1 Department of Orthopaedic Surgery, UCLA School of Medicine, Los Angeles, CA
90095, USA
2 Department of Human Genetics, UCLA School of Medicine, Los Angeles, CA 90095,
USA
3 Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA 90095,
USA
4 Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA
90095, USA
5 Department of Molecular Cell and Developmental Biology, UCLA, Los Angeles, CA
90095, USA
* Author for correspondence (e-mail: klyons{at}mednet.ucla.edu)
Accepted 1 October 2002
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SUMMARY |
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Key words: Bone morphogenetic protein, Endocardial cushion, Outflow tract septation, Persistent truncus arteriosus, Hypomorph, Semilunar valve
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INTRODUCTION |
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The potential importance of gene dosage in BMP signaling is highlighted by
the recent finding that, in humans, mutations in the BMPR2 gene,
thought to lead to haploinsufficiency, cause primary pulmonary hypertension
(PPH), a vascular disease that is caused by increased proliferation of
endothelial and smooth muscle cells in the pulmonary arteries
(Deng et al., 2000;
Lane et al., 2000
). We report
the generation of mice carrying a modified allele of Bmpr2,
Bmpr2
E2, which encodes a protein that lacks half of the
extracellular ligand-binding domain. Mice homozygous for this mutant allele
display a mild skeletal phenotype, which includes posterior transformation of
the last thoracic vertebra, consistent with a reduction of BMP signaling in
these mutants. The mutants die before birth with cardiovascular defects. As
opposed to most mouse models of congenital cardiac defects,
Bmpr2
E2 mutants have a fully penetrant, very
restricted phenotype, which is limited to the outflow tract. The defect
associates an absence of septation of the outflow tract of the heart with
interruption of the aortic arch, a condition known in humans as persistent
truncus arteriosus type A4 (Jacobs,
2000
). In addition, the semilunar valves, which prevent backflow
from the aorta and pulmonary trunk into the ventricles, do not form in
mutants. The signaling pathways responsible for the differentiation of the
conotruncal ridges into both the conotruncal septum and the semilunar valves
are very poorly understood. We show that an intact BMP signaling pathway is
required for maintenance of the conotruncal ridges and formation of the
semilunar valves.
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MATERIALS AND METHODS |
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RT-PCR
Total RNA was prepared from E12.5 embryos of each genotype using Trizol
(Gibco-BRL) according to the manufacturer's protocol. Reverse transcription
was carried out with a gene-specific primer located in exon 5 (E5R,
5'-GTG AAG ACC CTG TTT CCG GTC-3') with the ProStar kit
(Stratagene). A region encompassing exon 2 was amplified by PCR with
oligonucleotides E1F (5'-CTT CTT TGC TGG CCC AGG GA-3') and E3R
(5'-TGG TGT TGT GTC AGG GGG TG-3') or E5R. As a control, a region
downstream of the deletion was amplified with primers in exons 3 and 5 (E3F,
5'-GGT CTC ACA TCG GTG ATC CC-3'; E5R as above). Semi-quantitative
PCR on cDNA obtained from wild-type, mutant and heterozygous littermates was
performed with oligonucleotides E1F and E5R, by collecting aliquots of each
reaction after 12, 18, 22, 25, 28 and 30 cycles, run on gel, imaged, scanned
and quantified. No differences in levels of expression of Bmpr2 and
Bmpr2E2 were observed.
Generation of Bmpr2 and Bmpr2E2
expression constructs and transfections
cDNA was prepared from the above-described total RNA using Superscript II
reverse transcriptase and random hexamers (Gibco-BRL). Full-length coding
sequences for Bmpr2 and Bmpr2E2 were obtained by PCR
using the Bmpr2+/+ and
Bmpr2
E2/
E2 cDNAs, respectively, as
templates, and the following primers: 5'-ctgaattc TTC TTT GCT GGC CCA
GGG ATG AC-3' and 5'-tgtctaga AAC ATC TCA CAG ACA ATT CAT
TC-3' [lowercase letters indicate nucleotides added to the sequence
(e.g. to include restriction sites) that do not match the sequence of the gene
to be amplified]. The resulting amplification products were digested with
EcoRI and XbaI, and subcloned into pBluescript II KS
(Stratagene). The subclones were sequenced on both strands to verify that no
mutations were introduced. The sequence analysis also verified that exon 2 is
absent from the gene product of the mutant allele. The
EcoRI/XbaI fragments encoding Bmpr2 and
Bmpr2
E2 were introduced into pcDNA3 (Invitrogen) to generate
pcDNA-Bmpr2 and pcDNA-Bmpr2
E2.
Mv1Lu or P19 cells (ATCC) at 50-70% confluence were transfected, using the
Superfect transfection reagent (Qiagen), with 0.5 µg of msx2-Lux
(Liu et al., 1994;
Daluiski et al., 2001
), 0.5
µg of lacZ-encoding control plasmid and 0.05 µg of either
pcDNA-Bmpr2 or pcDNA-Bmpr2
E2 per well. After 3 hours, medium was
replaced by MEM + 1% FBS + non essential amino acids. Twenty-four hours
post-transfection, recombinant human BMP2 or BMP7 (Genetics Institute) was
added where indicated. After an additional 24 hours, cells were lysed with
Reporter lysis buffer (Promega) and luciferase activity measured with the
Luciferase Assay System (Promega). Results were obtained in triplicate for
each experiment and normalized to ß-galactosidase activity. The results
shown in Fig. 1F are normalized
for four and two independent experiments for BMP2 and BMP7, respectively.
Statistical significance was assessed using a t-test for correlated
samples using the Vassar website
(http://faculty.vassar.edu/lowry/VassarStats.html).
Ink injection
India ink was injected into the ventricles of the hearts of two litters of
E12.5 and three litters of E13.5 embryos with custom-made pulled glass
pipettes and an Eppendorf micro-injector. Each embryo was genotyped a
posteriori with genomic DNA made from its yolk sac. Ink was allowed to
circulate, and embryos were fixed into 4% paraformaldehyde (PFA) overnight,
dehydrated in an increasing series of methanol and cleared in benzyl
benzoate:benzyl alcohol (2:1).
Cleared skeletal preparation, histology and in situ
hybridization
Cleared skeletal preparations were performed as described
(Hogan et al., 1994). Embryos
were fixed in 4% PFA and embedded in paraffin wax, sectioned (7 µm) and
stained with Eosin/Hematoxylin according to standard techniques. TUNEL
analysis was performed on 7 µM paraffin wax-embedded sections with
Promega's Apoptosis Detection System according to the manufacturer's
instructions. Cell proliferation was assessed by immunohistochemistry with
antibodies against the Proliferating Cell Nuclear Antigen (PCNA, Zymed).
Immunohistology for smooth muscle actin was performed with the monoclonal
anti-
SMA clone 1A4 (Sigma). Whole-mount in situ hybridization was
performed as described (Hogan et al.,
1994
), with BM Purple (Boehringer Mannheim) as a substrate for
alkaline phosphatase. For in situ hybridization on sections, the embryos were
embedded into OCT medium (VWR), and 20 µm cryosections were cut and
processed as described (Hogan et al.,
1994
). The Bmp4 probe was as described
(Jones et al., 1991
). The
Pax3 probe is a gift from Dr P. Gruss. The probe for Ctgf
was made from IMAGE clone dbEST #723742. The periostin and Tbx1
probes were cloned by RT-PCR using whole-embryo RNA as described previously
(Kruzynska-Frejtag et al.,
2001
; Garg et al.,
2001
).
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RESULTS |
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Since the first four exons of Bmpr2 are in frame, we hypothesized
that deleting exon 2 would maintain the reading frame and produce a shorter,
but correctly translated, protein (Fig.
1D). To verify this, we performed RT-PCR using RNA extracted from
wild type, heterozygous and homozygous mutant embryos. For all genotypes, an
amplified product was obtained with primers downstream of the mutation
(between exons 3 and 5), as well as with primers spanning the deletion,
indicating that a stable mRNA is transcribed from the mutant allele
(Fig. 1E). Semi-quantitative
PCR confirmed that the levels of expression of the wild-type and mutant
alleles are not significantly different (not shown). The difference in size
between the mutant and wild-type PCR products corresponded to the size of exon
2. Cloning and sequencing of the full-length wild-type and
Bmpr2E2 cDNAs confirmed that exon 2 was deleted and
that exons 1 and 3 were in frame in the mutant.
Responsiveness to BMPs of cells transfected with the mutant
Bmpr2E2 cDNA was assayed with a reporter construct
that contains luciferase under the control of a BMP-responsive element from
the promoter of the Msx2 gene
(Liu et al., 1994
;
Daluiski et al., 2001
).
Addition of BMP2 to Mv1Lu cells led to a three- to fourfold stimulation of the
BMP pathways (Fig. 1F, columns
1 and 2). Transfection with the wild-type receptor increased the level of
induction to approx. fivefold (Fig.
1F, columns 3 and 4). However, in cells transfected with the
Bmpr2
E2 receptor, a significantly lower induction was
observed (columns 5 and 6; P<0.025), showing that BMP signaling is
reduced in the presence of the mutant receptor. Similar results were obtained
when transfecting P19 cells under the same experimental conditions (data not
shown).
BMP7, a ligand of a different subclass, was also able to induce luciferase
expression in Mv1Lu cells, albeit with less efficacy
(Fig. 1F, columns 1, 9 and 10).
Transfection with Bmpr2 did not lead to increased msx2-Lux induction
in response to 100 ng/ml BMP7 (Fig.
1F, columns 7 and 9), but a 1.4-fold increase was observed in
response to 300 ng/ml BMP7 (Fig.
1F, columns 8 and 10). By contrast, cells transfected with
Bmpr2E2 were unresponsive to BMP7 at either concentration
(Fig. 1F, columns 11 and
12).
These results indicate that Bmpr2E2 has reduced signaling
capacity compared with wild type Bmpr2. The stability, ligand binding and
signal transduction properties of the mutant receptor that contribute to the
defective signaling properties will be described in detail elsewhere. The
diminished signaling capacity of cells overexpressing the mutant receptor
compared with control cells (Fig.
1F, columns 2, 4 and 6) suggests that either
Bmpr2
E2 has dominant-negative properties, or that
overexpression of this altered receptor leads to sequestration of BMP ligands
and/or type I receptors into impaired signaling complexes. In fact, this
apparent dominant negative effect in vitro when overexpressed has been
observed for other Bmpr2 mutant receptors which do not have such
effects in vivo, and is thought to be an artifact due to interference with
intracellular trafficking in transfected cells
(Rudarakanchana et al., 2002
).
The observations that heterozygotes exhibit no apparent defects, and that the
phenotype of Bmpr2
E2/
E2 mutants (see below)
is much less severe than that of mice homozygous for the Bmpr2 null
allele (Beppu et al., 2000
),
strongly argue that Bmpr2
E2 is not a
dominant-negative or null allele but retains some signaling capacity and thus
is a hypomorphic allele.
Viability of the Bmpr2E2/
E2
mutants
Heterozygous males and females were viable and fertile, with no apparent
malformations. Intercrosses of heterozygotes produced no live homozygous
mutants, indicating an embryonic lethal phenotype. Up to E11.5, mutants were
found in expected Mendelian ratios (39
Bmpr2E2/
E2 out of 154 embryos at E11.5). Up
to this stage, mutants are alive and phenotypically indistinguishable from
wild-type or heterozygous siblings (not shown). Dissection of litters at later
gestational ages showed that, even within the same litter, mutants die at
various stages (Fig. 2A-C),
with the onset of lethality occurring between E12 and birth. Anomalies of
vascularization of the yolk sac, consisting of regions without apparent
vessels, were noted upon dissection (not shown). The avascular regions were
larger for embryos that had died before E12.5 than for embryos that died at
later stages, suggesting that yolk sac vascular anomalies could represent a
major contribution to the early lethality of the mutants. This aspect of the
phenotype will be reported elsewhere.
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Skeletal phenotype of Bmpr2E2/
E2
mutants
BMP signaling pathways are essential for skeletal patterning and growth
(Hogan, 1996), and Bmpr2 is
thought to be the common receptor for all osteogenic BMPs. Therefore, the
observation of a skeletal phenotype is predicted in animals with decreased BMP
signaling through Bmpr2
E2.
Whole-mount cleared skeletal preparations of whole litters aged E14.5-E18.5
confirmed that ossification was delayed in mutants compared with their
siblings. This was observed in both endochondral and membranous bones. It was
particularly severe in the lateral ossification centers of cervical vertebrae
C3-C6, the fourth sternebra (not shown), the ventral processes of cervical
vertebrae C1-C4 and the interparietal bone
(Fig. 2D,E). Whether this
results from a general developmental delay or from a specific defect of
osteogenesis is not immediately apparent. Although there was no overt growth
defect, a vascular defect could conceivably result in developmental delay of
the Bmpr2E2 mutants. However, the observation that
specific skeletal elements (e.g. atlas) are more severely affected than others
argues for a primary defect in skeletogenesis.
Loss of the 13th pair of ribs, associated with a posterior transformation of the 13th thoracic vertebra into a 7th lumbar vertebra, was observed in all the mutants, independent of the genetic background (n=9 homozygotes, Fig. 2F). Therefore, in addition to a role in skeletal growth, full activity of Bmpr2 is essential for skeletal patterning along the anteroposterior axis.
Cardiac septation defects in
Bmpr2E2/
E2 mutants
The onset of lethality over several days at late gestation stages suggested
a cardiovascular defect. The external aspect of the heart was normal in
mutants up to E12 (not shown). At later stages, two normally septated vessels,
the aorta and the pulmonary trunk were seen exiting the arterial pole of the
heart, but their abnormal relative position was noted
(Fig. 3A,B). Closer inspection
suggested that although the outflow tract was septated distally into aorta and
pulmonary trunk, the region proximal to the heart was not septated. Moreover,
a dimple at the apex of the ventricle, a common sign of incomplete ventricular
septation, was noted in mutants (Fig.
3B).
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To help visualize the anatomy of the outflow tract, we injected ink into the ventricles of embryos. At E13.5, when septation of the ventricle into a left and a right cavity is complete in wild-type embryos, injection of ink into the right ventricle of mutants immediately resulted in leakage of ink into the left ventricle, confirming a ventricular septal defect. The ink injections also documented abnormal septation of the proximal part of the outflow tract (OFT) of the heart, the conotruncus, with a single lumen and no separation between right and left vessels (Fig. 3D). Histological examination revealed that the ventricular septal defect observed in mutants is not due to a simple developmental delay, because it is observed at all stages of gestation (e.g. Fig. 4D,I-J). Histological examination at E14.5 and E16.5 also confirmed that OFT septation is abnormal. Massive absence of the conal cushions (Fig. 4J,K) was visible as early as E12.5, although one of the posterior conal cushions was present (arrowhead in Fig. 4D,K) in mutants.
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Interrupted aortic arch in
Bmpr2E2/
E2 mutants
In addition to abnormal septation of the conotruncus and a ventricular
septal defect, ink staining also revealed interruption of the aortic arch
(Fig. 3E-G), of varying
severity, between the roots of the left common carotid and the left subclavian
artery (IAA type B), resulting in extensive communication between the
pulmonary trunk and the descending aorta, via the ductus arteriosus. This
regression of the aortic isthmus (which derives from the left aortic arch 4)
was also observed histologically (Fig.
4H). The varying severity observed at early stages reflects a
progressive regression occurring at these stages of intense remodeling of the
aortic arches. The interruption of the aortic arch was complete (and fully
penetrant) in animals observed at later gestation stages.
Except for occasional retro-esophageal position of the left subclavian artery (Fig. 4B), the development of neighboring tissues was normal. The pulmonary arteries stemmed normally from the pulmonary trunk (Fig. 3F,G, Fig. 4N). In particular, other tissues affected in neural crest deficiency syndromes were unaffected: thymus (Fig. 4G,H) and thyroid (not shown) were present and no tracheo-esophageal fistula was observed (Fig. 4B).
The semilunar valves are absent in
Bmpr2E2/
E2 mutants
Semilunar valves prevent backflow of the blood from the aortic and
pulmonary trunks into the ventricles. They form at the junction of the
conotruncus and the aortic sac, hypothetically by remodeling of the top part
of the conotruncal ridges. No evidence for the presence of semilunar valves
was detected in the outflow tract of ink-injected mutant hearts
(Fig. 3C,D). Histology at
various gestational stages demonstrated that semilunar valve tissue is
completely absent in mutants after E12.5
(Fig. 4A-B,E-H,L). The defect
was limited to the outflow tract, and the atrioventricular (future mitral and
tricuspid) valves were grossly normal (Fig.
4C).
Absent septation of the OFT results from impaired growth of the
conotruncal ridges
Two major cell types contribute to the formation of the conotruncal
swellings that eventually fuse to septate the outflow tract: the endocardium
and the neural crest.
Starting around day E9.5 in the mouse, the resident endocardium, under the influence of inductive signals from the underlying myocardium, undergoes an epithelio-mesenchymal transformation (EMT), which can be followed by histological analysis. At E11.5, the swellings are of maximum thickness in wild-type mice and fill the entire lumen of the outflow tract (Fig. 5A,C). In the mutants, the EMT has occurred, and ridges have started to form (Fig. 5B,D), suggesting that intact BMP signaling is not required for induction of the EMT. However, in mutants, the swellings are much thinner than in wild type, and cell density is lower (see the zones of cell-free cardiac jelly in Fig. 5B,D). No increase of cell death was observed by TUNEL analysis (not shown). However, by E12.5, the swellings are no longer apparent (Fig. 4K), suggesting that BMP signaling is involved in the continued development and/or maintenance of the conotruncal cushion tissue.
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Cell proliferation rates in OFT cushions vary widely with the cell type,
the stage of differentiation, and the position of the cells within the cushion
(Kubalak et al., 2002).
Therefore, the very different shapes and sizes of the cushions in mutants
compared with wild-type embryos do not allow direct comparison of
proliferation rates of each region. However, PCNA staining in myocardial cells
was indistinguishable in wild-type and mutant littermates
(Fig. 5E-G). Abundant
PCNA-positive cells were found both in wild-type and mutant cushions, showing
that, despite the reduced size of the cushions at E11.5, mesenchymal cells
proliferate extensively in the mutant (Fig.
5E-G). However, as the two sections from the same mutant embryo
illustrate (Fig. 5F-G), high
variability was found in the extent of cell proliferation in different regions
of the mutant OFT cushions, which could reflect abnormal growth regulation of
these cells. This high variability in cell proliferation has also been noted
within cushions of Bmp6-/-;Bmp7-/-
mice (Kim et al., 2001
).
Neural crest cells originating from the posterior rhombencephalon migrate
through pharyngeal arches 3, 4 and 6, and contribute to the swellings
(Kirby, 1999;
Jiang et al., 2000
). An
essential role for cardiac neural crest cells in septation of the mammalian
outflow tract has been inferred from studies of various mutant mouse strains
(reviewed by Kirby, 1999
), but
the role these cells play is unknown. The best studied neural crest deletion
syndrome with conotruncal abnormalities is the DiGeorge/Velo-Cardio-Facial
spectrum. Most individuals with this condition harbor a large deletion of
chromosome 22q11, and the phenotype is thought to be caused by the
haploinsufficiency of the genes located in this region. Among the deleted
genes, TBX1 is the main candidate for the conotruncal defects
observed (reviewed by Botta et al.,
2001
). In the Bmpr2
E2 mutants, the
levels and sites of expression of Tbx1 were not affected
(Fig. 5H). Pax3 is a
transcription factor expressed in neural crest cells, including those that are
fated to populate the outflow tract (Jiang
et al., 2000
). Pax3-deficient mice present with
generalized neural crest defects including conotruncal abnormalities
(Conway et al., 1997
). To
examine whether the septation defects in mice arise as a consequence of
impaired migration of cardiac neural crest, we examined Pax3
expression. At E10.5, a stream of Pax3-expressing neural crest cells
is seen migrating toward the OFT (Fig.
5I). These cells are not affected in the
Bmpr2
E2 mutant
(Fig. 5J). Although previously
thought to be cells fated to the OFT, they are actually migrating toward the
hypoglossal muscle (Epstein et al.,
2000
). In addition, smooth muscle actin (SMA)-positive cells,
which represent at least a subset of the cardiac neural crest
(Epstein et al., 2000
), were
found to reach the outflow tract in mutant as in wild-type embryos
(Fig. 5K,L). These results,
showing that Tbx1, Pax3 and SMA expression is unaffected, along with
the absence of defects in other neural crest-derived tissues argue against a
general defect in neural crest migration. However, an effect of BMP signaling
on survival and/or differentiation of neural crest cells within the outflow
tract is possible.
The expression of genes implicated in EMT was examined to determine whether
this process is impaired in mutants. Bmp2 and Bmp4 are
expressed in the myocardium underlying the cushion-forming regions and have
therefore been considered candidates for an EMT-inductive myocardial signal
(Lyons and Hogan, 1993).
Bmp4 expression was not modified in the
Bmpr2
E2 mutants (data not shown). Connective tissue
growth factor (Ctgf) is an immediate early gene expressed in response
to TGFß treatment (Kothapalli et al.,
1997
). TGFßs induce EMT in vitro, and are expressed in
endocardial cells in regions where cushion formation takes place (reviewed by
Nakajima et al., 2000
). We
found that Ctgf was expressed in the conotruncal ridges at day E11.5
(not shown) and in the outflow tract vessels, above the valve level,
predominantly in the endothelium (Fig.
5M). Expression of Ctgf was not affected in
Bmpr2
E2 mutants
(Fig. 5N).
Finally, we tested the expression of periostin, a gene encoding an
extracellular matrix protein known to be responsive to BMP signaling in
osteoblasts (Ji et al., 2000).
Recently, periostin was shown to be expressed at high levels in all cells of
the cardiac endocardial cushions
(Kruzynska-Frejtag et al.,
2001
). Periostin expression was greatly diminished in the OFT
cushions of Bmpr2
E2 mutants
(Fig. 5O,P), while maintained
at high levels in other parts of the embryo, in particular in the ventral
mesenchyme (not shown).
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DISCUSSION |
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We found that signaling through BMP pathways is reduced in vitro in two
different cell lines, with two different classes of BMP ligand, when
transfected with the Bmpr2E2 mutant receptor. However, in
vitro elucidation of the precise mechanism(s) by which BMP signaling is
decreased in the presence of the mutant receptor is hampered by the
unavailability of cell lines that do not express endogenous wild-type
receptor. This limitation is exemplified in studies of the effects of
mutations in individuals with PPH. In this case, the molecular mechanism that
lead to disease is thought to be haploinsufficiency of BMPR2
(Deng et al., 2000
;
Lane et al., 2000
;
Thomson et al., 2000
;
Machado et al., 2001
). This
conclusion is based on the finding that the altered BMPR2 alleles in
many individuals with PPH encode severely truncated, nonfunctional products.
Moreover, some individuals with PPH do not have mutations within the
BMPR2 coding sequence, but nonetheless express BMPR2 at
diminished levels (Atkinson et al.,
2002
). Although the above findings strongly support
haploinsufficiency, an artifactual dominant negative effect, similar to the
one we observe, is seen in cell transfection experiments using mis-sense and
frameshift BMPR2 mutations associated with PPH. This effect is most
likely to be due to impaired intracellular trafficking, leading to reduced
expression of both the mutant and wild-type Bmpr2 at the cell surface
(Machado et al., 2001
;
Rudarakanchana et al., 2002
).
In addition to the in vitro data, several in vivo observations strongly argue
that Bmpr2
E2 is a hypomorphic allele.
Bmpr2
E2/
E2 embryos survive until
midgestation, whereas null mutants die at gastrulation
(Beppu et al., 2000
). The
dramatic difference in severity is not a result of strain-specific differences
since we examined mice on several genetic backgrounds, including the 129
strain reported by Beppu and colleagues. These results indicate that
Bmpr2
E2 retains partial activity in vivo.
The skeletal phenotype also brings strong evidence that BMP signaling is
decreased in Bmpr2E2 mutants. A general delay in
ossification was observed in homozygous mutants, as predicted for a reduced
function Bmpr2 allele. No such defects are seen in heterozygotes, arguing
against a dominant-negative mode of action. Interestingly, several skeletal
elements, such as the supraoccipital bone and ventral process of the atlas,
are more affected than others. This result suggests that either different
skeletal elements require different levels of BMP signaling for their
formation, or that the reduced level of signaling through the mutant Bmpr2 is
compensated in some skeletal elements by another type II receptor.
In addition to defects in bone formation,
Bmpr2E2 mutants exhibit defects in vertebral
patterning. The loss of the 13th thoracic vertebra, and its replacement with a
lumbar vertebra, is observed in Bmpr2
E2 mutants as
well as in follistatin-deficient mice
(Matzuk et al., 1995b
).
Follistatin is an activin antagonist. Hence, in
Bmpr2
E2 and follistatin mutants, the level of
signaling through activin pathways is expected to be increased relative to the
level of BMP signaling. Conversely, GDF11 and ActRIIB are components of
activin signaling pathways, and mice deficient in these genes exhibit extra
thoracic vertebrae (Oh and Li,
1997
; McPherron et al.,
1999
; Gamer et al.,
2001
). Along with our results, these findings suggest that
regulation of the relative levels of signaling through TGFß/activin and
BMP pathways may play a role in anteroposterior skeletal patterning.
Finally, periostin, a gene that is known to be a downstream target of the
BMP pathway in other tissues (Ji et al.,
2000), is downregulated in the outflow tract cushions of
Bmpr2
E2/
E2 hearts compared with wild-type
and heterozygous littermates. Taken together, the in vitro results, the
apparently recessive nature of the Bmpr2
E2 mutant
phenotype, the skeletal phenotypes, and the downregulation of a known target
of the BMP pathway are most consistent with a hypomorphic mode of action for
Bmpr2
E2.
The lethality of homozygotes for a probable null allele of Bmpr2
around the time of gastrulation (Beppu et
al., 2000) is reminiscent of that of null mutants for BMP2 or BMP4
(Winnier et al., 1995
;
Zhang and Bradley, 1996
),
suggesting that Bmpr2 is the major receptor for these ligands during
gastrulation. The fact that Bmpr2
E2/
E2 mice
undergo normal gastrulation, whereas Bmpr2 null homozygotes do not,
shows that the residual activity of Bmpr2
E2 is sufficient to
transduce the effects of BMP2 and BMP4 in early development. Similarly, the
fact that individuals with PPH with haploinsufficiency of BMPR2 are
viable into adulthood has suggested that 50% of normal BMP pathway activity is
sufficient for humans to undergo gastrulation
(Deng et al., 2000
).
Therefore, mesoderm formation during gastrulation does not require full BMP
signaling activity. By contrast, the phenotype of the
Bmpr2
E2 mutants shows that morphogenesis of the
cardiovascular and skeletal systems does.
Bmpr2E2 mutants: a model for type A4
persistent truncus arteriosus
The cardiovascular phenotype of Bmpr2E2 mutants
is summarized in the cartoon in Fig.
3H-I. In Bmpr2
E2 mutants, the
distalmost region of the outflow tract is normally septated, suggesting that
the wedge of neural crest cells that migrate into the aortic sac to form the
aorticopulmonary septum is, at least in part, present. However, the proximal
part of the aortic sac and the conotruncus constitute a single outflow vessel,
a condition known in humans as persistent truncus arteriosus (PTA) type A1 (as
opposed to type A2 where the total length of the outflow tract is
non-septated) (Jacobs,
2000
).
The absence of the conal cushion tissue, which during normal embryogenesis
fuses with the muscular part of the interventricular septum to complete the
separation of the ventricles, results in a ventricular septal defect (VSD).
Such a membranous VSD is almost always associated with PTA, where it is a
hemodynamic necessity (Jacobs,
2000).
In our mutants, the PTA is associated with regression of the aortic
isthmus, a tissue that derives from the left aortic arch 4. Such an
association of interrupted aortic arch and PTA is also a described entity in
humans, known as PTA type A4 (Jacobs,
2000), for which the Bmpr2
E2 mutants
are the first fully penetrant animal model. The form of interrupted aortic
arch (Type B), observed in Bmpr2
E2 mutants, with
resorption between the left common carotid and subclavian arteries, is the
same as that commonly observed in the DiGeorge/Velo-Cardio-Facial syndromes.
However, DiGeorge syndrome is associated with a variable constellation of
defects of the heart and neck region and face, including PTA and thymic
hypoplasia, that is thought to result from a widespread defect of neural crest
cells (Emanuel et al., 1999
),
a mechanism that is unlikely to cause the phenotype of
Bmpr2
E2 mutants (see below).
Embryological origin of the outflow tract defect: neural crest cells
abnormalities or defective epithelio-mesenchymal transformation of the
endocardium?
Distally, the single tube that comprises the outflow tract in early
midgestation embryos is septated into the aorta and the pulmonary trunk by a
structure that is purely neural crest in origin, the aortico-pulmonary septum.
This is the structure that is primarily affected in
DiGeorge/Velo-Cardio-Facial syndromes, resulting in type A2 PTA.
Proximally, however, septation occurs by a different, less well understood
mechanism that requires at least two processes. The first of these is a
transformation of endocardial cells into mesenchymal cells that populate the
cardiac jelly. BMP signaling may be directly involved in the inductive
interactions between myocardium and endocardium during EMT (reviewed by
Nakajima et al., 2000). In
vitro, BMP2 is not sufficient to trigger the onset of EMT but it can synergize
the inductive effect of TGFßs. Consistent with an essential role in vivo,
BMP2 and BMP4 expression in the heart is restricted to the regions of
myocardium that underlie the cushion-forming regions
(Lyons and Hogan, 1993
;
Nakajima et al., 2000
). In
Bmpr2
E2 mutants, we observe initiation of the EMT,
but, in the outflow tract, the cushions fail to progress and never reach their
maximum extension. This offers genetic evidence that intact BMP signaling is
not necessary for initiation of the EMT, but is required for normal growth and
maintenance of the conotruncal ridges.
The second process is an invasion of the conotruncal ridges by neural crest
cells that may contribute to the cell population forming the septae, and may
also be involved in the proliferation and/or survival of the cells that have
undergone the EMT. Several studies have shown that BMPs are required in vivo
for formation and/or survival of (non-cardiac) neural crest cells (reviewed by
Christiansen et al., 2000;
Délot et al., 1999
).
However, the role of cardiac neural crest cells in mammalian endocardial
cushion development is poorly understood, and investigations have long been
hampered by the absence of consensus molecular markers for the subpopulation
of neural crest cells that populate the outflow tract. In particular, the
percentage of cells labeled by various proposed neural crest markers is highly
variable, and even for a single marker the extent of labeling varies in
different lines of reporter transgenic mice
(Brown et al., 2001
).
The analysis of splotch (Pax3-/-) mutants, a mouse
model for both total neural crest ablation in chicks and DiGeorge syndrome in
humans, has suggested that the bulk number of neural crest cells migrating
into the heart is a determining factor for OFT septation
(Conway et al., 2000). In
Bmpr2
E2 mutants, neural crest migration, as
illustrated by Pax3 and smooth muscle actin expression, is not
massively affected, consistent with defects that are different than in models
of total neural crest ablation. In addition to the restriction of PTA to the
conus, the adjacent tissues that develop from branchial arches, such as
thyroid and thymus, appear normal in Bmpr2
E2
mutants. Therefore, the aortic arch remodeling defects and OFT septation
defect seen in Bmpr2
E2 mutants are unlikely to
result from widespread neural crest ablation. Moreover, cells expressing SMA
are present in the OFT of Bmpr2
E2 mutants,
indicating that at least this subset of cardiac neural crest cells migrates to
the OFT. Our results are, however, consistent with a role for BMP signaling in
cardiac neural crest cells. For example, BMP signaling could be necessary for
cell-cell interactions between the newly formed mesenchymal cells arising as a
result of EMT and the incoming neural crest cells. The pattern of expression
of Bmpr2 does not offer hints as to which cell type requires intact
BMP signaling, as it is ubiquitous throughout the embryo
(Roelen et al., 1997
) (E. C.
D. and K. M. L., unpublished). Fate mapping of the cardiac neural crest
(Epstein et al., 2000
;
Jiang et al., 2000
) in
Bmpr2
E2 mutants, as well as tissue-specific
targeting of the mutation will therefore be crucial to assess whether subsets
of cardiac neural crest cells have different roles in outflow tract septation
and valve formation.
Genetic control of valvulogenesis
Semilunar valves develop at the distal end of the conotruncal ridges,
hypothetically by remodeling (reviewed by
Pexieder, 1995). However, in
newborn mice and humans with PTA, although the septum that derives from those
ridges is absent, differentiated valves are usually present (albeit with an
abnormal number of leaflets). This suggests independent genetic control of
septation and valve formation.
Defective semilunar valvulogenesis in Bmpr2E2
mutants suggests that the duration and/or strength of BMP signals must be
tightly controlled. This is highlighted by the observation of semilunar valve
defects in mice deficient in other components of the BMP signaling pathway.
Mice deficient for Tll1, a mammalian homologue of the
Drosophila gene tolloid, which cleaves the BMP antagonist
chordin (Scott et al., 1999
),
have dysplastic semilunar valves (Clark et
al., 1999
). Mice mutant for Smad6, an inhibitory
intracellular mediator of BMP signaling, exhibit hyperplasia of the valves and
OFT septation defects (Galvin et al.,
2000
). The opposing valve phenotypes of
Bmpr2
E2 mutants and Smad6 mutants suggest
that Smad6 could be a downstream antagonist of Bmpr2-mediated signaling in the
endocardial ridges. More recently, double
Bmp6-/-;Bmp7-/- mutants have been shown to have
hypoplastic OFT cushions. However, mechanistic interpretation of this finding
was difficult as the mice seem to recover at later stages, and no OFT
septation abnormalities were described
(Kim et al., 2001
). Our
results demonstrate that modulation of the levels of BMP signaling is crucial
to the development of the semilunar valves, with too much signaling
(Smad6 mutants) leading to hyperplasia, and too little (our results,
Tll1 and Bmp6-/-;Bmp7-/- mutants)
leading to hypoplasia of the valves.
Interestingly, mutants for molecules of the EGF signaling pathway also
display enlargement of the valves, restricted to the semilunar valves
(Chen et al., 2000). Antagonism
of the BMP pathway by EGF signaling has previously been described in vitro
(Kretzschmar et al., 1997
) and
activation of Ras-dependent signaling suppresses EMT
(Lakkis and Epstein, 1998
).
Thus, the opposed phenotypes of mutants deficient in EGF (which can act
through Ras mediators) and Bmpr2 signaling raise the exciting possibility that
valve formation could be controlled by regulating the relative levels of
signal output from Ras- and BMP-dependent pathways.
In summary, our results show that the generation of Bmpr2 allele
encoding a protein with altered signal transduction properties can reveal
tissues the development of which requires wild-type levels of BMP signaling.
This approach is particularly useful for studying tissues for which Cre
transgenic strains are not available. Using this approach, we find that
septation and valvulogenesis of the mammalian OFT is crucially dependent upon
the level of BMP signaling. The finding that the OFT septation defect is
restricted in Bmpr2E2 mutants to the proximal
region provides further genetic evidence that mechanisms of septation of the
proximal and distal OFTs are distinct. Our findings show that EMT is initiated
and that at least some subpopulations of cardiac neural crest cells migrate
into the OFT. However, further development of the proximal OFT is impaired,
most probably due to defective cell-cell interactions that depend on BMP
signaling, as suggested by the downregulation of the BMP-responsive gene
periostin in the OFT. The use of Cre/loxP technology will determine which cell
populations require intact BMP signaling in order to mediate these important
cell-cell interactions.
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ACKNOWLEDGMENTS |
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