fig. 3F) and the structure of the mitral and tricuspid valves appear normal in Sema3C mutants (data not shown).
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In utero, blood is oxygenated in the placenta rather than the lungs and is diverted from the pulmonary circulation into the descending aorta through a shunt called the ductus arteriosus. The pulmonary and systemic circulations are separated when this shunt closes just after birth. The systemic circulation is maintained in a Sema3C mutant embryo before birth because blood can bypass the interruption of the aortic arch by flowing from the heart into the descending aorta through the ductus arteriosis. After birth, however, blood cannot enter the descending aorta once the ductus arteriosus closes. Sema3C mutant mice therefore die from cardiovascular defects that are incompatible with postnatal life.
Sema3C is expressed in the developing outflow tract
To better understand the etiology of this phenotype, we studied the
expression of Sema3C in the cardiac outflow tract during development.
Sema3C mRNA is expressed in the conotruncus as early as E10.5 and
persists through E12.5 (Fig.
4A-C). Thus, expression of Sema3C is contemporaneous with
the migration of cardiac neural crest cells into the outflow tract (Waldo et
al., 1998). More detailed
analysis of the expression pattern in sections at E10.5 demonstrates that
Sema3C is expressed in the mesenchyme surrounding the branchial arch
arteries (Fig. 4D). At this
stage, Sema3C is restricted to the left side of the conotruncus and
appears to be expressed in the myocardial cuff.
|
By E12.5, expression of Sema3C is restricted to the proximal outflow tract and the great vessels (Fig. 4E). Strong expression is also seen in the mesenchyme of the aorticopulmonary septation complex where neural crest cells form the developing septum (Fig. 4F). Sema3C is expressed in cells adjacent to neural crest migration pathways, suggesting a non cell-autonomous function. It is also expressed within the aorticopulmonary septation complex, possibly even within the neural crest itself. It is not uncommon to observe an axon guidance molecule to be expressed in neurons that are responsive to that cue. One dramatic example is the modulation of ephrin responsiveness by ephrin expression in retinal ganglion cells (Hornberger et al., 1999). It is possible that cardiac neural crest cells may both express and respond to Sema3C.
Abnormal migration of cardiac crest cells in Sema3C mutant
embryos
Sema3C has been shown to influence growth cone motility and guidance in
vitro. Cardiac neural crest cells are known to be required for proper
formation and septation of the developing cardiac outflow tract. We
hypothesized that Sema3C acts as a guidance cue for migrating cardiac crest
cells, that its loss prevents crest cells from populating the aortic arch or
the proximal outflow tract, and that the resulting absence of crest cells in
these structures is sufficient to induce the cardiac abnormalities in
Sema3C mutant mice.
We compared the migration of cardiac crest cells into the proximal cardiac
outflow tract in wild-type and mutant embryos. Cardiac crest cells were
visualized with a series of molecular markers including plexinA2. This
transmembrane molecule is a member of a family of receptors that is thought to
help mediate semaphorin signaling (Takahashi et al.,
1999; Tamagnone et al.,
1999
). In an accompanying
paper, transgenic and fate-mapping techniques demonstrate that plexinA2 is
expressed by neural crest cells that surround the aortic arches and migrate
into the cardiac outflow tract (Brown et al.,
2001
). The complementary
expression patterns of Sema3C and plexinA2 raise the possibility that plexinA2
is a receptor component for Sema3C. PlexinA2 expression
(Fig. 5E) is colocalized with
other cardiac crest cell markers, including Foxc1
(Fig. 5A) and endothelin
receptor A (EdnrA; Fig.
5C), in the developing heart of wild-type embryos. Crest cells in
wild-type embryos are seen to encase the branchial arches by E10.5 (not shown)
and they populate the endocardial cushions of the outflow tract by E12.5
Fig. 5A,C,E). Patterning of
neural crest cells in the outflow tract of the heart is altered in
Sema3C mutant littermates. Altered expression of Foxc1
(Fig. 5B), endothelin receptor
A (Fig. 5D), and plexinA2
(Fig. 5F) suggests that neural
crest cell invasion of the outflow tract is significantly impaired in mutant
embryos.
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The branchial arch arteries form normally in Sema3C mutant
embryos
The most dramatic and fully penetrant cardiac phenotype observed in P0
Sema3C mutant pups is interruption of the aortic arch. This anomaly
could arise either from a failure in formation of the fourth left branchial
arch artery, or from inappropriate regression of this artery segment during
the complex process that reconfigures the symmetric embryonic vasculature into
the asymmetric adult pattern. This remodeling process is known to require the
cardiac neural crest, although the mechanisms by which specific right or left
sided segments of branchial arch arteries are maintained or lost remain
obscure. Branchial arch remodeling takes place in the mouse between embryonic
days 10.5 and 13.5. Examination of sections of E10.5 and E11.5 Sema3C
mutant embryos demonstrates that the branchial arch arteries are present and
normal morphologically (Fig.
6A-H). The anomalies seen later must therefore arise from a
failure of the arch arteries to remodel correctly.
|
Consistent with our observation of impaired neural crest migration into the
cardiac outflow tract at E12.5, we first detected structural cardiovascular
abnormalities at E13.5. Histologic analysis of an E13.5 Sema3C mutant
embryo revealed a ventricular septal defect (VSD;
Fig. 6J) not seen in wild-type
litter mates (Fig. 6I). In the
mutant embryo, the aorta was seen to arise from the right ventricle
(Fig. 6K) resulting in a double
outlet right venticle. This malformation is seen after neural crest ablation
in chick embryos (Kirby et al.,
1983).
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DISCUSSION |
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Previous work from other laboratories has demonstrated that both remodeling
of the branchial arch arteries and septation of the outflow tract require a
subset of neural crest cells known as the cardiac crest. Transplantation of
quail neural crest cells into developing chicks has shown that cardiac neural
crest cells contribute to the tunica media of the aortic arch and to the
septum of the outflow tract (Waldo et al.,
1998). When the cardiac neural
crest is ablated in the chick, the resulting malformations are variable but
commonly include persistent truncus arteriosus and absence of various
combinations of branchial arch arteries, including those derived from
pharyngeal arches 3 and 4 (interrupted aortic arch) and pharyngeal arch 6
(absent ductus arteriosus; Kirby et al.,
1983
; Creazzo et al.,
1998
).
The similarity between the cardiovascular phenotypes in Sema3C mutant mice and crest ablated chicks suggests that the mouse phenotype reflects a requirement for Sema3C in some important aspect of cardiac neural crest cell development. Two additional observations are consistent with this hypothesis. First, branchial arch arteries form normally in Sema3C mutants. Subsequent interruption of the aortic arch must therefore be attributable to a failure of the branchial arches to reorganize correctly, a process known to require the cardiac neural crest. Second, incomplete septation of the outflow tract is evident very early in Sema3C mutant embryos and can be attributed to an impairment in crest cell migration into the proximal portion of the tract.
Several engineered and spontaneous mutant mouse lines have been described
with interruption of the aortic arch or improper septation of the conotruncus.
The cardiac defects in these lines have been ascribed to abnormalities in
cardiac neural crest development. In most of these mutant mice, either
septation of the conotruncus or remodeling of the branchial arch arteries is
separately disturbed. Pax3, retinoic acid receptors, and connexin 43 are all
expressed in cardiac neural crest cells (Franz,
1989; Mendelsohn et al.,
1994
; Reaume et al.,
1995
). Disruption of these
genes leads to defects in septation of the conotruncus. It has been
hypothesized that the cardiac defects arise in these knockout mice because the
specification of, or communication between, cardiac neural crest cells is
perturbed. Interruption of the aortic arch is not observed in mice with these
mutations.
Mutations in the endothelin pathway result in a different set of
cardiovascular anomalies. Disruption of this intercellular signaling pathway,
which mediates communication between the arterial endothelium and the
surrounding mesenchyme, leads to interruption of the aortic arch and abnormal
persistence of some vessels that normally regress. This has been demonstrated
by targeted disruption of endothelin-1, endothelin converting enzyme-1, or the
endothelin receptor A (Clouthier et al.,
1998; Kurihara et al.,
1995
; Yanagisawa et al.,
1998
). Mice null for
HoxA3 or the winged helix transcription factors, Foxc1
(Mf-1) or Foxc2 (Mfh-1), also die as the result of
aortic arch anomalies (Winnier et al.,
1999
; Chisaka and Cappechi,
1991
). Although PTA is observed
in 6% of EdnrA mutant mice and 10% of endothelin converting enzyme-1
mutant mice, septation of the conotruncus usually occurs normally in these
knockout lines.
In contrast to these other mouse mutants, disruption of the Sema3C
locus causes both aortic arch interruption and persistent truncus arteriosus
in the majority of mutant mice (Table 1B). One interpretation of this result
is that Sema3C might function upstream of these other genes in a pathway
regulating cardiac neural crest development. For example, loss of Sema3C
function could affect crest cell specification, block cardiac crest cell
migration, or reduce the expression levels of other genes known to be
important in crest cell directed remodeling of the cardiac outflow tract. We
have not exhaustively assayed the expression levels of genes that are
expressed in cardiac crest or those known to affect remodeling of the outflow
tract, but our data do show that three of them, Foxc1, the endothelin
A receptor, and plexinA2 (see Brown et al.,
2001) are all expressed in
Sema3C mutant mice in the regions of the branchial arches normally
populated by neural crest cells. These observations argue that cardiac crest
cells are properly specified and begin to migrate normally in Sema3C
mutant mice.
Neural crest cell migration into the proximal cardiac outflow tract,
however, appears to be impaired. Since Sema3C is normally expressed
in the myocardial cuff of the outflow tract, a plausible and attractive
interpretation of these results is that Sema3C acts as an attractant for
cardiac crest cells and helps to promote their entry into the outflow tract.
This proposed function would be analogous to its chemoattractant activity for
cultured cortical axons (Bagnard et al.,
1998). Alternatively, Sema3C
could promote entry indirectly, perhaps by inducing the expression of a
permissive or attractive signal in nearby tissues. While our results support
the hypothesis that Sema3C promotes the migration of cardiac crest
cells into the proximal outflow tract, we cannot eliminate the possibilities
that Sema3C affects cardiac crest cell proliferation, differentiation or
survival within the proximal tract.
Cardiac defects including persistent truncus arteriosus and interruption of
the aortic arch have been described in neuropilin-1 knockout mice
(Kawasaki et al., 1999). These
mice die between E12.5 and E13.5 and have multiple defects in several organ
systems. Neuropilin-1 is capable of acting as a coreceptor for the angiogenic
signals vascular endothelial growth factor (VEGF) and placenta growth factor 2
(PIGF) (Migdal et al., 1998
;
Soker et al., 1998
).
Cardiovascular defects present in neuropilin-1-/- embryos
have been interpreted to result from impaired angiogenic signaling. However,
neuropilin-1 can also serve as an obligatory coreceptor for a subset of class
3 semaphorins including semaphorin 3A and semaphorin 3C (He and
Tessier-Lavigne, 1997
;
Kolodkin et al., 1997
).
Defects in axon pathfinding in neuropilin-1-/- embryos can
be ascribed to a loss of sensitivity to these guidance cues. The striking
similarity between cardiac defects observed in the neuropilin-1
knockout and in the Sema3C mutant mice is consistent with the
hypothesis that neuropilin-1 functions as a receptor component for Sema3C in
cardiac neural crest. Our results strongly argue that the cardiac defects
observed in neuropilin-1-deficient embryos are due to the inability
of cardiac neural crest cells to respond to Sema3C.
The cardiovascular defects resulting from the disruption of the
Sema3C locus closely resemble heart defects seen in human infants
with congenital heart disease. Some of these arise as a consequence of
microdeletions within chromosome 22q11 (Driscoll,
1994), however, a majority of
infants with outflow tract and aortic arch defects do not have this particular
chromosomal abnormality (Goldmuntz et al.,
1998
). Semaphorins, their
receptors, and the signaling pathways they influence now constitute a new
class of candidate genes for human congenital cardiac disease.
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ACKNOWLEDGMENTS |
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