1 Developmental Biology Program, The Saban Research Institute of Childrens'
Hospital Los Angeles, Departments of Pathology and Surgery, Keck School of
Medicine, University of Southern California, Los Angeles, CA, USA
2 Cardiovascular Division, Department of Medicine and the Department of Cell and
Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA
* Author for correspondence (e-mail. vkaartinen{at}chla.usc.edu)
Accepted 8 April 2004
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SUMMARY |
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Key words: Bone morphogenetic proteins, ALK2, Outflow tract development, Mouse
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Introduction |
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The NC is a multipotent population of cells that originates from the dorsal
neural tube at all axial levels (LaBonne
and Bronner-Fraser, 1999). A subpopulation of NCCs called the
cardiac neural crest (CNC) delaminates from the rhombencephalon between the
mid-otic placode and the third somite
(Kirby et al., 1983
;
Kirby and Waldo, 1995
), and
migrates lateroventrally along the pharyngeal arch arteries into pharyngeal
arches 3, 4 and 6. Some CNC cells participate in patterning of pharyngeal arch
arteries, whereas others continue to migrate to the conotruncus, where they
contribute to formation of the aorticopulmonary septum
(Knecht and Bronner-Fraser,
2002
). BMPs have been shown to play an important role together
with other soluble factors, such as WNTs and FGFs, in NC specification,
possibly by serving a maintenance role in the induction process
(Gammill and Bronner-Fraser,
2003
). Misexpression of the BMP antagonist noggin (Nog) in the
premigratory NC disrupts early specification, although it is unclear if this
is a cell-autonomous effect (Ohnemus et
al., 2002
). A direct role for BMP signaling in later stages of
cardiac neural crest development has not been demonstrated.
Members of the TGFß superfamily, including BMP and TGFß proteins
signal through a heteromeric receptor complex of type I and type II
transmembrane Ser/Thr kinase receptors
(Massague, 1998). Upon ligand
binding, type II receptors, which are constitutively active kinases,
phosphorylate and activate type I receptors (also called ALKs)
(Massague, 2000
;
Derynck and Zhang, 2003
). It
has been shown that individual BMPs elicit distinct cellular responses and
bind to different type I receptors with different binding affinities. For
example, ALK3 and ALK6 are able to bind and transduce signaling from
structurally distinct BMPs (ten Dijke et
al., 1994
; Jamin et al.,
2002
). ALK2, however, is more specific and binds preferentially
the 60A subgroup of BMPs, i.e. BMP5, BMP6 and BMP7 in vitro
(Macias-Silva et al., 1998
).
Simultaneous inactivation of Bmp6 and Bmp7 in mice leads to
cardiac OFT, valve and septation defects
(Kim et al., 2001
).
Interestingly, Tgfb2 knockout mice display a plethora of phenotypes
including cardiac defects, such as double outlet right ventricle
(Sanford et al., 1997
), while
mice deficient in Tgfb1 or Tgfb3 do not display any cardiac
phenotypes (Shull et al.,
1992
; Kulkarni et al.,
1993
; Kaartinen et al.,
1995
; Proetzel et al.,
1995
). ALK2 was originally cloned as a TGFß type I receptor
(Ebner et al., 1993
), and,
indeed, it appears that in specific cell types, such as mouse mammary
epithelial cells and chick endocardial cushion cells, ALK2 can also mediate
TGFß signals (Miettinen et al.,
1994
; Lai et al.,
2000
). Although ALK2 is also able to bind activin in vitro
(Attisano et al., 1993
;
Tsuchida et al., 1993
), it
cannot transduce activin-like signals under physiological conditions
(Chen et al., 1997
;
Suzuki et al., 1997a
;
Macias-Silva et al.,
1998
).
Type I receptors are the primary determinants of the downstream signaling
specificity and therefore understanding their function is key to uncovering
molecular signaling mechanisms regulated by BMPs during embryogenesis.
Unfortunately, the early embryonic lethality at gastrulation has prevented the
use of conventional Alk2-knockout mice in assessing the role of ALK2
later in development (Gu et al.,
1999; Mishina et al.,
1999
). To circumvent this hurdle, we used the Cre-loxP strategy to
specifically abrogate Alk2 function in NCCs. The resulting ALK2
mutants display aortic arch and cardiac OFT defects reminiscent of common
forms of human congenital heart disease. Based on the presented results we
conclude that ALK2 plays an essential cell-autonomous role in the later stages
of cardiac neural crest development and differentiation.
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Materials and methods |
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Histological analyses
Embryonic tissues were fixed with 4% formaldehyde for 12 hours, dehydrated
and embedded in paraffin wax. Sections (5 µm) were stained with Hematoxylin
and Eosin. Embryos or dissected hearts were stained for ß-galactosidase
activity as described (Hogan et al.,
1994). Briefly, the specimens were fixed in 4% formaldehyde for 30
minutes at room temperature, washed three times for 10 minutes in the
detergent wash, and developed for 2-6 hours in the X-gal staining
solution.
Intracardiac ink injections
Indian ink was injected intracardially with custom made glass pipettes (12
µm opening) at E10.5 and E11.5. After injections, embryos were fixed in 4%
formaldehyde for 12 hours, dehydrated and cleared in benzyl benzoate:benzyl
alcohol (2:1).
In situ hybridization and immunohistochemistry
Radioactive in situ hybridization was performed as described
(Wawersik and Epstein, 2000).
Protocols are available at
www.uphs.upenn.edu/mcrc.
Whole-mount in situ hybridization on embryos was carried out as described
(Hogan et al., 1994
). Probes
specific for Bmp2 (Kim et al.,
2001
), Bmp4 (Kim et
al., 2001
), Bmp5
(Solloway and Robertson,
1999
), Bmp6 (Kim et
al., 2001
), Bmp7 (Kim
et al., 2001
), Tgfb2
(Blavier et al., 2001
),
Tgfb3 (Blavier et al.,
2001
), Msx1 (Furuta
et al., 1997
), Foxd3
(Labosky and Kaestner, 1998
),
Sox10 (Lioubinski et al.,
2003
), Plexin A2 (Plxna2 Mouse Genome
Informatics) (Brown et al.,
2001
) and Ednra
(Feiner et al., 2001
) were
used. For immunohistochemistry, fixed sections were stained with monoclonal
-smooth muscle actin antibody (DAKO) using Histomouse kit (Zymed)
according to the manufacturer's instructions.
Apoptosis and cell proliferation
Apoptotic cells were detected using the DeadEnd Fluorometric TUNEL system
(Promega). Cell proliferation was analyzed using the BrdU incorporation assay
(Zymed) or by immunostaining for phophohistone H3 (Cell Signaling). The
proliferation index was calculated as number of positively staining nuclei
divided by the total number of nuclei per cross-section of the OFT.
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Results |
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Alk2/Wnt1-Cre mutants display regression of the pharyngeal arch arteries
We examined pharyngeal arch arterial anatomy and patency at E10.5 and E11.5
using intracardiac India ink injections. At E10.5, the 3rd, 4th and 6th
pharyngeal arch arteries were indistinguishable between mutants and controls
(Fig. 4A,B). By contrast, at
E11.5, the 3rd and particularly 6th arteries of mutant embryos displayed
bilaterally inappropriate regression, while the 4th artery appeared relatively
normal (Fig. 4C,D). Recent
studies have demonstrated that NCCs populate the pharyngeal arch arteries and
differentiate into vascular smooth muscle cells
(Waldo et al., 1996;
Kochilas et al., 2002
).
Moreover, it has been shown that NCCs in a mouse model of DiGeorge syndrome
fail to differentiate appropriately in a process associated with aberrant
regression of pharyngeal arch arteries
(Lindsay et al., 2001
;
Kochilas et al., 2002
).
Therefore, we examined smooth muscle differentiation using an
-smooth
muscle actin (
SMA) antibody in Alk2/Wnt1-Cre embryos prior to
the time when inappropriate regression was apparent (E11.0). In controls,
SMA-positive cells formed a characteristic immunopositive ring around
the 3rd, 4th and 6th pharyngeal arch arteries
(Fig. 4E,G), while in
Alk2 mutants we consistently observed only weak and diffuse staining
around the 3rd and particularly 6th arteries
(Fig. 4F,I). Thus, the
pharyngeal arch arteries develop normally in Alk2/Wnt1-Cre mutants,
but the 3rd and 6th arteries regress inappropriately associated with defective
neural crest-derived smooth muscle cell differentiation.
|
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The detailed analysis of R26R reporter mice at E10.5 and E11.5
revealed that, although NCCs deficient in ALK2 populated the pharyngeal arch
mesenchyme and surrounded the forming aortic arch arteries
(Fig. 4H,J; Fig. 6A-F)
(Dudas et al., 2004), the
Alk2 mutants failed to demonstrate progression of NCCs into the
proximal OFT when compared with control littermates
(Fig. 6A-F). This finding was
verified by sectioning the specimens in the frontal plane
(Fig. 6G-L). In controls, the
mesenchyme of the OFT cushions was well formed and contained a large number of
ß-galactosidase-positive cells at all three levels. By contrast, the OFT
cushions in Alk2/Wnt1-Cre mutants were reduced in size. Moreover, the
proximal OFT was essentially devoid of NCCs, while the distal OFT displayed a
dramatic decrease in the amount of positively staining cells. At E13, when OFT
septation is complete, intense blue staining could be seen both in the aorta
and pulmonary trunk of control embryos, indicating neural crest derivatives.
Positively staining cells were also present in similar regions of the 7
Alk2 mutants studied, but the numbers of labeled cells were
smaller.
|
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The results of the present study imply that while cardiac NCCs deficient in Alk2 are capable of populating the conotruncus, they do not reach the OFT in sufficient numbers or do not otherwise function properly during the crucial period around E11.0 for OFT septation to occur normally. Thus, Alk2 is required cell-autonomously for successful NCC migration to the distal cardiac OFT and for appropriate remodeling of the aortic arch arteries.
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Discussion |
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Bmp6 and Bmp7 are both expressed in the cardiac OFT, and
Bmp6/;
Bmp7/ mutants display a delay in OFT and
ventricular septation (Kim et al.,
2001). Therefore, it is possible that these ligands transduce
their signals via ALK2 to induce OFT septation. However, defects displayed by
double knockouts are relatively mild compared with those seen in our
Alk2/Wnt1-Cre mutants. Hence, additional or distinct BMPs, such as
Bmp2 or Bmp5, are likely to be involved in ALK2 signaling
during cardiac OFT development. In addition, Bmp4 is expressed in the
OFT region and its abrogation specifically in cardiomyocytes has been shown to
lead to OFT abnormalities, such as double-outlet-right-ventricle and
atrioventricular canal defects (Jiao et
al., 2003
). Moreover, Delot and co-workers demonstrated that mice
homozygous for a hypomorphic BMP2 receptor (Bmpr2) display a failure
in aortico-pulmonary septation (Delot et
al., 2003
). However, these experiments did not involve a
tissue-specific gene inactivation approach, so it remains unclear whether this
defect reflects disruption of the same signaling pathway, as that reported
herein for ALK2. Interestingly, the defect in Bmpr2 hypomorphs was
detectable only below the valve level and was therefore distinctly different
from the defect seen in Alk2/Wnt1-Cre mutants. In addition to BMP
genes, TGFß genes, particularly Tgfb2 and Tgfb3, are
also strongly expressed in the cardiac OFT
(Fig. 1). Moreover,
Tgfb2 null mutants display aortic arch artery and OFT septation
defects (Sanford et al., 1997
;
Molin et al., 2002
). Based on
the phenotypic similarities between Tgfb2/
and Alk2/Wnt1-Cre mutants, we cannot exclude the possibility that
ALK2 is also involved in TGFß signaling during cardiac OFT development.
Interestingly, recent studies suggest that in endothelial cells, the TGFß
type I receptor (ALK5) mediates TGFß-dependent recruitment of ALK1, which
is closely related to ALK2, into a TGFß receptor complex
(Goumans et al., 2003
).
Whether similar heteromeric complexes between ALK2 and other type I receptors
occur in NCCs remains to be shown. However, our finding that expression of
MSX1 (a known BMP target) is significantly reduced in the distal OFT region
suggests that ALK2 probably mediates BMP signaling, but does not rule out
(additional) signaling through TGFßs. Alternatively, it is possible that
the differences in Msx1 expression are secondary and result from the altered
anatomy of the OFT.
It has been previously demonstrated that ALK2 is involved in cardiac
morphogenesis in amphibians and avians
(Ramsdell and Yost, 1999;
Lai et al., 2000
). Here, we
demonstrate that Alk2 is expressed in cardiac NCCs. This expression
pattern overlaps with another BMP1 receptor Alk3, which is
ubiquitously expressed, and required for atrioventricular cushion
morphogenesis (Mishina et al.,
1995
; Dewulf et al.,
1995
; Gaussin et al.,
2002
). Therefore, it is notable that ALK2 function in CNC cells
during OFT morphogenesis is essential and cannot be substituted by other type
I receptors, such as ALK3. It remains to be shown whether this reflects
differences in ligand preference, level of expression, or divergence in
downstream signaling mechanisms.
In mouse models of DiGeorge syndrome, the 4th and 6th arch arteries often
show inappropriate regression (Jerome and
Papaioannou, 2001; Kochilas et
al., 2002
). During normal development, the 3rd arch arteries give
rise to common carotid arteries, the 4th arch arteries contribute to the
formation of the distal part of the aortic arch, the brachiocephalic artery
and a proximal part of the right subclavian artery, while the sixth arch
arteries contribute to the ductus arteriosus and the proximal parts of the
pulmonary arteries. In Alk2/Wnt1-Cre mutants we detect inappropriate
regression of the 3rd and particularly the 6th arch arteries, whereas the 4th
arch arteries appear relatively normal. Despite this regression pattern, the
lungs and head appear to develop normally. This may reflect the substantial
capacity of embryos to overcome a localized arterial growth impairment as
previously suggested (Lindsay and Baldini,
2001
; Tallquist and Soriano,
2003
).
The fate of migrating NCCs is specified by the environment through which
they migrate (Trainor et al.,
2002). Consequently, the pharynx is a likely source of important
instructive signals for the migrating CNC cells. Interestingly, TBX1,
a gene associated with DiGeorge syndrome, is not expressed in the CNC, but in
the adjacent mesendoderm of the pharyngeal arches
(Garg et al., 2001
). Recent
studies have shown that expression of a secreted growth factor Fgf8
is diminished in Tbx1-expressing cells from Tbx1 mutant mice
(Vitelli et al., 2002
), and
that mice deficient in FGF8 display abnormal apoptosis of CNC cells and a
typical DiGeorge syndrome phenotype
(Abu-Issa et al., 2002
;
Brown et al., 2004
). However,
it is not currently known whether CNC cells are direct targets of FGF8 or
whether its effect is indirect. Although downregulation of BMP signaling in
pharyngeal endoderm seems to be a prerequisite for CNC cell survival
(Garg et al., 2001
;
Tiso et al., 2002
;
Bachiller et al., 2003
), our
present results demonstrate that signals transduced via ALK2 are required for
normal CNC cell differentiation and for successful colonization of the distal
OFT. Therefore, it is possible that FGF and TGFß superfamily signaling
pathways converge to control CNC cell fate during cardiac OFT morphogenesis.
Convergence of FGF and BMP signaling to orchestrate cell fate specification
has been demonstrated during limb and tooth development, and may be a common
theme during organogenesis (Peters and
Balling, 1999
; Zuniga et al.,
1999
; Capdevila and Izpisua
Belmonte, 2001
).
In addition to NCCs, the myocardium of the OFT is populated by cells from
the presumptive anterior (secondary) heart-forming field (AHF), which also
gives rise to the majority of cells in the right ventricle
(Kelly et al., 2001;
Yelbuz et al., 2002
;
Kelly and Buckingham, 2002
).
These two distinct populations of cells have been shown to be in close
apposition in the pharyngeal arch mesenchyme at E9, and therefore signaling
between them may play an important role in OFT morphogenesis
(Kelly and Buckingham, 2002
).
In accordance with this model, it has been demonstrated in the chick that NCCs
in the caudal pharyngeal arches are required for differentiation and function
of the myocardium, and it has been suggested that NCCs may regulate
availability of factors, such as FGF8, in the pharynx that control addition of
myocardium from the AHF (Waldo et al.,
1999
; Farrell et al.,
1999a
). In the present study, we show that Alk2/Wnt1-Cre
mice display OFT septation defects and significant lethality between E14 and
E18, and demonstrate remarkable hyperplasia of the right ventricle, a
phenotype not commonly seen in other mouse mutants with PTA. Therefore, it is
possible that signaling via ALK2 in NCCs is required for proper regulation of
cells in the AHF, and that disturbances in this process lead to defects not
only in the OFT, but also in the right ventricle. Alternatively, the
hyperplastic right ventricle may result from hemodynamic changes secondary to
PTA, although a similar degree of right ventricular hyperplasia is not seen in
other mouse PTA models.
In summary, tissue-specific knockout of the Alk2 gene in NCCs results in cardiac malformations reminiscent of common congenital heart defects seen in human newborns. As many newborns with aortic arch and/or outflow defects do not display characteristic deletions in the DGCR or mutations in TBX1, our results open the possibility that BMP signaling transduced via ALK2 in NCCs is part of a critical pathway involved in conotruncal development in humans. Moreover, the phenotypic characteristics of the Alk2/Wnt1-Cre mouse make it a valuable experimental model for the study of human conotruncal birth defects.
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ACKNOWLEDGMENTS |
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