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First published online 3 August 2006
doi: 10.1242/dev.02499
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1 Department of Pathology and Immunology, Washington University School of
Medicine, 660 South Euclid, St Louis, MO 63110, USA.
2 Developmental Biology Program, Washington University School of Medicine, 660
South Euclid, St Louis, MO 63110, USA.
3 Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, 660 South Euclid, St Louis, MO 63110, USA.
4 Molecular Developmental Biology Group, National Institute of Environmental
Health Sciences, Research Triangle Park, NC 27709, USA.
5 Genetics of Development and Disease Branch, NIDDK, NIH, Bethesda, MD 20892,
USA.
* Author for correspondence (e-mail: kchoi{at}wustl.edu)
Accepted 16 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hematopoiesis, Vasculogenesis, Endocardial cushion, FLK1 (KDR), ALK3 (BMPR1A), SMAD4, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Subsequently, its expression is confined to endothelial cells and to the
developing endocardial tube. Consistent with this expression pattern,
Flk1-deficient mice die between E8.5 and E9.5 with defects in
yolk-sac blood-island formation and vasculogenesis
(Shalaby et al., 1995). Cell
replating and transplantation experiments have demonstrated that
FLK1+ cells isolated from differentiated embryonic stem (ES) cells
or embryos generate hematopoietic, endothelial and vascular smooth muscle
cells. For example, ES- and embryo-derived FLK1+ cells, but not
FLK1- cells, are enriched for blast colony forming cells (BL-CFCs),
an in vitro equivalent of the hemangioblast
(Chung et al., 2002;
Faloon et al., 2000;
Huber et al., 2004). Ema et
al. (Ema et al., 2003) showed
that BL-CFCs are multipotential, such that blast colonies from single
FLK1+ cells could generate smooth muscle cells, as well as
hematopoietic and endothelial cells in vitro. Similarly, Yamashita et al.
(Yamashita et al., 2000)
reported that FLK1+ cells could generate both smooth muscle and
endothelial cells in vitro and in vivo. Subsequent fate-mapping studies of
FLK1+ cells by using Flk1+/Cre and Rosa26-LacZ
reporter (R26R) mice showed that FLK1+ cells could also contribute
to cardiac and skeletal muscle (Motoike et
al., 2003). Collectively, these studies indicate that
FLK1+ mesoderm generates cells of the circulation system.
Using the in vitro differentiation model of ES cells, we recently showed
that BMP4 was able to induce FLK1+ cells
(Park et al., 2004). In
serum-free conditions, BMP4 activated the SMAD1/5 pathway. Inhibition of the
SMAD1/5 pathway reduced the generation of FLK1+ cells. Consistent
with the notion that BMP4 is crucial for the generation of FLK1+
mesoderm, Bmp4-deficient mice die between E6.5 and E9.5 with defects
in mesoderm formation and patterning. Those that survive up to E9.5 show
severe defects in blood islands (Winnier
et al., 1995). Additionally, mice lacking the BMP receptor type IA
(ALK3; BMPR1A - Mouse Genome Informatics) fail to complete gastrulation, and
none survive past the E9.5 stage (Mishina
et al., 1995). Smad1-deficient mice display early
embryonic lethality and die between E9.5 and E10.5 owing to the failure of
chorioallantoic fusion (Lechleider et al.,
2001; Tremblay et al.,
2001). Smad5-deficient mice die between E9.5 and E11.5,
displaying anemia and disorganized vessels, despite the formation of the
primitive vascular plexus (Chang et al.,
1999; Yang et al.,
1999). Collectively, these studies have shed light on BMPs and
their downstream signaling in embryonic development. However, the early
lethality with complex abnormalities precludes placing the precise window in
which they play a role in hematopoietic and endothelial cell development.
To better understand the requirement of BMP signaling in blood and endothelial cell development, we inactivated Alk3 in FLK1+ mesoderm by using Flk1+/Cre knock-in mice. Our studies demonstrate an essential role for signaling through ALK3 in vessel remodeling and maturation. Mutant embryos died between E10.5 and E11.5, with defects in vessel remodeling and smooth muscle cell formation and/or recruitment to the dorsal aorta. Mutant embryos also displayed defects of endocardial cushion formation in the AVC. We propose that ALK3 signaling in FLK1+ mesoderm is crucial for proper vessel formation and heart development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The generation of Alk3floxed/floxed and
Smad4 floxed/floxed mice and genotyping were previously
described (Mishina et al.,
2002; Yang et al.,
2002).
Histological analysis
Embryos were fixed in 4% paraformaldehyde (PFA), paraffin embedded and
sagittally sectioned at 5 µm. For antigen retrieval, sections on the slides
were incubated with 0.1% trypsin for 15 minutes at 37°C and washed with
phosphate-buffered saline (PBS). The sections were immersed in blocking buffer
(5% normal goat serum and 0.1% Tween 20 in PBS) for 40 minutes at room
temperature, followed by overnight incubation at 4°C with anti-PECAM1
antibody (1:30, Pharmingen). After incubation with anti-biotinylated rat IgG
(1:200, Zymed), the sections were further incubated with anti-SMC actin
antibody (1:200, Sigma) and Cy5-conjugated streptavidin (1:400). For
cross-sections, frozen cryosections of 10 µm were stained with antibodies
as described above without an antigen retrieval step.
Whole-mount staining
Embryos were fixed overnight in Dent's fixative (methanol and DMSO, 4:1) at
4°C, followed by blocking in PBS containing 5% normal goat serum, 0.1%
Tween 20 and 2% non-fat skim milk) for 2 hours at room temperature. After
incubation with anti-PECAM1 antibody (1:300) and alkaline
phosphatase-conjugated anti-Rat IgG (1:250, Zymed), signals were detected with
the NBT/BCIP kit (Promega). For yolk sac, 4% PFA was used instead of Dent's
fixative, blocking and antibody incubation were performed in PBS containing 5%
normal goat serum and 1% DMSO (Morikawa
and Cserjesi, 2004).
Real time quantitative reverse transcription PCR
RNAs from tissues were extracted by using Trizol (Gibco). cDNA was
generated using the Invitrogen kit. Expression of genes indicated in the text
was measured by real-time qRT-PCR, and normalized by Gapdh
expression. Each experiment with duplicates was performed at least three
times. For primers, see Table
1.
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Hematopoietic colony assay
Hematopoietic colonies were generated as described previously
(Faloon et al., 2000;
Palis et al., 1999). Briefly,
yolk sacs were isolated, dissociated in collagenase with 20% FBS, and the
resulting cells were replated in 1% methyl cellulose containing hematopoietic
cytokines. The dissociated fetal liver cells from E10.5 embryos were replated
in Methocult M3434 (Stemcell Tech, CA). Hematopoietic colonies were counted
4-7 days later.
Statistics
The results of real-time quantitative reverse transcription PCR were
analyzed by Student's t-test. P<0.05 was considered
significant.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Flk1+/Cre mice, in which the
Cre gene is introduced into one allele of the Flk1 locus
(Motoike et al., 2003). The
expression of Cre recombinase in these mice is under the control of the
endogenous Flk1 promoter, providing the specific deletion of floxed
genes in FLK1+ cells. To generate Alk3 CKO mice, we first
set up matings between Flk1+/Cre and
Alk3floxed/floxed mice.
Flk1+/CreAlk3+/floxed mice were subsequently
mated with Alk3floxed/floxed mice to generate
Flk1+/CreAlk3floxed/floxed CKO mice
(Fig. 1A). The expected ratio
of obtaining Alk3floxed/floxed, Alk3+/floxed,
Flk1+/CreAlk3+/floxed,
Flk1+/CreAlk3floxed/floxed mice is 1:1:1:1,
respectively. Genomic DNA PCR was used to verify the expected genotypes
(Fig. 1B).
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; Johansson and Wiles,
1995; Kanatsu and Nishikawa,
1996; Liu et al.,
2003; Park et al.,
2004), it was expected that Alk3 CKO embryos might
display defects in hematopoiesis. Initial observation of the E9.5 mutants,
however, indicated that red blood cells were present
(Fig. 1D). To determine if the
level of hematopoietic progenitors present in the mutant yolk sacs was
diminished, we performed hematopoietic progenitor assays. Yolk sacs from E9.5
embryos were isolated, dissociated and plated into methylcellulose
supplemented with hematopoietic cytokines. As shown in
Fig. 2A, similar numbers of
erythroid and macrophage colonies developed from the Alk3 CKO and
control yolk sacs. The hematopoietic colonies that developed from the
Alk3 CKO yolk sacs were confirmed by Giemsa staining (not shown). To
determine whether incomplete Alk3 deletion could have resulted in the
generation of hematopoietic colonies from the Alk3 CKO yolk sac,
FLK1+ cells or TER119+ (LY76+ - Mouse Genome
Informatics) cells (erythrocytes) from E9.5 yolk sacs were FACS sorted and
subjected to genomic PCR. As shown in Fig.
2B, the correctly excised band was detectable in DNA obtained from
the Alk3 CKO yolk sac-derived FLK1+ cells or
TER119+ cells. By densitometry, we estimated that over 95% of the
floxed exon 2 was correctly deleted in the Alk3 CKO cells. Moreover,
when the Alk3 CKO yolk sac-derived hematopoietic colonies were picked
and subjected to genomic PCR, a PCR band corresponding to the floxed exon 2 of
Alk3 was present in the controls, but not in the mutants,
demonstrating that Alk3 was correctly deleted in all mutant-derived
hematopoietic colonies (Fig.
2C). Previous studies indicate that E9.5 yolk sacs contain
predominantly definitive hematopoietic progenitors
(Palis et al., 1999). To
further validate that definitive hematopoiesis was indeed unaffected in the
Alk3 CKO embryos, we dissected out E10.5 fetal liver and performed
hematopoietic colony assays. As shown in
Fig. 2D, Alk3 CKO
fetal liver generated a similar number of hematopoietic colonies compared with
controls. Collectively, the relatively normal frequency of hematopoietic
progenitors present in E9.5 mutant yolk sacs and in E10.5 mutant fetal livers
argues that ALK3 signaling is not constitutively required for hematopoietic
development.
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) and
Flk1+/CreSmad4+/floxed (see Table S1 and Fig.
S1 in the supplementary material). SMAD4 is the common SMAD that mediates
signaling by all members of the TGFß superfamily. Smad4 CKO
mutants died earlier than Alk3 CKO mutants, between E9.5 and E10.5
(see Table S1 in the supplementary material). Performing a hematopoietic
progenitor assay at E8.5 revealed that the number of hematopoietic colonies in
Smad4 CKO embryos was decreased by about 50% compared with controls
(Fig. 2E, upper). E9.5 yolk sac
replating generated similar results (Fig.
2E, lower). Hematopoietic colonies that developed from the
Smad4 CKO and wild-type yolk sacs were indistinguishable in size and
morphology. A complete deletion of floxed exon 8 of Smad4 was
confirmed by genomic PCR on yolk sac-derived hematopoietic colonies (see Fig.
S1 in the supplementary material). Collectively, these studies demonstrate
that BMP, TGFß or activin signaling contributes to hematopoietic
differentiation from FLK1+ mesoderm; however, such signaling is not
mediated by ALK3.
Defective vessel development in Alk3 CKO embryos
The Alk3 CKO mutants that died by E10.5 exhibited anemic yolk sacs
(Fig. 1F'). As
hematopoiesis was undisturbed, vessel development might be defective. To test
this possibility, we analyzed yolk sac vasculature by whole-mount staining
using anti-PECAM1 (CD31) antibody, a marker for endothelial cells. As shown,
wild-type embryos displayed large vessels with extensive branching and a
well-developed capillary network (Fig.
3A). However, Alk3 CKO E10.5 yolk sacs showed only the
primary vascular plexus of poorly developed vascular channels
(Fig. 3B). Similarly, abnormal
CD31 staining was obvious in the Alk3 CKO mutant brain vasculature
(Fig. 3D). From these
observations, we conclude that ALK3 is required for vessel remodeling.
To better understand the molecular mechanisms involved in ALK3-mediated
vessel remodeling, we examined the expression of members of the Id family
(inhibitor of DNA binding/differentiation). Id genes are important for
angiogenesis and are well-known direct downstream targets of BMP signaling
(reviewed by Ruzinova and Benezra,
2003). We first evaluated the expression of all Id genes in the
yolk sac using quantitative real-time reverse transcription PCR (qRT-PCR). As
shown in Fig. 3E, Id2
was expressed at high levels in E8.5-E10.5 yolk sac, while Id4 was
the least expressed. Importantly, the expression of all Id genes in
Alk3 CKO yolk sacs was decreased by 
40-60% compared with control
yolk sacs, suggesting that Id genes are downstream targets of ALK3 signaling
in mediating vessel remodeling (Fig.
3F). Additionally, we observed that the expression of
urokinase-type plasminogen activator (Plau) and peptidase inhibitor 1
(Serpine1), both of which are important mediators of angiogenesis,
was upregulated in Alk3 CKO yolk sacs
(Fig. 3F).
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-actin
antibody, a marker for vascular SMCs. We examined at least 15 different
regions in cross-sections (Fig.
4E-J) and five different regions in sagittal sections (not shown)
along the dorsal aorta per embryo. As discussed earlier, mutant embryos
appeared to have no obvious defects in vessel structure itself, as judged by
CD31 staining (Fig. 4I).
However, Alk3 CKO mutants appeared to have fewer SMCs around the
dorsal aorta, compared with wild-type embryos
(Fig. 4J). Subsequently, E11.5
dorsal aortas were isolated, RNA was prepared and subjected to qRT-PCR.
Alk3 CKO mutants expressed similar levels of Cd31 when
compared with controls (Fig.
4K, upper panels). However, they all expressed decreased levels of
both SMC -actin and myosin heavy chain (Mhc) when compared
with the controls, confirming a decreased number of SMCs around the dorsal
aorta.
To better characterize the blood vessel defects in Alk3 CKO
embryos, we examined E10.5 wild-type and mutant embryos (excluding those that
showed the pale yolk sac phenotype) by transmission electron microscopy (TEM)
(Fig. 4L). TEM studies revealed
obvious abnormalities in the mutant dorsal aortas. Consistent with SMC
-actin staining and qRT-PCR, the SMC number was diminished.
Furthermore, the SMCs that were present did not make close contacts with
adjacent endothelial cells. Although we observed relatively normal CD31
staining, there were frequent breaks in the endothelial cell layer in the
mutants, while no breaks were found in the wild-type dorsal aortas
(Fig. 4L). We further examined
genes involved in vessel stability/integrity using the qRT-PCR. Angiopoietin 1
and 2 (Angpt1 and Angpt2), and Tie2 expression
levels were comparable between control and Alk3 CKO embryos
(Fig. 4K, middle panels and not
shown). However, the expression of integrin V and ß3 was reduced
in the mutants, compared with controls
(Fig. 4,K lower panels).
Importantly, we found decreased expression of Pten, but increased
expression of Vegf in the Alk3 CKO embryos
(Fig. 4K, middle and lower
panels). All Smad4 CKO mutants displayed angiogenesis defects, as
only the primary vascular plexus was observed in E9.5 and E10.5 yolk sacs
(Fig. 5A). In these
Smad4 CKO yolk sacs, the expression of all Id genes was greatly
reduced compared with controls (Fig.
5B). The level of reduction was even greater in Smad4 CKO
yolk sacs compared with Alk3 CKO yolk sacs. Collectively, we conclude
that ALK3, SMAD4 and ID proteins are crucial mediators of vessel remodeling.
Moreover, defects in SMC recruitment and/or differentiation, reduced
interactions between endothelial cells, and/or increased VEGF could contribute
to the abdominal hemorrhage in Alk3 CKO embryos.
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). Thus,
we examined the hearts of Smad4 and of Alk3 CKO embryos by
Hematoxylin and Eosin staining. At E9.5, trabeculae in the ventricles and
endocardial cushion in the AVC begin to form
(Fig. 5C). As shown in
Fig. 5D, trabeculae in the
ventricles was much thinner and AVC endocardial cushion was not present in
Smad4 CKO hearts. At E10.5, trabeculae develop further and
endocardial cushions can be detected in both AVC and outflow track (OFT)
(Fig. 6A). When E10.5
Alk3 CKO hearts (excluding those with the pale yolk sac phenotype)
were examined, we detected no cushion cells in the AVC
(Fig. 6A). However, development
of the OFT cushion appeared to be normal. This observation raised the issue
whether FLK1+ cells contribute differently to the cushion cells of
the AVC and OFT. To address this, Flk1+/Cre mice were
crossed with Rosa26R-LacZ mice (Soriano et al., 1999). At E10.5, the majority
of lacZ-positive cells in the AVC were present in the cushion cell
layer and in the endocardium. In the OFT, lacZ-positive cells were
also found in the endocardium (Fig.
6B). By E12.5, all AVC-cushion cells were lacZ positive,
while OFT-cushion cells were composed of both lacZ positive and
negative cells. These results indicate that AVC cushion cells are derived
solely from FLK1+ cells, while OFT cushion cells could originate
from non-FLK1+ cells, presumably from the neural crest.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ALK3 in hematopoiesis
There is great interest in identifying inductive signals that initiate the
hematopoietic program. Xenopus animal cap explants, avian, mouse
ES/EB cell culture and knockout studies have suggested that members of
TGFß and fibroblast growth factor (FGF) families are crucial for
hematopoietic development (Adelman et al.,
2002; Chang et al.,
1999; Dickson et al.,
1995; Faloon et al.,
2000; Flamme et al.,
1995; Flamme and Risau,
1992; Huber et al.,
1998; Johansson and Wiles,
1995; Kanatsu and Nishikawa,
1996; Liu et al.,
2003; Maeno et al.,
1996; Oshima et al.,
1996; Pardanaud et al.,
1996). Among the members of the TGFß superfamily of growth
factors, numerous studies indicate that BMP4 is important for hematopoietic
development. We recently demonstrated that BMP4 is crucial for induction of
brachyury+ mesoderm from ES cells and FLK1+ cells from
brachyury+ cells. In combination with VEGF, BMP4 significantly
enhanced the production of SCL+ hematopoietic cells
(Park et al., 2004). Even
though these studies suggested that BMP4 contributed to hematopoietic
development, we could not position whether BMP4 functioned in the induction of
FLK1+ mesoderm or whether BMP4 was independently required for the
induction of SCL+ hematopoietic progenitors from FLK1+
mesoderm. To further elucidate the function of BMP/BMP receptor-mediated
signaling during the embryonic organogenesis, we generated Alk3 CKO
mice in which Alk3 was inactivated in FLK1+ cells. The
analyses of Alk3 CKO mutant yolk sacs and fetal livers indicated that
blood cells developed normally in these mutants. Although we cannot exclude
the possibility that other BMP type I receptors, such as ALK2 or ALK6,
compensated for the loss of ALK3 activity, our studies provide genetic
evidence that the BMP4-ALK3 axis is not crucial for hematopoietic development.
Our observation is consistent with a recent study that hematopoietic
development occurred normally in zebrafish when ALK3 signaling was blocked by
an inducible dominant-negative ALK3 expression after the onset of gastrulation
(Pyati et al., 2005).
The Alk3 CKO hematopoietic phenotype suggested that non-BMP
signaling, perhaps by signaling through TGFß or activin receptors could
contribute to hematopoietic cell development. Tgfb1-/- or
Tgfbr2-/- mice show defects in yolk sac hematopoiesis,
although Tgfbr1-deficient mice display enhanced hematopoiesis
(Dickson et al., 1995;
Larsson et al., 2001;
Oshima et al., 1996).
Additionally, activin A could increase hematopoietic development in the
presence of BMP4 and VEGF (Park et al.,
2004). To determine if any members of the TGFß growth factor
superfamily and their receptors are involved in hematopoietic specification,
we analyzed hematopoietic development in the Smad4 CKO embryos, and
found that mutant embryos were morphologically normal at E8.5. However, E8.5
and E9.5 mutant yolk sacs contained one half as many hematopoietic progenitors
as controls. This clearly indicates that SMAD4-dependent signaling contributes
to the formation of hematopoietic cells from FLK1+ mesoderm.
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;
Yamaguchi et al., 1994).
Subsequently, we demonstrated that Fgfr1-/- ES cells
differentiated up to the Brachyury+ cell stage but failed to
express normal level of FLK1, suggesting a role for FGFR1 in the early stages
of hematopoiesis (Faloon et al.,
2000). Treatment of wild-type ES cells with FGF2, a ligand for
FGFR1, enhanced the generation of FLK1+ cells
(Faloon et al., 2000). To
determine if the remaining hematopoietic activity in the Smad4 CKO
mutant is due to FGF signaling, we inactivated Fgfr1 or
Fgfr1 and Fgfr2 in FLK1+ cells. We found that
Fgfr1 CKO or Fgfr1/2 double CKO mice were born alive and
that E13.5 Fgfr1/2 double CKO fetal liver contained similar levels of
hematopoietic progenitors when compared with wild-type controls (see Fig. S2
in the supplementary material). Collectively, we conclude that FGF receptor
signaling is required to generate FLK1+ mesoderm, but is
dispensable for subsequent hematopoietic development. In the future, the
nature of the residual hematopoietic activity in Smad4 CKO embryos
still needs to be elucidated.
ALK3 in vessel development
Vessel formation in the developing embryo requires vasculogenesis and
angiogenesis with successful recruitment of supporting cells such as smooth
muscle cells (SMCs) and pericytes. Herein, we demonstrate that Alk3
CKO embryos display defects in vessel remodeling, as well as vessel
maturation/integrity. It is not clear why some of the mutants showed such a
severe angiogenesis phenotype, while the remaining mutant embryos showed
defects in vessel maturation and/or integrity. We suggest that it could be due
to mixed genetic background or due to efficiency and/or timing of the
recombination of Alk3 floxed alleles. Nevertheless, Alk3 CKO
mutant embryos that died by E10.5 all formed a primary vascular plexus, but
failed to complete angiogenesis. This observation provides the first genetic
evidence that ALK3 is crucial for vessel remodeling. Our results are
consistent with previous observations that BMP6 or BMP2 could promote
endothelial tube formation and migration of endothelial cells in vitro
(Langenfeld and Langenfeld,
2004; Valdimarsdottir et al.,
2002). Activation of ALK3 induced tube formation and the migration
of endothelial cells, in part, by stimulating the expression of Id1
(Valdimarsdottir et al.,
2002). Indeed, we found that expression of Id genes
(Id1-Id4) was decreased by 40-60% in the mutant yolk sacs, compared
with the controls. We propose that ALK3-mediated Id gene activation in
endothelial cells is crucial for vessel remodeling in the yolk sac.
Alk3 CKO mutant embryos that died by E11.5 appeared to develop
normal blood vessels in the yolk sac and in the embryo proper. Nonetheless,
these mutants displayed dilated vessels in the brain and abnormal branching in
the trunk and obvious vessel abnormalities when examined by TEM. The most
striking phenotype was that all the Alk3 CKO embryos showed abdominal
hemorrhage by E11.5, suggesting that vessel integrity is defective. It has
been reported that several pathways, including VEGF, PDGF/PDGFR, ANG1/TIE2 and
ALK-1/endoglin have important functions in vessel integrity. PDGFB could
recruit mural cells to endothelial cells
(Hirschi et al., 1999), and
mice lacking Pdgfb or Pdgfrb show a reduced degree of
vascular SMCs and pericytes in vessels
(Hellstrom et al., 1999;
Lindahl et al., 1997). More
recently, inactivation of Pdgfb in endothelial cells led to decreased
recruitment of pericytes to vessels
(Bjarnegard et al., 2004). In
the present study, staining with antibodies to CD31 and SMC- actin
revealed that the endothelial cell layer in the dorsal aortas of Alk3
CKO mutants was surrounded by a reduced number of SMCs. This observation was
confirmed by TEM, which showed that SMCs in the Alk3 CKOs did not
make close contacts with endothelial cells. More importantly, we found
frequent breaks in the endothelial cell layer on the dorsal aorta wall in the
Alk3 CKO mutants, while there were no obvious breaks found in the
dorsal aorta of the controls. Expression of integrin V and ß3 was
decreased in the mutants, although we did not find differences in angiopoeitin
1, angiopoeitin 2 and Tie2 expression. Importantly, Alk3 CKO
embryos expressed decreased Pten with a concomitant increase in
Vegf expression. There is emerging evidence that BMP signaling acts
in the upstream of the PTEN/PI3K/VEGF pathway. Treatment of BMP2 can stabilize
PTEN protein (Waite and Eng,
2003). Loss of Alk3 not only results in the inhibition of
PTEN activity, but also the activation of AKT, downstream of PI3K
(He et al., 2004). PTEN can
inhibit VEGF expression by inhibiting the PI3K/AKT pathway
(Jiang et al., 2000;
Pore et al., 2003).
Furthermore, a recent study has demonstrated that abrogation of Pten
in TIE2+ cells leads to defects in angiogenesis and that these
mutants display increased level of genes involved in vessel development
including Vegf (Hamada et al.,
2005). Collectively, we propose that ALK3 in FLK1+
cells may regulate vessel integrity through the PTEN/PI3K/VEGF pathway.
As for SMC defects in Alk3 CKO embryos, it is not clear whether
the Alk3 CKO vessel phenotype is due to Alk3 deficiency in
endothelial cells or whether SMC generation per se is defective. One possible
explanation would be that Alk3-deficient endothelial cells fail to
recruit SMCs to the dorsal aorta. However, we did not observe misplaced SMCs
around the dorsal aorta. Furthermore, Pdgfb was equally expressed in
wild-type and mutant dorsal aorta (not shown), although we cannot exclude the
possibility that other pathways could be regulated by ALK3. Alternatively,
absence of Alk3 could lead to defective SMC generation, resulting in
insufficient number of SMCs for vessel integrity. Indeed, we found decreased
expression of SMC -actin and myosin heavy chain in mutant dorsal aorta.
Recent studies have shown that vascular endothelial cells or FLK1+
endothelial cells can (trans)differentiate into SMCs during development
(DeRuiter et al., 1997;
Ema et al., 2003;
Frid et al., 2002;
Yamashita et al., 2000).
Therefore, ALK3 could be required for SMC generation and/or
transdifferentiation from endothelial cells.
Mice lacking Tgfb1, Tgfbr1 (Alk5), Alk1 or
Tgfbr2 all display varying degrees of yolk sac angiogenesis defects
(Dickson et al., 1995;
Larsson et al., 2001;
Oh et al., 2000;
Oshima et al., 1996). A
similar phenotype has also been reported for Smad5-/- mice
(Chang et al., 1999;
Yang et al., 1999).
Intriguingly, recent studies provide compelling evidence that ALK5 is
exclusively expressed on SMCs (Seki et
al., 2006). Thus, angiogenesis defects seen in Alk5
mutant mice are most probably due to SMC defect indirectly affecting
endothelial differentiation. Vessel remodeling and vessel maturation/integrity
defects observed in Alk3 CKO mutant mice are most similar to vessel
abnormalities observed in Alk1 knockout mice. Notably, mice deficient
in Alk1 showed dilated vessels. The Alk1-deficient dorsal
aorta had significantly reduced levels of SMC
(Oh et al., 2000), suggesting
that a close interaction between endothelial cells and supporting cells is
crucial for maintaining vessel integrity. Moreover, the expression of
Vegf, Plau1 and Serpine1 was increased in both Alk3
CKO and Alk1 mutant mice. Additionally, all Id genes were
downregulated in Alk3 CKO mutants. As ALK1 mediated signals in
endothelial cells were shown to activate Id genes
(Goumans et al., 2002), it is
of interest to see if Id gene expression is also downregulated in
Alk1 mutant mice. And yet, yolk sac vascular defects of Alk1
knockout mice appear to be more severe compared with Alk3 CKO
embryos. For example, remodeling defects in Alk1 knockout mice are
obvious as early as E9.5, at which time point Alk3 CKO embryos cannot
be distinguished from the control embryos. To this end, it is important to
note that all Smad4 CKO embryos show angiogenesis defects.
Collectively, we propose that vessel remodeling and maturation is achieved by
redundant as well as specific signaling mediated by ALK1 versus ALK3.
ALK3 and AV cushion formation
One of the most distinct features of heart morphogenesis is an
endothelial-mesenchymal transformation (EMT) (reviewed in
Person et al., 2005). During
this process, endocardium in the AVC and OFT differentiates into mesenchymal
cells, are delaminated, and eventually invade the cardiac jelly, the
extracellular matrix between the endocardium and the myocardium. The
mechanisms involved in cushion formation or its further morphogenesis have
been extensively studied. By using an in vitro explant culture system, it has
been shown that EMT could be induced by myocardium underlying the AVC
(Mjaatvedt et al., 1987;
Runyan and Markward, 1983).
BMP2 and TGFß2 could also induce EMT in endocardium explant culture
(Sugi et al., 2004).
Furthermore, Tgfb2-/- mice showed defects in the OFT, but
initiation of endocardial cushion formation seemed to be normal
(Sanford et al., 1997).
Consistently, conditional inactivation of Bmp2 in cardiomyocytes
using the Nkx2.5-Cre system results in endocardial EMT defects
(Ma et al., 2005). Conditional
deletion of Alk3 in cardiomyocytes using the Mhc-Cre system
results in heart abnormalities involving the interventricular septum,
trabeculae and AV cushion defects (Gaussin
et al., 2002). In these mice, Tgfb2 expression is greatly
reduced, implying that TGFß2 is crucial for AV cushion morphogenesis.
Outflow tract defects are not observed in these mice. Intriguingly,
conditional inactivation of Bmp4 in cardiomyocytes using the
Nkx2.5-Cre system results in defective outflow tract septation and
abnormal morphogenesis of branchical arch arteries
(Liu et al., 2004), while
Bmp4 inactivation using the rat troponin T (Tnnt)-Cre
results in severe AVC defects (Jiao et
al., 2003). Collectively, these studies emphasize the importance
of myocardium in inducing EMT. It is important to note that
Alk3-mediated signaling in the cardiomyocytes is important for
myocardium proliferation and for AVC morphogenesis.
Herein, we demonstrate that ALK3 signaling in the endocardium is
intrinsically required for AV cushion formation. The selective absence of the
endocardial cushions in the AVC raised the issue of whether the origin of
endocardium in the AVC is different from that of the OFT. However, we found
that FLK1+ cells can contribute to both AVC and OFT endocardial
cushions. Given the finding that neural crest cells also contribute to the
OFT, but not to the AV cushion (Epstein et
al., 2000; Gitler et al.,
2003), it is possible that ALK3 signaling in neural crest cells in
the OFT rescued the Alk3 deficiency in the FLK1+ cells.
Recently, Wang et al. (Wang et al.,
2005) demonstrated that conditional inactivation of Alk-2
using the Tie-2-Cre system resulted in selective defects in the AV
superior cushion. The AV inferior cushion appeared to be normal in these mice.
At E14.5, the surviving Alk-2 CKO mutant embryos showed a range of
ventricular septal defects. The differences in AV cushion defects observed in
our studies compared with Wang et al.
(Wang et al., 2005) could be
due to the nature of cells that express Cre (FLK1+ versus
TIE2+ cells). However, the specificity of the ALK2 versus ALK3 in
superior versus inferior cushion formation cannot be ruled out. As for the
timing of the Alk3 inactivation, it is worth noting that about a
third of Alk3 conditional mutants using the Tie2-Cre system
developed relatively normal AV cushion (Ma
et al., 2005). Importantly, conditional inactivation of
Alk3 in neural crest cells using the Wnt1-Cre system results
in outflow tract defects including a septal failure and reduced conotruncal
length (Stottmann et al.,
2004). Moreover, mice homozygous for a hypomorphic allele of
Bmpr2 displayed defects in the formation of the OFT valve, but the
AVC cushion formation was normal, despite the ubiquitous expression of
Bmpr2 in the heart (Delot et al.,
2003; Roelen et al.,
1997).
During the AV cushion EMT, AV endocardium loses the characteristics of
endothelial cells and cell-cell interaction, while activating genes for
mesenchymal cells. Snai1, a Slug family member, and Twist1,
a basic HLH transcription factor is known to promote the EMT by downregulating
VE-cadherin and/or E-cadherin (Timmerman
et al., 2004; Yang et al.,
2004). We found a reduced expression level of Snai1 and
Twist1 with sustained expression of VE-cadherin in Alk3 CKO
embryos. Inactivation of Alk3 in Tie2 Cre mice showed a loss
of Twist1 and a mild defect in VE-cadherin, although Snail1
expression was not affected (Ma et al.,
2005). SOX9, a high-motility group transcription factor, is
expressed in the mesenchymal cells of AV and OFT cushion
(Akiyama et al., 2004). SOX9
has been implicated in neural crest cell delamination and AVC cushion
formation (Akiyama et al.,
2004; Cheung and Briscoe,
2003). Conditional knockout of Sox9 in the
Wnt1-Cre system showed ectopic expression of Nfatc1, leading
to a failure of cushion cell development
(Akiyama et al., 2004). We
observed that Sox9 expression was downregulated, while
Nfatc1 expression was not affected in Alk3 CKO hearts. These
results suggest that ALK3 signaling in the AV endocardium is crucial for the
initiation stage of the AV EMT. As Snail and Sox9 are important regulators of
Notch and Wnt/ß-catenin, respectively, it will be interesting to see if
ALK3 signaling interacts with these pathways in the AV EMT. Collectively, we
show that ALK3 signaling is required for the AV cushion formation. Further
studies on redundant as well as distinct signaling mediated by ALK3 versus
ALK2 are warranted.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/17/3473/DC1
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ACKNOWLEDGMENTS |
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