1 Alkek Institute of Biosciences and Technology, Texas A&M System Health
Science Center, 2121 Holcombe Blvd, Houston, TX 77030, USA
2 Department of Anatomy and Developmental Biology, St. George's Hospital Medical
School, University of London, Cranmer Terrace, London SW17 ORE, UK
Author for correspondence (e-mail:
jmartin{at}ibt.tamu.edu)
Accepted 24 July 2002
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SUMMARY |
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Key words: Cardiac development, Homeobox, Morphogenesis, Mouse
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INTRODUCTION |
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Although Pitx2-null mice have severe cardiac phenotypes that are
similar to those observed in humans with laterality defects, the
Pitx2-null phenotype suggests that Pitx2 function is
important after looping morphogenesis, as Pitx2 mutant hearts loop
correctly to the right (Gage et al.,
1999; Kitamura et al.,
1999
; Lin et al.,
1999
; Liu et al.,
2001
; Lu et al.,
1999b
). Analysis of individuals with laterality defects has
revealed a spectrum of associated cardiac septation and valve anomalies,
including abnormalities in conotruncal and right ventricular development,
atrial lateralization and atrioventricular (AV) septation
(Brown and Anderson, 1999
;
Icardo and Sanchez de Vega,
1991
). These observations suggest that the genetic pathways
regulating left-right asymmetry may also directly regulate valve and cushion
morphogenesis and that subtle defects in left-right asymmetry may be a common
etiologic factor for congenital heart disease.
In common with human patients, Pitx2-null mice display atrial
septal defects (ASD), abnormal AV septation (resulting in complete AV canal)
and abnormal arterioventricular connections
(Kitamura et al., 1999;
Liu et al., 2001
).
Pitx2 mutants also have a hypoplastic right ventricle. Although
Pitx2 mutant mice have severe cardiac anomalies, the primary function
of Pitx2 in heart development remains unclear as the Pitx2
mutant heart phenotypes could be secondary to delayed looping morphogenesis or
embryonic rotation.
In this work, we have used a combination of gene expression analysis and gene targeting approaches to investigate Pitx2 function in cardiovascular development in more detail. Our data demonstrate that the Pitx2c isoform is expressed in a presumptive secondary heart field that invades the heart after looping morphogenesis. Pitx2c was expressed in a subpopulation of left branchial arch and splanchnic mesoderm apposed to forming branchial arch arteries (BAAs) and in left aortic sac mesothelium. An isoform-specific deletion of Pitx2c, generated by gene targeting in embryonic stem cells, revealed that Pitx2c functions to regulate asymmetric BAA remodeling and to pattern the outflow tract (OFT). Fate-mapping studies with a Pitx2 cre knock-in allele revealed that Pitx2 daughter cells invade the AV cushions and valves in a Pitx2-dependent fashion, suggesting a role for Pitx2 in local cell movement or survival within the heart. Our results provide insight into Pitx2 function in post-looping cardiac morphogenesis and in BAA remodeling.
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MATERIALS AND METHODS |
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After homologous recombination, the Pitx2 c neo allele
resulted in deletion of the majority of exon 4, including all coding sequences
within this exon. The Pitx2
c neo targeting vector was
electroporated into AK7 ES cells, targeted clones identified by Southern blot,
and injected into 3.5 dpc C57BL/6J mouse embryos to generate chimeras. To
induce recombination between the two loxP sites and remove PGKneomycin
cassette, we crossed Pitx2
c neo chimeras to CMVCre
recombinase deleter strain. Pitx2
c neo and
c
alleles were maintained on a mixed 129/SvxC57BL/6J genetic
background.
The Pitx2 abccreneo will be described elsewhere.
Briefly, to generate this allele, an IRES cre PGKneomycin cassette was
introduced into the PvuII and NruI sites of Pitx2
exon 5, that encodes part of the homeodomain, generating a null allele of
Pitx2. Wholemount in situ with a cre recombinase
(cre) probe confirmed that expression of cre recapitulated
endogenous Pitx2 expression pattern.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described
(Lu et al., 1999b). The
Pitx2c probe was a 1 kb genomic fragment containing exon 4 that was
linearized with XhoI and transcribed with T7 polymerase. The
semaphorin 3c probe has been described previously
(Brown et al., 2001
) and the
cre probe was a cDNA fragment that was linearized with EcoRI
and transcribed with T7 polymerase.
lacZ staining and histology
For histology, embryos were fixed overnight in buffered formalin,
dehydrated through graded ethanol and paraffin embedded. Sections were cut at
7-10 µm and H&E stained. lacZ staining was described
(Lu et al., 1999a).
Corrosion cast and casting dye injections
Injection of casting dye: 18.5 dpc embryos were harvested and sternum
removed. Yellow casting dyes (Connecticut Valley Biological Supply) were
injected into right ventricles using a capillary pipette, followed by blue dye
into left ventricle. Corrosion casts: 18.5 dpc embryos were isolated and the
heart exposed by a thoracic incision. Batson number 17 acrylic (Polysciences)
was injected into right and left ventricles until great arteries were filled.
After hardening overnight in distilled water at 4°C, tissues were removed
with Maceration Solution at 50°C for 24 hours without shaking.
India Ink Injections
Embryos were dissected and placed in ice cold PBS. Individual embryos were
placed in warm PBS to facilitate ventricular contractions. Using a pulled
glass pipette, India Ink was injected into ventricles until ink penetrated
small vessels. Embryos were post fixed in 10% formalin and cleared in benzyl
alcohol:benzyl benzoate (2:1).
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RESULTS |
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Because of the correct dextral looping in Pitx2 null embryos, we
hypothesized that Pitx2 functioned in a cell population that
contributed to the heart during or after cardiac looping. Experiments
performed in chick and mouse embryos have revealed that cells outside the
primary heart field contribute to conotruncal development. One cell population
originates in the splanchnic and branchial arch mesoderm, and migrates into
the OFT and right ventricle of the looped heart
(Kelly et al., 2001;
Mjaatvedt et al., 2001
;
Waldo et al., 2001
). We found
that cells within this presumptive secondary heart field express
Pitx2c asymmetrically at 9.5 dpc both as they migrate and after
populating the OFT and right ventricle, revealing that this cell population
has laterality.
Whole-mount in situ using a Pitx2c-specific probe on 9.5 dpc embryos revealed left-sided Pitx2c expression in splanchnic mesoderm at the level of and just caudal to the OFT (Fig. 1A,B). Serial sectioning showed that Pitx2c expression in left splanchnic mesoderm was continuous with expression in left aortic sac mesothelium and OFT myocardium (Fig. 1C-F). Expression of the Pitx2a and Pitx2b isoforms was not detected in the presumptive secondary heart field or developing OFT (not shown).
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We examined coronal and parasagittal sections through 9.5 and 10.5 dpc embryos to investigate in more detail the Pitx2c expression pattern during these timepoints prior to remodeling of the BAA. Ventral coronal sections through 9.5 dpc embryos showed left-sided Pitx2c expression in aortic sac mesothelium near the junction of the aortic sac and the BAA (Fig. 1F). More dorsal coronal sections revealed low levels of Pitx2c expression in left branchial arch and splanchnic mesoderm in proximity to the third BAA (Fig. 1G). Serial parasagittal sections through 10.5 dpc embryos showed Pitx2c expression in ventral branchial arch and splanchnic mesoderm that was continuous with Pitx2c expression in left branchial arch mesoderm evident on more lateral sections (Fig. 1H,I). The parasagittal sections at this timepoint also revealed the diminished intensity in Pitx2c expression dorsally towards the dorsal aorta (Fig. 1I). Our expression studies also showed Pitx2c expression in the left atrium, primary interatrial septum, left dorsal mesocardium, and right ventricular and interventricular myocardium (Fig. 1J-M). From these studies, we conclude that Pitx2c is asymmetrically expressed in a subpopulation of the presumptive secondary heart field that contributes to the OFT and right ventricular myocardium after cardiac looping, suggesting that Pitx2c provides laterality to the OFT and right ventricle myocardium. Moreover, asymmetric Pitx2c expression in ventral branchial arch and splanchnic mesoderm, with higher levels ventrally near the junction of the BAA and aortic sac, suggests a role for Pitx2c in formation of the BAAs.
Pitx2c mutants survive gestation and turn normally
The Pitx2c isoform is encoded by exons 4, 5 and 6, and uses a
distinct promoter from that of the Pitx2a and Pitx2b
isoforms (Shiratori et al.,
2001). The Pitx2 exon 5 and exon 6, which encode the
homeodomain, are common to all Pitx2 isoforms in mice
(Fig. 2A,B). To directly
investigate Pitx2c function using a loss-of-function approach in
mice, we constructed a targeting vector that replaced the
Pitx2c-specific exon 4 with a PGKneomycin LoxP cassette, the
Pitx2
c neo targeting vector
(Fig. 2B,C). Upon germline
transmission, the Pitx2
c neo allele was crossed to the
cmv cre recombinase deletor strain to remove the PGK
neomycin cassette and generate the final Pitx2
c allele
(Fig. 2C,D). Both Pitx2
c neo and Pitx2
c-/- mutants were
obtained at the Mendelian ratio at 18.5 dpc. Mutant neonates were born alive
but quickly became cyanotic and died a few minutes after birth. We noted that
Pitx2
c-/- mutants turned normally, suggesting that
the Pitx2a and Pitx2b isoforms have redundant function with
Pitx2c in turning or body wall closure as Pitx2a, Pitx2b
homozygous mutant embryos also turn normally
(Liu et al., 2001
)
(Fig. 2E).
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Pitx2c patterns the aortic arch vessels
To determine if Pitx2c had a role in patterning the great vessels
of the aortic arch, we performed casting dye and corrosion cast experiments on
18.5 dpc Pitx2 c-/- embryos. Among the 21 mutant
embryos examined, 57% (12 out of 21) had the wild-type pattern of left aortic
arch with right innominate artery while 29% (six out of 21) had right aortic
arch with left innominate artery (Fig.
3A-D). In addition, 14% (three of 21) showed double aortic arch
without innominate artery (Fig.
3E-J). Of the double aortic arches, two were right dominant and
the other left dominant. Thus, of all arches examined, 62% (n=13)
were left dominant and the remaining 38% (n=6) were right dominant.
In addition, all Pitx2
c-/- embryos had double
outlet right ventricle (DORV) in which both the aorta and pulmonary artery
drain the right ventricle (Fig.
3O,P). Blood exited the left ventricle of the Pitx2
c-/- embryos through a ventricular septal defect.
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The corrosion casting experiments revealed that Pitx2c had an important role in patterning of the BAAs. To determine if Pitx2c had a role in the initial formation of the BAAs or was important in BAA remodeling, we performed India ink injections at 11.0 and 11.5 dpc at the initiation of BAA remodeling. At these timepoints, all Pitx2c mutant embryos (n=6) formed symmetric BAAs that were indistinguishable from wild type littermates (Fig. 3K-N). From this, we conclude that Pitx2c functions in remodeling of the BAA. The very discrete, asymmetric Pitx2c expression pattern within the region of the forming BAAs also supports the idea that Pitx2c would have a role in modulating BAA remodeling rather than in the initial endothelial tube assembly.
Cardiac neural crest migrates normally in Pitx2c
mutants
Great vessel remodeling and patterning of the conotruncal region, both
defective in Pitx2c mutants, require normal development of the
cardiac neural crest. To determine if cardiac neural crest contributed to the
conotruncal region of Pitx2 c-/- embryos, we
performed a fate-mapping experiment with the wntl cre transgenic line
that directed cre expression to the precursors of the cardiac neural
crest and the Rosa26 reporter line
(Jiang et al., 2000
;
Soriano, 1999
). cre
expression will induce recombination at the Rosa26 locus resulting in
expression of lacZ in all descendents of Wntl-expressing
cells that include the cardiac neural crest. At both 11.5 and 12.5 dpc, we
found that cardiac neural crest contributed normally to the conotruncal region
and aortic and pulmonic valves of Pitx2
c-/-
embryos suggesting that Pitx2 function in conotruncal cushion
morphogenesis occurred subsequent to neural crest migration into the
Pitx2 mutant heart (Fig.
4A-F). Analysis of sections through the branchial arch arteries of
10.5 dpc wild type and Pitx2
c-/- embryos showed
similar amounts of mesenchyme surrounding the arteries further supporting the
idea that cardiac neural crest was correctly deployed in Pitx2
c-/- embryos (Fig.
4G-J).
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To determine how loss of Pitx2 affected development of OFT
myocardium, we examined the expression of semaphorin 3c (Sema3c), an
OFT myocardial marker, in wild type and Pitx2c mutants
(Brown et al., 2001;
Feiner et al., 2001
). Although
at 10.5 and 11.5 dpc, Sema3c was expressed normally in
Pitx2c mutant OFT myocardium (Fig.
5A-D), this expression was downregulated by 12.5 dpc
(Fig. 5E,F). These data
suggested that OFT myocardium was correctly specified and that migration of
OFT myocardial precursors was intact in Pitx2c mutants.
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To establish this more firmly, we used a Pitx2 cre knock-in allele
(Pitx2 abccreneo), an allele of Pitx2 that
expresses cre in the endogenous Pitx2 expression domain, to
mark cells fated to express Pitx2. The Pitx2
abccreneo allele has a cre
recombinasePGKneomycin cassette introduced into Pitx2 exon 5 to
generate a null Pitx2 allele, removing function of all Pitx2
isoforms (see Materials and Methods, and
Fig. 5G,H). Moreover,
expression of cre from the Pitx2
abccreneo
qualitatively recapitulates the endogenous Pitx2 spatiotemporal
expression pattern (not shown). At both 10.5 and 12.5 dpc, spatial expression
of cre was similar in the OFT of the control
abccreneo heterozygotes and
abccreneo;
abcnull Pitx2-null
mutant embryos, supporting the idea that Pitx2 patterns the OFT
myocardium after it is established (Fig.
5I,J and not shown). Taken together, these data support the notion
that Pitx2 functions in branchial arch and splanchnic mesoderm, a
developmental field that is distinct from cardiac neural crest. Moreover,
downregulation of Sema3c expression in Pitx2 mutants
suggests that Pitx2 has a role in maintenance of gene expression in
OFT myocardium.
Pitx2 daughter cells contribute to OFT, inner curvature
myocardium and valves
In addition to cardiac neural crest, OFT and inner curvature myocardium
invades the cardiac cushions (van den Hoff
et al., 1999; van den Hoff et
al., 2001
). To determine if descendents of
Pitx2-expressing myocardium populated the cardiac cushions, we used
the Pitx2
abccreneo allele and the Rosa26
reporter allele to follow the fate of Pitx2 daughter cells after
Pitx2 expression had been extinguished. At timepoints when
Pitx2c is actively expressed in the heart, 9.5 dpc until 12.5 dpc,
distinctions between Pitx2 daughter cells and newly labeled
Pitx2-expressing cells can be made in regions of the heart that never
express Pitx2c. For example, at 9.5 and 10.5 dpc
Pitx2-expressing cells are restricted to the left side of the forming
OFT (Fig. 1C-E). By contrast,
lacZ-positive cells were detected on both sides of the OFT tract
myocardium, suggesting that labeled Pitx2 daughter cells, found on
the right side of the OFT, had moved from the left side
(Fig. 6A). The distribution of
lacZ-positive cells in the OFT tract in Pitx2 null embryos
was similar to that of the wild type, suggesting that Pitx2 is not
required for movement of the myocardial precursors from branchial arch
mesoderm into the OFT (Fig.
6A,B). We noted that the number of lacZ-labeled cells in
the OFT myocardium of 10.5 dpc embryos was less than what would be expected
from the Pitx2c expression pattern. This may reflect the delay
between cre transcription and Cre-mediated excision that requires the
accumulation of adequate levels of Cre protein. Moreover, the delay in the
readout is also lengthened by the need for transcription and translation of
lacZ from the Rosa26 locus
(Nagy, 2000
).
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At 12.5 dpc, when Pitx2c is still expressed in the heart, the
Pitx2 abccreneo allele cannot distinguish between
newly labeled lacZ-positive cells and Pitx2c descendents
that are no longer expressing Pitx2c. At this timepoint,
lacZ-labeled cells were predominantly found in the myocardium
overlying the interventricular groove with some cells found in the proximal
OFT (Fig. 6C,D). By 14.5 dpc
and 16.5 dpc, when Pitx2c expression is extinguished
(Fig. 6F,H), there was an
increase of lacZ-positive Pitx2c descendents over the medial
aspect of the heart, suggesting an outward expansion of Pitx2c
descendents from the right ventricular and inner curvature myocardium
(Fig. 6E,G). Sections through
16.5 dpc hearts, after Pitx2c expression had been extinguished,
demonstrated that lacZ-positive Pitx2 daughter cells
populated the myocardium of the proximal OFT, as well as the remodeled
membranous and muscular ventricular septum and atrial septum
(Fig. 6I,J).
Analysis of the fate of Pitx2 daughter cells in the Pitx2
abccreneo;
c mutant embryos, that turned
normally and survived longer than Pitx2 null embryos, revealed that
fewer lacZ-positive cells were found in the right ventricular and
inner curvature myocardium of Pitx2 mutant embryos at both 14.5 dpc
(Fig. 6K,L) and 18.5 dpc
(Fig. 6M,N). Serial transverse
sections through the 18.5 dpc hearts revealed that in Pitx2
abccreneo heterozygous hearts lacZ-labeled
cells were found at the inferior border of the heart near the cardiac apex
(Fig. 6O,Q). By contrast, in
the Pitx2
abccreneo;
c mutants
lacZ-labeled cells were not found at the inferior boundary of the
heart (Fig. 6P,R). Moreover,
sections through 12.5 dpc hearts revealed that lacZ-positive cells
were found in the central AV cushion of Pitx2
abccreneo heterozygous embryos, revealing that
Pitx2 descendents contributed to the cushion mesenchyme
(Fig. 6S). By contrast, in both
12.5 dpc and 14.5 dpc Pitx2-null mutant embryos,
lacZ-positive cells were excluded from the central AV cushion
mesenchyme suggesting that Pitx2 function was required for invasion
of Pitx2 daughters into the AV cushion
(Fig. 6T,U). As Pitx2c
expression is never detected in endocardium, this fate mapping data suggests a
myocardial source for the lacZ-labeled cells in the AV cushion.
Defective valve morphogenesis is a common feature in human patients with
laterality defects and Pitx2-null embryos
(Brown and Anderson, 1999;
Icardo and Sanchez de Vega,
1991
; Liu et al.,
2001
). At 16.5 dpc, lacZ-positive Pitx2
descendents were detected in the AV valve leaflets of Pitx2
abccreneo heterozygotes but were excluded from the
valve leaflets of Pitx2
abccreneo;
c mutants
(Fig. 6V,W).
Defective pulmonary and caval vein morphogenesis in Pitx2
mutants
Corrosion casting and scanning electron microscopy was used to analyze the
morphology of the pulmonary veins in Pitx2 mutant embryos. The left
superior caval vein (LSCV) normally flows into the coronary sinus, while the
right superior caval vein (RSCV) and inferior caval vein (ICV) are connected
to the right atrium (RA) by thin strips at the valves. The left and right
pulmonary veins join to a common pulmonary vein (PV) that drains into the left
atrium (LA) (Fig. 7A,C). In
most Pitx2 c-/- embryos, morphology of these
structures was defective, with all these veins running together into a common
medial venous sinus (Fig.
7B,D). Consistent with this phenotype, fate mapping with the
Pitx2
abccreneo allele showed that
lacZ-positive Pitx2 daughter cells were observed bilaterally
in the pulmonary veins of Pitx2
abccreneo
heterozygous embryos but were severely reduced in the pulmonary veins of
Pitx2
abccreneo;
c mutant embryos
(Fig. 7E-H).
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DISCUSSION |
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Pitx2 functions in the presumptive secondary heart field
derived from branchial arch and splanchnic mesoderm
Recent advances have revealed that the primary heart field receives
contributions from a number of secondary fields. Functional studies have
implicated the cardiac neural crest in patterning of the aortic arch vessels
and conotruncus of the heart. For example, mice with mutations in components
of the endothelin signaling pathway
(Yanagisawa et al., 1998),
forkhead genes (Iida et al.,
1997
; Kume et al.,
2001
; Winnier et al.,
1999
) and splotch mutant mice
(Epstein et al., 2000
) have
defective arterioventricular connections secondary to cardiac neural crest
abnormalities. Moreover, inactivation of Sema3c and neuropilin 1
leads to faulty conotruncal cushion formation as a result of aberrant cardiac
neural crest migration (Brown et al.,
2001
; Feiner et al.,
2001
; Kawasaki et al.,
1999
). By contrast, our data reveal that Pitx2c has an
important role in patterning a separate heart field derived from the branchial
arch and splanchnic mesoderm.
We found that Pitx2c is expressed asymmetrically in the left
branchial arch and splanchnic mesoderm within cells that will contribute to
the OFT myocardium. Moreover, Pitx2c is also expressed in OFT
myocardium and right ventricular myocardium, regions of the heart that are
populated by cells derived from branchial arch and splanchnic mesoderm, but
not by cardiac neural crest (Jiang et al.,
2000; Kelly et al.,
2001
; Mjaatvedt et al.,
2001
; Waldo et al.,
2001
). Our fate-mapping studies with the Wnt1 cre and
Rosa26 reporter mice also show that cardiac neural crest migrates
normally in Pitx2 mutants.
Our data suggest that Pitx2c is not required for the initial
migration of branchial arch mesoderm into the outflow tract. Analysis of
Sema3c expression in Pitx2c mutants suggests that the defect
in Pitx2c mutants occurs relatively late in conotruncal development.
Moreover, cre expression in Pitx2 abccreneo;
abcnull mutants was similar to that observed in the
abccreneo heterozygous OFT. One idea to explain
these data is that Pitx2 functions to maintain signaling between the
outflow tract myocardium and underlying endothelium and forming conotruncal
cushions. This model would be similar to what has been proposed for
Pitx2 in craniofacial development where Pitx2 has a role in
epithelial-mesenchymal signaling important for tooth organogenesis
(Lin et al., 1999
;
Lu et al., 1999b
). Another
possibility, based on the ventricular myocardial defect observed in
Pitx2 mutant embryos (see below), is that Pitx2 regulates
local expansion of OFT myocardium. We favor the first hypothesis as we do not
detect differences in the number or localization of lacZ-labeled
cells in the OFT of wild-type and Pitx2 mutant embryos. Nonetheless,
it is still formally possible that subtle differences in OFT myocardial
expansion could be responsible for the conotruncal defect observed in
Pitx2 mutants. Taken together, our findings support the idea that
Pitx2 patterns the branchial arch mesoderm and OFT myocardium to
support normal development of the conotruncus.
Pitx2c and asymmetric remodeling of the branchial arch
arteries
Development of the branchial arch arteries and subsequent remodeling into
the mature aortic arch arteries involves a series of paracrine signaling
events (Fishman and Kirby,
1998; Hanahan,
1997
; Yancopoulos et al.,
2000
). The forming BAA endothelial tubes are located within the
branchial arch mesoderm in close proximity to surface ectoderm and the
endoderm-derived epithelium of the branchial pouches. Signaling from
endothelium to mesenchyme is thought to be important for recruitment of
supporting cells, such as smooth muscle precursors and pericytes, which are
important for stabilization of the forming endothelial tubes
(Hanahan, 1997
).
Vascular remodeling involves local disruption of the critical interaction
between endothelium and support cells with resulting regression of the
endothelium. In one system, endothelial regression occurs by programmed cell
death secondary to loss of survival factors
(Meeson et al., 1996;
Meeson et al., 1999
). The
mechanisms underlying asymmetric remodeling of the BAAs, resulting in
left-sided aortic arch, are poorly understood.
Cell ablation studies in chick embryos and loss-of-function experiments
performed in mice have defined a role for cardiac crest in maintaining the
integrity of branchial arch arteries (Brown
et al., 2001). However, recent fate mapping experiments using
Wnt1 cre and Rosa26 reporter mice, while confirming the
importance of the cardiac crest in mouse BAA formation, suggest that cardiac
neural crest does not provide the signal for asymmetric remodeling of the BAAs
(Jiang et al., 2000
).
The important role of branchial arch endoderm in BAA development has been
illustrated by phenotypes of individuals with DiGeorge syndrome and mouse
models of this syndrome that include severe defects in aortic arch artery
formation (Lindsay et al.,
2001; Merscher et al.,
2001
). Importantly, defects were observed more commonly in the
right fourth BAAs of a haploinsufficent mouse model for DiGeorge Syndrome
(Lindsay and Baldini, 2001
).
The gene implicated in these events, Tbx1, is expressed in branchial
arch endoderm, suggesting that endoderm-derived signals may have a role in
asymmetric remodeling of BAA.
Pitx2c is expressed asymmetrically in a very discrete population of cells in proximity to the left aortic sac and left BAAs. Despite this restricted expression, there is a strong BAA phenotype in Pitx2c mutants. These observations suggest that Pitx2c may have a role in recruitment or maintenance of supporting cells to the left BAAs and aortic sac. In wild-type embryos, Pitx2c may be important for stabilization of left-sided BAAs, such as the sixth BAA, that will form the left-sided ductus arteriosus. In the absence of Pitx2c function, maintenance of the sixth BAA would be impaired, resulting in formation of a right-sided ductus arteriosus in some embryos. This alteration would initiate a cascade, perhaps resulting from the altered hemodynamics of the persistent right-sided sixth BAA, to alter remodeling of the other BAAs. Although these ideas will need to be verified in future experiments, our data provide new information about the role of Pitx2 in asymmetric remodeling of the BAA.
Pitx2 in cushion and valve morphogenesis
Our data suggest that Pitx2 has a greater role in AV cushion
morphogenesis when compared with formation of the conotruncal cushions.
Pitx2-null embryos have severe defects in the central mesenchymal
mass that forms the AV cushions and valves resulting in complete AV canal
(Kitamura et al., 1999;
Liu et al., 2001
). The
conotruncal phenotype is a failure of rotation of the truncus arteriosus and
conotruncal cushion dysmorphology
(Kitamura et al., 1999
;
Liu et al., 2001
). Genetic
evidence from mice implicates Bmp-signaling in conotruncal cushion
morphogenesis (Kim et al.,
2001
). Noggin overexpression experiments performed in chick
embryos revealed that Bmp signaling in conotruncal cushion formation
functioned through a mechanism involving regulation of cardiac neural crest
migration (Allen et al., 2001
).
Less is known about the signaling pathways that regulate AV cushion
morphogenesis, although recent experiments suggest that Bmp-signaling has a
central role (Gaussin et al.,
2002
).
Data from zebrafish suggest that composition of matrix is of crucial
importance in the initial formation of valves and implicate Wnt and Bmp
signaling in these events (Walsh and
Stainier, 2001). In vitro studies suggest that the action of
matrix metalloproteases on cushion mesenchyme is required for migration of
mesenchyme into the forming cushions (Song
et al., 2000
). This epithelial-mesenchymal transition that leads
to cushion deposition requires Tgfß signaling
(Brown et al., 1996
;
Brown et al., 1999
). In
addition, Tgfß2 null mice have multiple defects in valve and septal
morphogenesis, implicating this signaling pathway in cushion morphogenesis
(Bartram et al., 2001
;
Sanford et al., 1997
). Our
data reveal that Pitx2 has a role in regulating cellular movement
into the formed AV cushion, a late step in cushion morphogenesis. One idea to
explain these data is that Pitx2c is required for the myocardial
invasion of AV cushion mesenchyme. Pitx2c is expressed in the inner
curvature myocardium that surrounds the AV cushion and these myocardial cells
have been shown to invade the AV cushion mesenchyme
(van den Hoff et al., 2001
).
However, another possible source of cells that invade the AV cushion is dorsal
mesocardium that also expresses Pitx2c. Further experiments are
currently under way to elucidate the exact source of invading Pitx2
daughter cells.
Pitx2 function in the venous pole
The data presented here extend our previous understanding of Pitx2
function in development of the venous pole of the heart. Previous studies have
demonstrated an important role for Pitx2 in patterning of the atrial
appendages and atrial septation (Kitamura
et al., 1999; Liu et al.,
2001
). Analysis of the Pitx2c mutants also reveal a role
for Pitx2 in morphogenesis of the pulmonary and caval veins. Fate
mapping suggests a direct role for Pitx2 in vein morphogenesis as
Pitx2 daughters populate pulmonary and caval veins. Moreover,
diminished contribution of Pitx2 daughters to the Pitx2
mutant pulmonary vein suggests a role for Pitx2 in cell movement or
cell sorting that may be similar to Pitx2 function in AV cushion
morphogenesis. Alternatively, Pitx2 may function to regulate
proliferation or survival of pulmonary vein and AV cushion progenitors.
Pitx2 function in expansion of ventricular myocardium
Pitx2c is expressed in the right ventricular and inner curvature
myocardium (Campione et al.,
2001; Schweickert et al.,
2000
). Our fate mapping experiment revealed that Pitx2
daughter cells expand to extensively populate both right and left ventricular
myocardium. In Pitx2 mutant embryos, fewer Pitx2 daughters
are observed contributing to ventricular myocardium. Moreover, analysis of
cre expression in Pitx2 mutants, that marks the right
ventricle, suggested that the size of the right ventricle was reduced in
Pitx2 mutants. One interpretation of these data is that
Pitx2 functions in growth of the right ventricular myocardium.
Further experiments will be required to distinguish between defective movement
of precursors into the right ventricle and failure of the right ventricular
myocardium to proliferate.
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ACKNOWLEDGMENTS |
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Footnotes |
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