1
Cardiovascular Division, Department of Medicine, University of Pennsylvania,
Philadelphia, PA 19104, USA
2
Department of Neurobiology, University of Pennsylvania, Philadelphia, PA
19104, USA
*
Author for correspondence (e-mail:
epsteinj{at}mail.med.upenn.edu
)
Accepted 30 March 2001
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SUMMARY |
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Key words: Neural crest, Heart, Semaphorin, Plexin, Mouse
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INTRODUCTION |
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Cardiac neural crest also is likely to mediate, through unknown mechanisms, the complex pattern of regression and persistence of right and left sided aortic arch segments that allows for remodeling of the great vessels and the production of the adult asymmetric vasculature. Little is known about how cardiac neural crest cells mediate pathfinding and are constrained to appropriate migratory pathways. Surprisingly, few molecular markers identify post-migratory crest cells in the heart providing few candidates for mediating these cellular functions.
In mammals, a critical role for neural crest during cardiac development has
been implicated by analogy to avian models (Waldo et al.,
1999) and by the description
of several spontaneous or engineered mutations in genes expressed by neural
crest cells that give rise to congenital heart disorders affecting the outflow
tract and great vessels. These genes are generally expressed by premigratory
or migrating cardiac neural crest, or in tissues adjacent to migration
pathways. Examples include Pax3 (Epstein,
1996
), Endothelin receptor A
(Ednra; Yanagisawa et al.,
1998
), Neurofibromatosis type
1 (Nf1; Brannan et al.,
1994
; Jacks et al.,
1994
), Connexin 43
(Cx43; also called Gjal; Lo et al.,
1999
), Foxc1 and
Foxc2 (formerly Mf1 and Mfh1; Iida et al.,
1997
; Winnier et al.,
1999
). However, mutations in
several of these genes affect multiple aspects of cardiovascular development
and specific expression by postmigratory neural crest cells in the heart has
not been clearly documented for any of these genes. The roles that they play
in neural crest migration, differentiation and survival remain unclear. In
fact, despite the description of these mutant phenotypes, the presence of
neural crest cells in the mammalian heart remained undocumented until very
recently (Jiang et al., 2000
;
Li et al., 2000
; Waldo et al.,
1999
).
The first evidence that neural crest cells populate the mammalian heart, in
a pattern similar to that seen in avians, came from observations of
ß-galactosidase expression in transgenic mice expressing lacZ
from a 6.5 kb proximal upstream region of the Cx43 gene (Lo et al.,
1997). Expression was noted in
the dorsal neural tube and neural crest derivatives including dorsal root
ganglia. Labeled cells were identified in the 3rd, 4th
and 6th pharyngeal arches and in the outflow tract of the heart.
The pattern of expression in the heart was remarkably similar to the
distribution of neural crest cells in chick embryos suggesting, by analogy,
that this transgene labels mammalian cardiac neural crest cells. Of note,
Cx43 itself is expressed widely throughout the developing heart and
is not restricted to neural crest, indicating that important regulatory
elements mediating Cx43 expression lie outside the 6.5 kb proximal
upstream region used to create this transgene (Waldo et al.,
1999
).
More recently, several groups have performed fate-mapping experiments in
the mouse using tissue-specific transgenes and Cre-lox technology. Promoter
elements from the P0, Wnt1 and Pax3 (Jiang et al.,
2000; Li et al.,
2000
; Yamauchi et al.,
1999
) genes that direct
expression to pre-migratory neural crest cells were used to direct expression
of Cre recombinase. These transgenic mice were crossed to Cre-reporter mice in
which expression of ß-galactosidase occurs only in cells that express Cre
(Soriano, 1999
; Tsien et al.,
1996
). Since the activation of
lacZ expression involves a somatic cell genomic rearrangement, cells
that express the Cre transgene are indelibly labeled for their lifetime, as
are all descendants of those cells. These studies have identified P0, Wnt1 and
Pax3 derivatives in the developing heart with apparently similar, but not
identical, patterns of expression. These patterns are generally similar to
neural crest patterning in avian hearts, suggesting that most or all labeled
cells are neural crest in origin. However, it should be emphasized that P0,
Wnt1 and Pax3 themselves are not expressed by neural crest cells in the heart,
and the use of the Cre-lox labeling method to track neural crest cells in
various mutant backgrounds is cumbersome and expensive. Nevertheless, these
studies confirm that in mammals, as in birds, cardiac neural crest cells
populate the outflow tract of the heart suggesting that cell-type-specific
expression of genetic programs exist that mediate neural crest function during
cardiac morphogenesis.
We demonstrate that PlexinA2 is expressed by migrating and postmigratory
cardiac neural crest cells in the mouse heart. PlexinA2 (Kameyama et al.,
1996) is a member of a large
family of receptors that recognize secreted semaphorin signaling molecules
involved in axon guidance and growth cone collapse in the central nervous
system (Tamagnone et al.,
1999
). We confirm the
specificity of PlexinA2 as a cardiac neural crest marker by comparison with
available transgenic lines, and we compare and contrast the available labeling
techniques for cardiac neural crest. In light of recent data that semaphorin
3C (Sema3C) is required for normal development of the aortic arches
and for septation of the cardiac outflow tract (see Feiner et al.,
2001
), we have examined the
expression of PlexinA2 in relation to Sema3C, and in
Sema3C-deficient embryos and in other mouse models with cardiac
neural crest defects (Feiner et al.,
2001
). We document that
PlexinA2-expressing cardiac neural crest is patterned abnormally in
these mutant embryos. These data suggest a model in which PlexinA2 plays a
functional role in cardiac neural crest migration by acting as a co-receptor
for class 3 semaphorin signaling molecules.
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MATERIALS AND METHODS |
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For in situ hybridization on paraffin sections, embryos were fixed as above, dehydrated in a graded ethanol series, paraffin embedded and sectioned at 10 µm. Concentrations for digoxigenin riboprobes were determined empirically by serial dilution in control experiments before use. Sections were counterstained with nuclear fast red and mounted in Vecta Mount (Vector laboratories, Burlingame, CA).
For radioactive in situ hybridizations, 35S-labeled sense and
antisense riboprobes were synthesized with SP6, T7 or T3 RNA polymerase and
35S-UTP as previously described (Lutz et al.,
1994). Hybridization was
carried out at 55°C overnight. Successful hybridization was assessed by
overnight exposure of slides to Kodak X-OMAT film. Slides were dipped in Kodak
NTB-2 emulsion, exposed for 5-7 days a 4°C, developed and fixed in Kodak
Dektol developer and fixer. Cell nuclei were counterstained with Hoechst 33258
(Sigma, St. Louis, MO) and mounted in Canada balsam/methyl salicylate.
Sections were digitally photographed on a Zeiss Axioplan 2 microscope. Images
were processed with Adobe Photoshop.
Transgenic and mutant mice
Splotch mice were obtained from the Jackson Labs and maintained on
a C57/B16 inbred background. Cx43-lacZ transgenic mice were obtained
from Dr Cecilia Lo (Lo et al.,
1997). Cx43-lacZ mice
are a stable transgenic line expressing a nuclear ß-galactosidase from a
6.5 kb Cx43 promoter in neural crest cells. P3pro-Cre mice were
generated by our laboratory (Li et al.,
2000
). P3pro-Cre
consists of a 1.6 kb proximal Pax3 promoter upstream of Cre recombinase.
Wnt1-Cre mice were obtained from Dr Andrew P. McMahon (Danielian et
al., 1998
). The Rosa reporter
mice, R26R, were obtained from Philippe Soriano (Soriano,
1999
).
Sema3C+/- mice were maintained on a CD1 background (Feiner
et al., 2001
).
X-gal staining
Embryos expressing ß-galactosidase transgenes were harvested into cold
PBS, and fixed for 2 hours in 2% PFA. To optimize tissue fixation and
penetrance of X-gal substrate (Roche Molecular, Indianapolis, IN), the chest
wall was opened before fixation and in some cases the heart was removed and
incubated in color substrate. Embryos were incubated in X-gal substrate (5 mM
K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM
MgCl2, 0.01% NP-40, 0.1%deoxycholate, 0.1% X-gal in PBS) at
37°C. For high resolution analysis of ß-galactosidase expression
patterns, embryos were paraffin embedded, sectioned and counterstained with
nuclear Fast Red.
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RESULTS |
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We investigated the expression of potential Sema3C receptor components
during mid gestation when cardiac neural crest migrates to and populates the
heart. Class 3 semaphorin receptors are composed of heteromultimers including
neuropilin and class A plexin subunits (Tamagnone and Comoglio,
2000).
Fig. 1 shows the expression of
candidate receptor subunits by in situ hybridization at E12.5 in the neural
tube, the aortic arches and the outflow tract of the heart. PlexinA2
(Fig. 1A-C) is expressed in the
dorsal neural tube (arrow, A) and along the aortic arches (B) with a
continuous stream of expressing cells extending into the outflow tract of the
heart (arrow, C). This pattern suggests expression by cardiac neural crest.
PlexinA1 (Fig. 1D-F)
is expressed at low levels or not at all at E12.5. PlexinA3
(Fig. 1G-I) is expressed in the
ventricular zone of the neural tube (G) and diffusely throughout the
pharyngeal arch mesenchyme (H) and adjacent to the aortic and pulmonary trunks
(I). Neuropilin 1 (Nrp1,
Fig. 1J-L) is expressed in the
ventral neural tube (J) and in the endothelium and surrounding mesenchyme of
the aortic arches (K,L) while neuropilin 2 (Nrp2,
Fig. 1M-O) is expressed in the
dorsal and ventral neural tube (excluding the ventricular zone, M) and in the
mesenchyme surrounding the ascending aorta (N,O). In summary,
PlexinA2 displayed a pattern of expression most consistent with
cardiac neural crest, while PlexinA3, Nrp1, and Nrp2 had
partially overlapping expression patterns consistent with possible functions
in cardiac neural crest and/or in adjacent tissues.
|
In light of these results, we focused our attention on PlexinA2
during cardiovascular development. Fig.
2A shows a whole-mount in situ hybridization of an E11.5 embryo
revealing two prongs of PlexinA2-expressing cells within the outflow
tract of the heart (arrows, Fig.
2A). In avians, two prongs of neural crest cells invade the
truncus arteriosus in a similar pattern (Waldo et al.,
1998). By whole-mount in situ
hybridization, this pattern of expression is distinct from that seen with
Sema3C (Fig. 2B) which
is expressed by the myocardium of the outflow tract adjacent to the outflow
endocardial cushions (see below). Since Sema3C encodes a secreted
ligand, and Sema3C mutant embryos have cardiovascular abnormalities
suggestive of neural crest-related defects, this complementary pattern of
expression further suggested that PlexinA2 might serve as a receptor component
on neural crest cells.
|
PlexinA2 is expressed by cardiac neural crest cells
The pattern of cardiac gene expression shown in
Fig. 2A has, to our knowledge,
not been previously documented in mammals. While the pattern is reminiscent of
neural crest population of the heart in birds, no other specific molecular
markers for postmigratory cardiac neural crest have been available in mice.
Pax3, CRABP and others have been suggested previously to label these
cells (Conway et al., 1997b),
but our studies have found these markers to be extinguished prior to neural
crest invasion of the cardiac region, or to be expressed by other cells types
(Epstein et al., 2000
).
Therefore, we sought to compare the expression of PlexinA2 in the
heart using recently developed transgenic mice that allow for labeling of
cardiac neural crest by fate mapping techniques.
Fig. 3 shows a comparison of
in situ hybridization for PlexinA2 at E12.5 (A,E,I) with X-gal
stained E12.5 hearts revealing Pax3-expressing neural crest derivatives
(B,F,J), Wnt1-expressing derivatives (C,G,K), and cells expressing the 6.5 kb
Cx43-promoter-lacZ transgene (D,H,L). Fate mapping of Pax3-expressing
neural crest precursors relies on the ability of a neural crest-specific
element in the Pax3 promoter to direct expression of Cre recombinase in
premigratory neural crest cells (Li et al.,
2000). Crossing these
transgenic mice to Cre-reporter mice results in ß-galactosidase
expression by neural crest derivatives. A similar approach is used to fate map
Wnt1-expressing neural crest percursors (Jiang et al.,
2000
). The use of
Cx43-lacZ transgenic mice relies upon the persistent expression of
this transgene in neural crest derivatives (Waldo et al.,
1999
). In all cases, streams
of labeled cells can be seen to invade the outflow tract and to reach the
level of the outflow endocardial cushions (large arrows,
Fig. 3A-D). No labeled cells
were seen using any of these techniques, within the atrioventricular
endocardial cushions (Fig. 3
and data not shown). However, variable and scattered labeling of cells within
the myocardium of either or both ventricles was seen in all cases (arrowheads,
Fig. 3A-D). Whether these cells
represent neural crest derivatives rather than ectopic expression of the
various transgenes remains to be determined, though neural crest derivatives
within the myocardium have been noted in other vertebrates (M. Kirby and J.
Yost, personal communication).
|
Examination of PlexinA2 expression reveals strikingly similar patterns of expression compared to transgenic labeling of neural crest in frontal (Fig. 3E-H) and cross (I-L) sections. In all cases, labeled cells were noted adjacent to the proximal pulmonary artery and aorta and formed a `saddle' within the mesenchymal portion of the outflow endocardial cushions (arrows, E-H). In cross section, the lower extremes of this `saddle' appeared as distinct clusters of labeled cells within the endocardial cushions (I-L). In slightly more rostral sections, these two clusters merged to form the septum between the aorta and pulmonary arteries.
PlexinA2 expression within the endocardial cushions
(Fig. 3I) most closely
resembled the pattern of Pax3 derivatives
(Fig. 3J), while Wnt1
derivatives appeared to occupy a larger portion of the cushion mesenchyme
(Fig. 3K). This is consistent
with earlier reports (Jiang et al.,
2000), although these
differences were at the time attributed to different sectioning techniques.
Whether this difference represents labeling of different populations of
cardiac neural crest by these various systems, or whether ectopic transgene
expression accounts for the broader Wnt1 expression domain remains to
be determined. In avians, fate mapping studies have yielded results more
consistent with the condensed populations of cardiac neural crest revealed by
PlexinA2 expression (Fig.
3I) and seen in the Pax3 fate-mapping studies
(Fig. 3J; Waldo et al.,
1998
). Together, these studies
confirm that PlexinA2 is expressed by postmigratory cardiac neural
crest.
Earlier expression of PlexinA2 is also consistent with a role for
it in cardiac neural crest migration and function. PlexinA2
expression was detected as early as E8.5 in the neural folds prior to closure
of the neural tube (Fig. 4A),
although expression was not localized to dorsal neural tube at this timepoint.
Shortly thereafter, by E9.5, intense expression was noted in the dorsal neural
tube at the level of the first three somites, the region from which cardiac
neural crest emerges (arrow, Fig.
4B). Expression in the roof plate persisted through E10.5 (arrow,
Fig. 4C) and broad signal was
detected in the mesenchyme of the branchial arches surrounding the aortic
arches (arrows, Fig. 4D and
data not shown). This is the region populated by migrating cardiac neural
crest. At E10.5, expression was first noted in the outflow tract of the heart
(arrow, Fig. 4E), consistent
with our recent identification of cardiac neural crest cells arriving in this
location at this stage of development (Epstein et al.,
2000). At E11.5,
PlexinA2 expression persists in tissue surrounding the forming aorta
and pulmonary trunks and in the outflow tract (arrow,
Fig. 4F). Expression is also
first noted in the myocardium of the left ventricle. These data suggest that
in addition to other expression domains, PlexinA2 is expressed by
pre- and postmigratory cardiac neural crest cells during mid gestation, when
septation of the outflow tract and repatterning of the aortic arches is taking
place.
|
PlexinA2 and Sema3C are expressed in distinct and
overlapping domains
Since Sema3C deficiency leads to cardiovascular abnormalities
attributable to neural crest defects (see Feiner et al.,
2001), and since plexins can
function as components of semaphorin receptors, we examined the relative
expression of Sema3C and PlexinA2 during cardiac
development. A careful analysis of PlexinA2 and Sema3C
expression in adjacent sections of mid gestation embryos reveals a striking
complementary pattern in many tissues, while expression overlaps in some
important areas. Most notably, both the secreted ligand Sema3C and
the putative receptor component PlexinA2 are expressed in the roof
plate of the neural tube (large arrows,
Fig. 5A,B,G,H). Both genes are
also expressed in the condensed mesenchyme of the cardiac outflow tract, a
region populated by neural crest (arrows,
Fig. 5B,H). However,
Sema3C is expressed somewhat more broadly in this region
(Fig. 5H-J) such that only a
subset of the Sema3C expression domain overlaps with that of
PlexinA2 in this tissue (Fig.
5I,J). Elsewhere in the neural tube, heart and great vessels the
pattern of expression is reciprocal. For instance, PlexinA2 is
expressed in the neural tube excluding the ventral horns
(Fig. 5A,B), while
Sema3C is restricted to the ventral horns
(Fig. 5G,H). PlexinA2
surrounds the ductus arteriosus (Fig.
5A) and dorsal aortae (Fig.
5A,B) while Sema3C is in the myocardium of the outflow
tract and in the proximal pulmonary trunk
(Fig. 5G). This reciprocal
pattern of expression is seen in other tissues including the intestine, where
PlexinA2 is present in smooth muscle
(Fig. 5C) while Sema3C
is expressed by intestinal epithelium (Fig.
5D). Similarly, in the lungs, PlexinA2 is expressed in
the bronchial smooth muscle (Fig.
5E) and Sema3C is in the underlying endothelium
(Fig. 5F). In summary,
PlexinA2 and Sema3C are both expressed in the roof plate of
the neural tube and in the condensed mesenchyme of the cardiac outflow
endocardial cushions. Expression is complementary and in opposing tissues in
the conotruncal region and in many other tissues including neural tube, lung
and intestine.
|
PlexinA2 expression in Sema3C-/-
embryos
Sema3C-/- embryos have conotruncal cardiac defects and
interruptions of the aortic arch suggestive of cardiac neural crest defects
(Feiner et al., 2001). We
determined the expression of PlexinA2 in wild-type and
Sema3C-/- littermates during the critical time points of
conotruncal septation. In mutant embryos at E12.5,
PlexinA2-expressing cells were seen migrating along the aortic arches
and populating the outflow tract of the heart
(Fig. 6B). This result
indicates that Sema3C is not required for neural crest migration to
the cardiac region. However, within the conus, PlexinA2 patterning
was different in Sema3C mutants compared with controls.
PlexinA2-expressing cells did not migrate as far distally within the
outflow mesenchyme in mutant embryos and did not form the localized clusters
of condensed mesenchyme (Fig.
6, see also Fig. 5
in Feiner et al., 2001
).
Expression within the endocardial cushions was reduced and more diffuse in
mutant embryos. It is worth noting that this area of abundant
PlexinA2 expression is directly adjacent to the normal
Sema3C expression domain in the myocardial cuff of the outflow tract
(Fig. 2B), a signal that is
missing in Sema3C-/- embryos. These results suggest that
Sema3C/PlexinA2 signaling may function to modulate final positioning of neural
crest cells within the outflow tract while other pathways affect more proximal
regions of cardiac neural crest migration.
|
PlexinA2 expression in Pax3-deficient
Splotch embryos
We also determined the location of PlexinA2-expressing neural
crest cells in another model with cardiac conotruncal defects. Pax3
is a transcription factor expressed by pre-migratory neural crest cells in the
dorsal neural tube. Mutations in the Pax3 locus result in the
Splotch mouse which exhibits failure of outflow tract septation and
other neural crest defects (Auerbach,
1954; Franz,
1989
). A poorly developed
myocardium with a thinned ventricular wall and ventricular septal defects are
also present. Pax3 is also expressed in limb muscle progenitors where
it functions to regulate hypaxial myoblast migration at least partially by
directly regulating the transcription of the c-Met gene encoding a
tyrosine kinase receptor that is required for myoblast migration (Bladt et
al., 1995
; Bober et al.,
1994
; Daston et al.,
1996
; Epstein,
1996
). c-Met is a
member of the Plexin family (Artigiani et al.,
1999
). Interestingly,
PlexinA2 expression was noted in the hypaxial musculature in our
studies (Fig. 7C), a conclusion
that was supported by the absence of PlexinA2 expression in these
domains that are absent in Splotch embryos
(Fig. 7F). These regions
included limb musculature (Fig.
7C,F) and the diaphragm (data not shown), tissues that are
deficient in Splotch. We sought to test whether Pax3 might modulate
PlexinA2 expression in neural crest cells in a manner analogous to
Pax3 modulation of c-Met in myoblasts. However, it is worth
emphasizing that Pax3 is not absolutely required for cardiac neural crest
migration (Epstein et al.,
2000
), despite clear evidence
that it is required for limb myoblast migration (Daston et al.,
1996
), indicating important
differences in Pax3 function in these tissues.
|
Patterning of PlexinA2-expressing neural crest cells was abnormal
in Splotch. While PlexinA2-expressing cells were seen
diffusely in the outflow endocardial cushions of Splotch embryos
(arrow, Fig. 7E), a localized
collection of expressing cells in the condensed mesenchyme seen in control
embryos (arrow, Fig. 7B) was
missing. However, it is important to note that PlexinA2-expressing
cells were clearly present in the outflow tract of Splotch embryos
(Fig. 7D,E), consistent with
our previous studies showing migration of neural crest cells to the hearts of
these mutant embryos (Epstein et al.,
2000). This result indicates
that Pax3 is not required for PlexinA2 expression in cardiac neural
crest, though we cannot rule out a role for Pax3 in regulation of
PlexinA2 expression not apparent in these studies because of
functional redundancy of other genes (e.g. Pax7).
Interestingly, we noted dramatic up-regulation of PlexinA2 in the
myocardium of the left ventricle of Splotch embryos by both
whole-mount (Fig. 7D,
arrowhead) and radioactive (Fig.
7E, arrowhead) in situ hybridization. While Pax3 is not expressed
in the ventricular myocardium, morphological and functional defects are
present in this tissue in Splotch embryos (Conway et al.,
1997a; Creazzo et al.,
1998
; Li et al.,
1999
). Hence, we conclude that
Pax3 deficiency results in a secondary elevation in PlexinA2
expression in the myocardium perhaps functionally related to these
morphological and contractile defects.
In summary, analysis of PlexinA2 expression in Splotch embryos is consistent with the existence of secondary alterations in myocardial gene expression in the setting of primary neural crest defects. These data also support the conclusion that neural crest cells can migrate to the heart in Splotch embryos, but cannot coalesce to form a functional septation complex.
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DISCUSSION |
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Plexins are known to function as co-receptors for secreted semaphorin
ligands and can be divided into at least 4 classes (for review see Tamagnone
et al., 1999; Tamagnone and
Comoglio, 2000
). Members of
the class A Plexins form functional multimeric receptor complexes with
neuropilin 1 (Nrp1), neuropilin 2 (Nrp2) or both in order to bind class 3
semaphorin ligands (Chen et al.,
1997
; Kolodkin et al.,
1997
; Tamagnone and Comoglio,
2000
). The recent observation
that mice homozygous null for Sema3C exhibit cardiovascular defects
(Feiner et al., 2001
),
combined with the data presented here, suggest that PlexinA2 may form part of
a functional receptor for Sema3C on cardiac neural crest cells. Our expression
data indicates that both Nrp1 and Nrp2 are expressed in
appropriate locations during cardiac development to participate in formation
of functional receptor complexes with PlexinA2, though this hypothesis will
require experimental validation. In vitro binding experiments demonstrate that
Sema3C can recognize both PlexinA2/Nrp1 and PlexinA2/Nrp2 complexes (Kolodkin
and Ginty, 1997
).
Interestingly, PlexinA2/Nrp2 complexes do not bind the related ligand Sema3A,
and PlexinA1/neuropilin complexes that bind Sema3A bind Sema3C poorly and do
not signal (Tamagnone and Comoglio,
2000
). Thus, specificity of
the cellular response to a particular semaphorin ligand results from the
specific Plexin co-receptor present in the complex. Neuropilins appear to
subserve less-specific functions in determination of ligand specificity. This
is consistent with our expression data suggesting that expression of PlexinA2
by cardiac neural crest may make these cells uniquely responsive to
Sema3C.
Sema3C null mice die shortly after birth and display an interrupted aortic arch and persistent truncus arteriosus. These defects suggest abnormalities of cardiac neural crest, though Sema3C itself is expressed predominantly in the myocardium surrounding the outflow tract during cardiac development consistent with its role as a secreted ligand affecting cardiac neural crest. Peripartum lethality is likely due to the normal closure of the ductus arteriosus at birth, which results in the loss of blood supply to the descending aorta in the setting of an interrupted aortic arch. Interestingly, other neural crest derivatives, such as the dorsal root ganglia, are normal in Sema3C-/- embryos indicating that cardiac neural crest are particularly susceptible to loss of Sema3C signals. This suggests that a specific Sema3C receptor or signaling complex is expressed by cardiac neural crest cells.
Further support for a role of semaphorin signaling in neural crest-mediated
cardiovascular development comes from the recent description of cardiovascular
phenotypes in Nrp1 (Kawasaki et al.,
1999) null mice.
Nrp1-deficient embryos die around E13.5 with severe cardiovascular
defects including an interrupted aortic arch, persistent truncus arteriosus
and a poorly developed vasculature. Analysis of the Nrp1 phenotype is
complicated by the fact that Nrp1 is also a co-receptor for vascular
endothelial growth factor (VEGF). VEGF is known to be required for vascular
development (Ferrara and Henzel,
1989
; Keck et al.,
1989
; Risau,
1997
) and the loss of
functional VEGF receptors accounts for at least part of the Nrp1 null
phenotype (Kawasaki et al.,
1999
). However, cardiac
outflow tract and aortic arch defects are suggestive of neural crest
deficiencies. In light of our data implicating semaphorin signaling in cardiac
neural crest development, it seems likely that a portion of the Nrp1
null phenotype is related to deficient Sema3C signaling.
Defects in Sema3C and Nrp1 null mice are reminiscent of
the spectrum of cardiovascular defects including interrupted aortic arch and
PTA that are seen in the human DiGeorge and Velocardiofacial syndromes
(Goldmuntz and Emanuel, 1997).
While many DiGeorge syndrome patients have chromosome 22q11 deletions, many
other patients with DiGeorge and related neurocristopathies do not have these
deletions. Our analysis of semaphorin signaling molecules during cardiac
neural crest development provides additional genes potentially responsible for
human congenital cardiovascular disease.
In addition to Sema3C, another class 3 semaphorin ligand, Sema3A, functions
during cardiac development (Behar et al.,
1996; Taniguchi et al.,
1997
). Sema3A acts as a
repulsive signal affecting neural crest migration in the chick (Eickholt et
al., 1999
). Sema3A signals via
a receptor complex composed of at least PlexinA1 and Nrp1, but it can also
bind PlexinA2/Nrp1 complexes (Takahashi et al.,
1999
). In the chick, cells
from neural crest explants from both the hindbrain and the trunk regions of
the embryo selectively avoid growth on Sema3A stripes in vitro, and individual
cells bind Sema3A and undergo a morphological shape change consistent with a
negative response to Sema3A (Eickholt et al.,
1999
). In mice, two
Sema3A-deficient lines were generated independently. Taniguchi et al.
(Taniguchi et al., 1997
)
demonstrated a non-lethal phenotype with defects in peripheral nerve
projections while Behar et al. (Behar et al.,
1996
) described perinatal
lethality characterized by hypertrophy of the right ventricle, dilation of the
right atrium and bone and cartilage abnormalities. In both cases, the defects
seen in Sema3A null mice are not suggestive of cardiac neural crest
defects. Nrp2 null mice display defects in selective axon pathfinding
during neurogenesis, but display no obvious defects in neural crest or cardiac
development (Chen et al.,
1997
). These results suggest a
lack of requirement for Sema3A and Nrp2 during cardiac
neural crest development, or the existence of functional redundancy with other
genes.
Taken together, the existing data suggest that the most likely receptor complex expressed by cardiac neural crest cells that mediates Sema3C signaling includes PlexinA2 and Nrp1.
By analogy to semaphorin signaling during neurogenesis, semaphorin
signaling in the cardiac neural crest may regulate pathfinding and migration.
Subtle abnormalities of neural crest positioning may result in congenital
heart disease. Consistent with this hypothesis, we have previously observed
defects in positioning of cardiac neural crest cell populations in the outflow
tract cushions in the Pax3 mutant Splotch (Epstein et al.,
2000). In Splotch
embryos, neural crest cells reach the outflow tract but are positioned in
inappropriate lateral domains within the truncus arteriosus (Epstein et al.,
2000
). This suggested to us
that Pax3 might regulate expression of PlexinA2 in cardiac neural
crest, a hypothesis that was also appealing because of the known function of
Pax3 to modulate expression of c-Met (a plexin-related gene) in
myoblasts. However, we found that PlexinA2 was expressed by cardiac
neural crest in the absence of Pax3. Thus, factors other than
PlexinA2 are required for proper neural crest positioning in the
heart.
Like Splotch embryos, Sema3C-deficient embryos have neural crest-related cardiac defects. Abnormalities of outflow tract septation in Sema3C nulls present a spectrum of defects ranging from little or no connection between the pulmonary trunk and aorta in mildly affected embryos, to complete failure of septation (persistent truncus arteriosus). We have noted a similar variability in the expression of PlexinA2 in Sema3C mutants. In some Sema3C null embryos, there is a dramatic decrease in PlexinA2 expression in the outflow tract cushions, while expression in other embryos appears close to normal. This phenotype is particularly interesting in that only a subset of the cardiac neural crest, those that populate the 4th aortic arch and a subset that enter the outflow tract, are affected. The ductus arteriosus, a 6th arch derivative develops normally. Thus, signaling between Sema3C and PlexinA2 appears to be critical for only a subset of cardiac neural crest. The expression of Sema3C in the myocardial wall of the outflow tract may represent a guidance signal for migrating neural crest cells directing proper positioning in the outflow tract. Additional roles for semaphorin signaling in neural crest differentiation, proliferation or survival remain to be determined.
We have demonstrated that PlexinA2 and Sema3C are
expressed in both complementary and overlapping domains in the developing
embryo consistent with a ligand-receptor interaction. In the central nervous
system, there are important parallels between the ephrin and semaphorin
families, both of which mediate axon pathfinding cues. In the case of the
ephrin family, examples exist in which a given cell type both expresses and
responds to a specific secreted ligand (Hornberger et al.,
1999). Thus, it is plausible
that cardiac neural crest cells both secrete Sema3C and also respond to Sema3C
that is secreted in an autocrine and paracrine fashion.
Expression of Sema3C and PlexinA2 partially overlap at E11.5 in the pharyngeal mesenchyme surrounding the aortic arch arteries. If co-expression of receptor and ligand is required for proper maintenance of the aortic arches, absence of the Sema3C ligand may result in a loss of arch positional identity or survival cues. Subsequent inappropriate arch regression would result in interruptions of the mature arch. Misexpression and transgenic rescue experiments using tissue-specific promoter elements should help to test this hypothesis.
In summary, our data provide evidence suggesting a critical role for semaphorin signaling during cardiac neural crest-mediated heart development. PlexinA2 is expressed by cardiac neural crest cells and likely functions as a component of a receptor for Sema3C. The importance of semaphorin signaling in both cardiac morphogenesis and neural patterning in the central nervous system suggests parallels between molecular and cellular pathways mediating cardiac neural crest migration and axon guidance. This paradigm suggests new avenues of research relevant to the understanding of neural crest development and the etiology of congenital heart disease.
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ACKNOWLEDGMENTS |
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