Msx1 and Msx2 regulate survival of secondary heart field precursors and
post-migratory proliferation of cardiac neural crest in the outflow tract
Yi-Hui Chen, Mamoru Ishii, Jingjing Sun, Henry M. Sucov, Robert E. Maxson Jr.⁎
Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center and Hospital,
University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA
Received for publication 20 December 2006; revised 21 May 2007; accepted 29 May 2007
Available online 4 June 2007
Msx1 and Msx2 are highly conserved, Nk-related homeodomain transcription factors that are essential for a variety of tissue–tissue interactions
during vertebrate organogenesis. Here we show that combined deficiencies of Msx1 and Msx2 cause conotruncal anomalies associated with
malalignment of the cardiac outflow tract (OFT). Msx1 and Msx2 play dual roles in outflow tract morphogenesis by both protecting secondary
heart field (SHF) precursors against apoptosis and inhibiting excessive proliferation of cardiac neural crest, endothelial and myocardial cells in the
conotruncal cushions. During incorporation of SHF precursors into the OFT myocardium, ectopic apoptosis in the Msx1−/−; Msx2−/− mutant
SHF is associated with reduced expression of Hand1 and Hand2, which from work on Hand1 and Hand2 mutants may be functionally important
in the inhibition of apoptosis in Msx1/2 mutants. Later during aorticopulmonary septation, excessive proliferation in the OFT cushion
mesenchyme and myocardium of Msx1−/−; Msx2−/− mutants is associated with premature down-regulation of p27KIP1, an inhibitor of cyclin-
dependent kinases. Diminished accretion of SHF precursors to the elongating OFT myocardium and excessive accumulation of mesenchymal cells
in the conotruncal cushions may work together to perturb the rotation of the truncus arteriosus, leading to OFT malalignment defects including
double-outlet right ventricle, overriding aorta and pulmonary stenosis.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Msx1, Msx2; Aorticopulmonary septation; Apoptosis; Cardiac neural crest; Conotruncus; Myocardium; Outflow tract malalignment; Proliferation;
Secondary heart field
The heart is composed of several cell types with distinct
lineage origins. These include cells of the primary and
secondary heart fields, which contribute to the myocardium,
and cardiac neural crest (NC) cells, which contribute to the
aorticopulmonary septum and membranous ventricular septum
(Mikawa, 1999). Perturbation of interactions between these
cardiac progenitors can result in congenital heart malforma-
tions, the most predominant of which are impaired alignment
and septation of the conotruncal region, or the outflow tract
(OFT) (Hoffman and Kaplan, 2002). The cardiac OFT is an
intricate structure formed by a series of complex morphogenetic
processes, including elongation, rotation, alignment and septa-
tion. OFT elongation requires accretion of extracardiac cells,
including myocardial precursors from the secondary heart field
(SHF) and NC cells, to the primitive heart tube, which is
composed of myocardium and endocardium originating from
the primary heart field (Kirby, 2002; Restivo et al., 2006). SHF
precursors migrate progressively through the aortic sac and
become incorporated into the OFT at different times during
cardiac looping (Cai et al., 2003; Kelly et al., 2001; Mjaatvedt et
al., 2001; Waldo et al., 2001). NC cells are required for
lengthening of the OFT prior to cardiac looping as well as
rotation of the OFTafter looping (Bajolle et al., 2006; Restivo et
al., 2006; Yelbuz et al., 2002). Elongation of the OFT is a
prerequisite for correct cardiac looping as well as complete
rotation and proper alignment during aorticopulmonary
As NC cells colonize the OFT mesenchyme to build up a
spiral configuration of the septating conotruncal cushions
Developmental Biology 308 (2007) 421–437
⁎Corresponding author. Fax: +1 323 865 0098.
E-mail address: firstname.lastname@example.org (R.E. Maxson).
0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
(Fananapazir and Kaufman, 1988; Waldo et al., 1999), the SHF-
derived myocardium of the distal OFT must undergo a complete
rotation to ensure the alignment of the outflow septum anlage
with the interventricular septum (Lomonico et al., 1986; Yasui
et al., 1997). A dramatic increase in the rate of growth of the
distal OFT relative to the heart facilitates the counterclockwise
rotation of the great arteries (Lomonico et al., 1986). Incomplete
or arrested rotation of the OFTat different developmental stages
causes different OFT malalignment defects including transposi-
tion of the great arteries (TGA), double-outlet right ventricle
(DORV) and tetralogy of Fallot (TOF) (Bostrom and Hutchins,
1988; Lomonico et al., 1986).
Cell proliferation, differentiation and apoptosis in the
developing OFT myocardium and cushions are finely tuned
to ensure timely remodeling of the OFT (Barbosky et al.,
2006; Cai et al., 2003; Kioussi et al., 2002; Martinsen, 2005;
Risebro et al., 2006; Trinh et al., 2005; Watanabe et al., 1998).
To achieve the exquisite regulation of OFT remodeling,
different cell populations in the developing OFT interact and
develop coordinately through a complex network of signaling
molecules and transcription factors (Brand, 2003; Hutson and
Kirby, 2003; Restivo et al., 2006; Srivastava, 2006; Srivastava
and Olson, 2000). Although this network is understood in
broad outline, a detailed picture of its components and
function is lacking.
Msx1 and Msx2, members of the highly conserved Nk family
of homeodomain transcription factors, play important roles in
tissue–tissue interactions and organogenesis during vertebrate
development (Ishii et al., 2003, 2005; Maxson et al., 2003).
Single mutants of Msx1 and Msx2 as well as Msx1−/−; Msx2−/−
double mutant mice all exhibit impaired development of cranial
neural crest-derived structures (Ishii et al., 2005; Satokata et al.,
2000; Satokata and Maas, 1994). Defects in double mutants are
much more severe than in single mutants (Ishii et al., 2005;
Satokata et al., 2000; Satokata and Maas, 1994). Interestingly,
while neither Msx1−/− nor Msx2−/− mutant mice have any
cardiac defect (Satokata et al., 2000; Satokata and Maas, 1994),
OFT malformations including DORV, TOF and persistent
truncus arteriosus (PTA) (Ishii et al., 2005 and this study),
suggesting functional redundancy between Msx1 and Msx2
during OFT morphogenesis.
Here we address the roles of Msx1 and Msx2 in OFT
morphogenesis by elucidating the mechanistic basis of OFT
malformations in Msx1−/−; Msx2−/− mutant mice. We found
that a single allele of Msx1 or Msx2 is sufficient for
cardiogenesis. Msx1 or Msx2 is required for the survival of a
subpopulation of SHF precursors at the time of migration into
the OFT myocardium. An Msx gene is also required to
restrict the expansion of the post-looping OFT, possibly by
maintaining a high level of p27KIP1, an inhibitor of cyclin-
dependent kinases, during early aorticopulmonary septation.
Our study thus uncovers two distinct roles for Msx1 and
Msx2 in OFT morphogenesis, one in the survival of a
subpopulation of OFT myocardial precursors in the SHF and
the other in the post-migratory expansion of the conotruncal
Materials and methods
Mouse strains and genotyping
All mice used in this study were maintained in a mixed genetic background
of BALB/c and CD-1. Msx1−/− and Msx2−/− single knockout and Msx1−/−;
Msx2−/− double knockout mice were described previously (Ishii et al., 2005;
Satokata et al., 2000; Satokata and Maas, 1994). The noon copulation plug was
counted as embryonic day 0.5 (E0.5). Genomic DNA was extracted from yolk
sac (embryos) or tails (postnatal mice) for genotyping. PCR primers and
conditions for Msx1 and Msx2 knockout alleles as well as the Wnt1-Cre and
R26R transgenes were as described (Jiang et al., 2000; Satokata et al., 2000;
Satokata and Maas, 1994).
Cryostat sectioning and section in situ hybridization
E10.5 embryos were prefixed overnight at 4 °C in 4% paraformaldehyde
(PFA) in phosphate-buffered saline (PBS) followed by cryopreservation in
sucrose solutions with increasing concentrations and freezing in OCT (Jiang et
al., 2000). Cryostat sections were cut at 10-μm thickness and RNA in situ
hybridization was performed usingthe In Situ Hybridization kit (BioChain) with
the protocol modified according to Dijkman et al. (www.roche-applied-science.
com/PROD_INF/MANUALS/InSitu/pdf/ISH_181-188.pdf). All RNA probes
were generated as described: Isl1 (Cai et al., 2003), Mef2c (von Both et al.,
2004), Hand1 (McFadden et al., 2005), Hand2 (McFadden et al., 2005) and
Pitx2 (Lu et al., 1999).
Whole embryos were prefixed for 30 min at room temperature in 4% PFA in
PBS and stained with X-gal as described (Jiang et al., 2000). Stained embryos
were postfixed in 4% PFA in PBS overnight at 4 °C, dehydrated through graded
ethanol and then embedded in paraffin. Sections were cut at 5-μm thickness,
fixed in acetone and counter-stained with nuclear fast red.
Immunostaining and detection of cell proliferation
Cryostat sections were immunostained with the following primary
antibodies: mouse monoclonal anti-α-smooth muscle actin (1:800, Sigma),
mouse monoclonal anti-AP-2α (1:4, Developmental Studies Hybridoma Bank)
(Zhang et al., 1996), rabbit polyclonal anti-β-galactosidase (1:100, MP
Biomedicals), biotinylated goat polyclonal anti-BMP-2/4 (1:100, R&D
Systems), mouse monoclonal anti-bromodeoxyuridine (BrdU) (1:100, Sigma),
rabbit polyclonal anti-cleaved Caspase 3 (1:100, R&D Systems), mouse
Outflow tract and vascular malformations in Msx1−/−; Msx2−/− double
Tetralogy of Fallot
RERSCA, right dAo
4th aortic arch
adAo, dorsal aorta; DORV, double-outlet right ventricle; PTA-A4, persistent
truncus arteriosus type A4 (see text for details); RERSCA, retroesophageal right
422 Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
monoclonal anti-Connexin 43 (1:50, Chemicon), goat polyclonal anti-p21CIP1
(1:100, Santa Cruz Biotechnology), mouse monoclonal anti-p27KIP1(1:100,
Zymed), rabbit polyclonal anti-phospho-Smad1/5/8 (1:50, Cell Signaling),
guinea pig polyclonal anti-Pitx2 (1:200, a kind gift from Dr. Kioussi) (Kioussi et
al., 2002) and mouse anti-proliferating cell nuclear antigen (PCNA) (in the
Zymed PCNA staining kit).
Immunostaining using anti-PCNA or anti-BrdU antibodies was carried
out to detect cell proliferation. BrdU (Sigma) in 1× PBS was injected
intraperitoneally into the pregnant mouse (200 μg/g of body weight) 2 h
prior to embryo harvesting. Harvested embryos were fixed, washed and
cryosectioned as described above followed by immunostaining with the
anti-BrdU antibody (Sigma). The Quantity-One program (Bio-Rad) was
used to count cells from all sections of an OFT per embryo (on average 4–
5 sections for embryos at E10, 6–7 sections at E11, and 8 sections at
E12). CNC cells were labeled by the Wnt1-Cre/R26R two-component
transgenic reporter and were detected by the presence of β-galactosidase
(Jiang et al., 2000). CNC cell proliferation indices were represented by the
percentage of BrdU+/β-galactosidase+ double positive cells in the whole
population of β-galactosidase+ cells (i.e., CNC cells). Littermates of each
Msx1−/−; Msx2−/− mutant embryo were used as the controls, and no
Fig. 1. Cardiac outflow tract defects in Msx1/2 double mutant embryos. (A–C) A series of transverse sections (ordered rostral to caudal) of one control (Msx1+/−)
embryo illustrate the normal alignment of the pulmonary trunk with the right ventricle and the ascending aorta with the left ventricle, which is separated from the right
ventricle by a normal interventricular septum continuous with the conotruncal tissue. (D, E, G) Transverse sections of one Msx1/2 mutant embryo (number 5 in
Table 1) ordered rostral to caudal in the G-D-E sequence indicate persisting right-sided dorsal aorta (G) as well as the common origin of the ascending aorta and
pulmonary trunk from the right ventricle (double-outlet right ventricle; compare panels D and E with panels A and B), although the valves of each vessel are distinct.
The white arrow indicates the connection between the ascending aorta and the right ventricle. (F, I) Transverse sections of Msx1/2 mutant embryo 1 (Table 1) display
ventricular septal defect (F) and double-outlet right ventricle with pulmonary stenosis (I). Note the normal morphology and size of the ascending aorta in spite of a
severely reduced size of the pulmonary trunk, which may result from the obstruction by the aorta (I). (H) Malaligned persistent truncus arteriosus in Msx1/2 mutant
embryo 7 reveals the origination of the common truncus arteriosus from the right ventricle, illustrating combination of the malalignment and septation defects in the
cardiac outflow tract. Ao, ascending aorta; ct, conotruncal tissue; IVS, interventricular septum; LA and LV, left atrium and ventricle; pa, pulmonary arteries; PT,
pulmonary trunk; RA and RV, right atrium and ventricle; rdAo, right-sided dorsal aorta; TA, truncus arteriosus; VSD, ventricular septal defect. Scale bars in panels A
(for A–C), D (for D, E, G), F (for F, I) and H are 0.5 mm.
423 Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
424 Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
discernable variation in either gene expression or cell proliferation/survival/
differentiation was detected between the controls from the same litter and
with different genotypes.
Cardiac outflow tract defects in mice with combined
deficiencies of Msx1 and Msx2
In our previous study, we noted the presence of conotruncal
abnormalities in a small number of Msx1−/−; Msx2−/− double
mutant embryos, but did not evaluate these in detail (Ishii et al.,
2005). We have now analyzed eight embryos in total (Table 1).
Five Msx1/2 mutant embryos had both OFTs originating
completely from the right ventricle (i.e., DORV). This
phenotype occurs when the OFT does not lengthen sufficiently
to allow the juxtaposition of the OFT with the septating
Of these five embryos, in two the aorta and pulmonary trunk
were oriented in a nearly side by side fashion (conventional
DORV; Figs. 1D, E) whereas in the other three the aortic valve
was located left and anterior to the pulmonary valve
(transposition type DORV (Bostrom and Hutchins, 1988);
data not shown). One embryo of the eight had an overriding
aorta, which, compared to DORV, is a less severe manifestation
of incomplete OFT elongation.
One embryo exhibited tetralogy of Fallot (TOF). The
pulmonary trunk had regressed due to atresia, and both
pulmonary arteries were supplied retrograde from a normal
ductus arteriosus originating from the dorsal aorta (data not
shown). TOF results from obstruction of the early OFTsuch that
the pulmonary channel does not form, and in this case, the
obstruction was complete: only a single outflow vessel was
apparently leaving the heart. The diagnosis of TOF as compared
to truncus arteriosus was possible because of the retrograde
origin of the pulmonary arteries from the dorsal aorta.
In the remaining embryo with PTA, there was a hypoplastic
left 4th aortic arch artery (PTA type A4 (Van Praagh and Van
Praagh, 1965)). Interestingly, the PTA was also malaligned
since the truncus originated predominantly from the right
ventricle (Fig. 1H).
All eight Msx1/2 null mutants had a membranous
ventricular septal defect (VSD) (Fig. 1F) due to malorientation
of the OFT (conus) septum and subsequent imperfect fusion
with the interventricular (conoventricular) septum (Bostrom
and Hutchins, 1988; Yasui et al., 1997). Thus, our analyses
reveal that the absence of both Msx1 and Msx2 gene products
results in a range of OFT malformations, all consistent with
problems in OFT elongation and alignment.
Histological analyses revealed normal cardiac development
in Msx1−/− and Msx2−/− single mutants (n=22) and in Msx1–
Msx2 homozygous–heterozygous compound mutants (n=20).
Although Msx1−/− mutant pups did not survive to term due to
cleft palate, mutant mice of the other genotypes including
Msx1+/−, Msx2+/−, Msx1+/−; Msx2+/−, Msx2−/− and
Msx1+/−; Msx2−/− have all been raised to adulthood without
any apparent cardiac manifestation. Therefore, our analyses
indicate that a single allele of either Msx1 or Msx2 is sufficient
to support normal cardiogenesis.
Msx1 and Msx2 are required for survival of a subpopulation of
secondary heart field cells whose normal fate is to be
incorporated into the outflow tract myocardium
Since the OFT malalignment defects in Msx1/2 null mutants
are consistent with a disruption of the SHF (Hutson and Kirby,
2003; Ward et al., 2005), we sought to address the roles of Msx1
and Msx2 in SHF development. We first examined the
expression patterns of Msx1 and Msx2 in the SHF and OFT
between embryonic days 9.5 and 11.5 (E9.5–E11.5), spanning
the phases of cardiac looping and aorticopulmonary septation.
The patterns of Msx1 and Msx2 expression largely overlapped
in most sites, including pharyngeal endoderm and mesoderm,
splanchnic mesoderm, somatic mesoderm, epicardium as well
as subpopulations of the OFT myocardium, endocardium and
cushion mesenchyme (Figs. 2A–F). This overlap supports the
hypothesis that Msx1 and Msx2 are functionally redundant in
OFT morphogenesis. In the right ventricle, however, Msx1
expression was confined to a subpopulation of myocardial cells
at E10.5 and was significantly reduced at E11.5 compared with
Msx2, which spanned a broader range in the ventricular
myocardium (Figs. 2B, C, E, F).
We showed previously that Msx1 and Msx2 are required for
the survival of NC subpopulations in the region of the
trigeminal ganglion and in the first pharyngeal arch (Ishii et
al., 2005). To investigate whether Msx1 and Msx2 promote the
survival of cardiac progenitors, we performed immunostaining
for cleaved Caspase 3 in transverse sections of E9.0–E9.5
Msx1−/−; Msx2−/− double mutants and control littermates at
the levels of the second and third pharyngeal arches.
Fig. 2. Section in situ hybridization to analyze marker gene expression in the Msx1/2 mutant SHF and OFT. (A–F) Overlapping expression of Msx1 (A–C) and Msx2
(D–F) in the pharyngeal endoderm and mesoderm, splanchnic and somatic mesoderm, epicardium, cardiac neural crest as well as the endocardium and myocardium of
the OFTand right ventricle in wild-type embryos at E9.5 (A, D), E10.5 (B, E) and E11.5 (C, F). Black arrows in panels B and C denote the absence of Msx1 expression
in a subpopulation of myocardial cells in the right ventricle at E10.5 and almost no Msx1 expression in the right ventricular myocardium at E11.5 (compare with Msx2
expression in the right ventricular myocardium at E10.5 and E11.5: black arrows in panels E and F). (G–N) Comparable expression of Isl1 (G–J) and Mef2c (K–N)
between Msx1/2 null mutants and littermate controls in the pharyngeal endoderm and mesoderm, splanchnic and somatic mesoderm as well as OFT myocardium.
Asterisks indicate Isl1 expression in the motor neurons (Pfaff et al., 1996). (O–V) Significantly decreased expression of Hand1 (O, P, S, T) and Hand2 (Q, R, U, V) in
the pharyngeal mesoderm (triangle arrowheads), splanchnic and somatic mesoderm (notched arrowheads), cardiac neural crest, OFT myocardium and epicardium (for
Hand1 only) in Msx1/2 null mutants compared with littermate controls at both E9.5 (O–R) and E10.5(S–V). Note a greater degree of reduction of Hand1/2 expression
in the splanchnic and somatic mesoderm (notched arrowheads) compared with the pharyngeal mesoderm (triangle arrowheads) in Msx1/2 mutants at both E9.5 (P, R)
and E10.5 (T, V). AoS, aortic sac; APS, aorticopulmonary septum; ba, branchial (pharyngeal) arch; CV, common ventricle; FG, foregut; LA and LV, left atrium and
ventricle; OFT, outflow tract; PM, pharyngeal mesoderm; RA and RV, right atrium and ventricle; SoM, somatic mesoderm; SpM, splanchnic mesoderm. Scale bars:
0.2 mm in panels C, F, H (for G–J), L (for K–N) and T (for S–V); 0.1 mm in panels D (for A, D), P (for O, P) and R (for Q, R).
425Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
While we did not detect cleaved Caspase 3-positive cells in
the control SHF at E9.0 or E9.5 (red signals in Figs. 3A, C, E,
G; green signals in Figs. 3I, K), we did observe apoptotic cells
in the Msx1/2 null mutant SHF. The signal, most extensive at
E9.0, spanned distal OFT myocardium, somatic mesoderm
and a subpopulation of splanchnic mesoderm adjacent to
pharyngeal mesoderm (green signals in Figs. 3J, L; red signals
in Figs. 3B, D, F, H).
Cai and colleagues (2003) documented extensive ectopic
apoptosis of SHF precursors in embryos deficient for the LIM
homeobox transcription factor, Isl1, at the same developmental
stages (Cai et al., 2003). However, apoptotic cells in Isl1 null
mutants were confined to SHF subpopulations distinct from
those in Msx1/2 null mutants: they were located in pharyngeal
endoderm and ectoderm as well as in a subpopulation of
splanchnic mesoderm adjacent to pharyngeal endoderm (Cai
et al., 2003).
Consistent with these results, we did not detect a change
in the expression of Isl1 or its downstream effector, Mef2c,
an MADS box transcription factor (Dodou et al., 2004), in
Msx1−/−; Msx2−/− mutant embryos (Figs. 2G–N). We also
analyzed cell proliferation in the Msx1/2 mutant SHF and OFT
at the same developmental stages and did not detect any
significant change (red signals in Figs. 3I, J).
To confirm that the apoptotic cells in the Msx1/2 mutant
splanchnic mesoderm are indeed SHF precursors whose normal
fate is to be added to the elongating OFT, we performed double
immunostaining with anti-Pitx2 and anti-cleaved Caspase 3
antibodies. Pitx2 is expressed in the left SHF. Its function is
required for the development of SHF but not cardiac NC cells
(Ai et al., 2006; Liu et al., 2002).
We found that apoptotic cells in left splanchnic mesoderm of
Msx1/2 null mutants were all positive for Pitx2 (Figs. 3B, D, F,
H). Immunostaining at E10.5 revealed dramatically reduced
numbers of Pitx2-positive cells in the pharyngeal mesoderm,
left somatic and splanchnic mesoderm as well as the left distal
OFT myocardium of Msx1/2 double mutants (Figs. 3M–P and
data not shown), indicating that Pitx2-expressing cells in these
Fig. 3. Ectopic apoptosis of a subpopulation of Pitx2-expressing cells in the Msx1/2 mutant SHF and distal OFT. (A–P) All nuclei were visualized by DAPI counter-
staining (blue fluorescence). Panels C, D, G, H, K, L, O andP are enlarged views of the boxes focusing on the splanchnicand somatic mesodermin panels A, B, E, F, I,
J, M and N, respectively. (A–L) Immunofluorescent staining for cleaved Caspase 3 demonstrates ectopic apoptosis in the Msx1/2 mutant splanchnic and somatic
mesoderm at both E9.0 (A–H) and E9.5 (I–L), with more apoptotic cells present at E9.0 (compare panels D and H with L). (A–H) Double immunostaining for Pitx2
(green FITC fluorescence) and cleaved Caspase 3 (red Rhodamine fluorescence) reveals that Pitx2-expressing cells undergo apoptosis in the left SHF and distal OFT
myocardium of the Msx1/2 null mutant but not in the control (orange merged signals indicated by white notched arrowheads in panels B and F; see also panels D and
H). White arrows in panels B and F indicate apoptotic cells in the right SHF and distal OFT myocardium of Msx1/2 null mutants, which do not express Pitx2. (I–L)
Double immunostaining for proliferating cell nuclear antigen (PCNA; red Rhodamine fluorescence) and cleaved Caspase 3 (green FITC fluorescence) demonstrates
ectopic apoptosis in the Msx1/2 mutant splanchnic and somatic mesoderm (indicated by the white arrow on the right side and by notched arrowheads on the left side in
panel J) but no discernable change of cell proliferation in the double mutant SHF or OFT. (M–P) Pitx2 immunostaining (green FITC fluorescence) at E10.5 reveals a
greatly reduced number of Pitx2-expressing cells in the pharyngeal mesoderm, left somatic and splanchnic mesoderm as well as the left distal OFT myocardium of the
Msx1/2 null mutant (indicated by the white arrow and notched arrowheads in panel N) compared with the control (M). AoS, aortic sac; CV, common ventricle; FG,
foregut; LV, left ventricle; OFT, outflow tract; RV, right ventricle; SoM, somatic mesoderm; SpM, splanchnic mesoderm. Scale bars: 0.1 mm in panels A (for A, B),
E (for E, F) and I (for I, J); 0.2 mm in panel M (for M, N).
426Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
regions died at an earlier developmental stage. In contrast, the
level of Pitx2 in the Msx1/2 mutant right ventricular myo-
cardium was normal (Figs. 3M, N), in agreement with the
possibility that myocardial cells in the Msx1/2 mutant proximal
OFT and common ventricle were not dying at E9.0 or E9.5
(Figs. 3B, F, J). Therefore, Msx1 or Msx2 is required for the
survival of a subpopulation of SHF precursors that are
ultimately incorporated into the myocardium of the OFT but
not of the right ventricle.
To identify potential Msx1/2 downstream targets that might
play a role in inhibiting ectopic apoptosis of cardiogenic
precursors, we first focused on the expression of Hand genes in
Msx1/2 null mutants. Hand genes were of interest for two
reasons. First, the expression patterns of Hand1 and Hand2
overlap with those of Msx1 and Msx2 in pharyngeal arches and
the heart (McFadden et al., 2005; Togi et al., 2004; Yanagisawa
et al., 2003). Second, a Hand-related gene was shown to inhibit
apoptosis of cardioblasts and pericardial cells in Drosophila
(Han et al., 2006).
We found that both Hand1 and Hand2 exhibited signifi-
cantly reduced expression in the Msx1/2 mutant somatic
mesoderm, SHF and OFT (Figs. 2O–V). Interestingly, ectopic
apoptosis in the Msx1/2 mutant SHF was predominant in the
regions with a greater reduction of Hand1 and Hand2
expression (compare Figs. 2P, R with Figs. 3B, F, J).
Since ectopic apoptosis of OFT myocardial precursors might
be associated with a shortened OFT, we next asked if there was
any cardiac looping defect in Msx1−/−; Msx2−/− mutant hearts.
All Msx1/2 null mutants analyzed at E9.5 (n=9) displayed
correct dextral cardiac looping, and there was no significant
alteration in the length of the distal OFT (Anderson et al., 2003)
(data not shown). Nonetheless, in comparison with control
hearts, Msx1/2 mutant hearts appeared less convoluted and had
a reduced space of the inner curvature between the inflow and
outflow limbs, features that were demonstrated in embryos with
a shortened OFT (Yelbuz et al., 2002) (Figs. 4A–D). In
addition, the Msx1/2 mutant ventricles were slightly displaced
ventrally and were relatively narrow when compared with the
control ventricles. This set of features was also characteristic of
a shortened OFT (Yelbuz et al., 2002) (Figs. 4A–D).
At E10.5, there was an even more severe cranial displace-
ment of the OFT and ventral displacement of ventricles in
Msx1/2 null mutants, which was accompanied by an apparent
rightward turn of the mutant cardiac apex (Figs. 4E–H).
Therefore, reduced accretion of SHF precursors into the Msx1/2
mutant OFT was associated with an altered looping configura-
tion that could contribute to an abnormal alignment of the OFT.
Msx1 and Msx2 negatively regulate post-migratory cell
proliferation in the outflow tract during aorticopulmonary
Previously we documented a delay in NC migration into
the pharyngeal region of E9.5 Msx1/2 null mutants (Ishii et
al., 2005). We also showed that overexpression of Msx2 is
associated with a reduced contribution of NC to the OFT of
the Splotch mutant mouse (Conway et al., 1997; Kwang et
al., 2002). To further investigate the effect of loss of function
of Msx1 and Msx2 on the contribution of NC to the septating
OFT, we traced the colonization of NC cells in the Msx1/2
mutant conotruncal regions during aorticopulmonary septation
using the Wnt1-Cre/R26R system. Transverse sections across
Fig. 4. Altered looping configuration of Msx1/2 double mutant hearts. (A–D)
Lateral views of control and Msx1/2 null mutant hearts at E9.5 reveal that the
dextral cardiac looping in double mutants is less convoluted compared with
control hearts (indicated by white dashed curves), leading to diminished space
between inflow and outflow limbs in double mutants. In addition, the width of
the common ventricle (indicated by red dashed lines) is relatively narrow in
Msx1/2 null mutants compared with controls probably because of ventral
displacement of the ventricle resulting from a shortened and cranially displaced
OFT. (E–H) Ventrolateral views of control and Msx1/2 null mutant hearts at
E10.5 demonstrate more obvious cranial displacement of the right ventricle and
ventral displacement of the left ventricle, concomitant with an apparent cranial
and rightward displacement of the cardiac apex (red asterisks). The red dashed
curves indicate the direction of displacement of the whole heart in Msx1/2
mutants resulting from a shortened and cranially displaced OFT. ba, branchial
(pharyngeal) arch; CV, common ventricle; LV, left ventricle; M, mandible; OFT,
outflow tract; RV, right ventricle; T, tail. Scale bars in panels A (for A–D), E (for
E, F) and G (for G, H) are 0.2 mm.
427Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
428 Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
E10.5 conotruncal regions revealed comparable numbers of
NC cells colonizing the OFT cushions of Msx1/2 null mutants
and littermate controls (Figs. 5A, B). However, at E12.5,
significantly increased numbers of NC cells were evident in
the OFT cushion mesenchyme of Msx1/2 null mutants,
indicating a dramatic expansion of the NC population in
conotruncal cushions after its migration into the OFT was
completed (Figs. 5C–J).
To assess whether reduced apoptosis or increased prolifera-
tion contributes to the overpopulation of post-migratory cardiac
NC cells, we performed TUNEL and BrdU double labeling. We
detected only sparse apoptotic signals and no significant
difference in the number of apoptotic cells between Msx1/2
null mutants and littermate controls (n=4; Figs. 6A–F). In
contrast, BrdU labeling indices were significantly higher in
Msx1/2 null mutants within the OFT region (n=4, Pb0.05,
Student's t test; Figs. 6A–F, M; and Table 2). Following the
elevated proliferation, the total number of cells was increased
significantly within the Msx1/2 mutant OFT at E12.0 (n=4,
Pb0.05, Student's t test; Table 2). In the SHF and 4–6th
pharyngeal arches, however, the level of cell proliferation was
comparable between controls and double mutants at all
developmental stages examined (Figs. 3I, J and 6A–F, and
data not shown). Therefore, aberrantly elevated cell prolifera-
tion in Msx1/2 null mutant hearts was confined to the OFT.
To further determine whether cardiac NC cells in the Msx1/
2 mutant OFT undergo excessive post-migratory proliferation,
we performed double immunostaining to colocalize lacZ-
expressing NC cells and BrdU-labeled proliferating cells in
embryos carrying both the Wnt1-Cre and R26R reporter
transgenes. In Msx1/2 mutant OFTs, the increased proliferation
indices were statistically significant for NC cells at all
developmental stages examined (n=4, Pb0.05, Student's
t test; Figs. 6G–L, N, and Table 2), but were statistically
significant for non-NC cells at E10.0 and E11.0 only (n=4,
Pb0.05 at E10.0 and E11.0, PN0.05 at E12.0, Student's
t test; Figs. 6G–L, O, and Table 2). In agreement with the
differentially increased cell proliferation, we observed sig-
nificantly increased total cell numbers for the NC population
but not for non-NC populations in Msx1/2 mutant OFTs at
E12.0 (n=4, Pb0.05 for NC cells, PN0.05 for non-NC cells,
Student's t test; Table 2). The results of these quantitative
analyses are consistent with our observation of excessive NC
cells in the Msx1/2 mutant conotruncal cushions at E12.5
(Figs. 5C–L). Taken together, our findings indicate (i) that
between E10.0 and E12.0 Msx1 and Msx2 negatively regulate
cell proliferation in the septating OFT, and (ii) that this effect is
predominantly on the cardiac neural crest.
Since Msx2 was shown to regulate the differentiation of
mesoangioblast stem cells into smooth muscle cells (Brunelli
et al., 2004), and since some neural crest cells normally
differentiate into smooth muscle cells prior to the formation of
the aorticopulmonary septum (Beall and Rosenquist, 1990), we
tested whether differentiation of cardiac NC cells into smooth
muscle cells was perturbed in the Msx1−/−; Msx2−/− mutant
OFT. At both E11.5 and E12.5, immunostaining for α-smooth
muscle actin (α-SMA) revealed a normal level of α-SMA
surrounding the left and right 4th arch arteries and the ductus
arteriosus as well as in the OFT of Msx1−/−; Msx2−/− mutants
(Figs. 7A–D). These results indicate that cardiac NC cells
differentiated normally into smooth muscle cells in Msx1/2 null
Premature down-regulation of p27KIP1 and increased Bmp2/4
signaling in Msx1−/−; Msx2−/− mutant outflow tracts during
To unravel the molecular mechanisms leading to increased
cell proliferation in Msx1−/−; Msx2−/− double mutant OFTs,
we asked whether deficiencies of Msx1 and Msx2 alter the
activity of specific growth control genes in the septating OFT.
Previous studies have demonstrated that both Msx1 and Msx2
regulate the G1/S transition of the cell cycle in cultured
mesenchymal and epithelial progenitor cells by activating
Cyclin D1 expression and Cdk4 activity (Hu et al., 2001), while
Msx1 also down-regulates the expression of p19INK4d, an
inhibitor of cyclin-dependent kinases (CDKs),in the developing
dental mesenchyme (Han et al., 2003a). Because the INK family
of CDK inhibitors (CKIs) is not expressed in the developing
heart (Koh et al., 1998; Quelle et al., 1995), we asked whether
the CIP/KIP family of CKIs was regulated by Msx1 and Msx2
in the cardiac OFT.
Immunostaining revealed that p21CIP1levels were similar in
control and Msx1/2 null mutant OFTs from E10.0 to E12.0
(Figs. 8A–F and data not shown), whereas p27KIP1levels were
significantly lower in Msx1/2 mutant OFTs at both E10.0 and
E11.0 (n=4, Pb0.05, Student's t test; Figs. 8G–J). We also
noticed a much greater reduction in p27KIP1levels in NC cells
than in non-NC cells in Msx1/2 mutant OFTs (n=4, Pb0.01 for
NC cells, 0.01bPb0.05 for non-NC cells, Student's t test; Figs.
8M, N), corresponding to our observation of a greater increase
of proliferation rates in NC cells than in non-NC cells (Figs. 6N,
O). Interestingly, in control OFTs, the p27KIP1level declined
dramatically between E11.0 and E12.0, consistent with an
increase of proliferation rates from E11.0 to E12.0 (Figs. 6M–O
and Table 2).
Fig. 5. Excessive post-migratory expansion of cardiac NC in the Msx1/2 double mutant conotruncal cushions. (A–J) Demonstration of cardiac NC colonization in the
conotruncal region by LacZ staining on transverse sections of mouse embryos carrying the Wnt1-Cre and R26R reporter transgenes. (A, B) At E10.5, comparable
amounts of LacZ-expressing cardiac NC cells colonize the control and Msx1/2 double mutant OFT cushions (indicated by black arrows). (C–J) At E12.5, a massive
excess of cardiac NC cells accumulates in the conotruncal tissue separating the ascending aorta and the right ventricle in Msx1/2 null mutants (indicated by red arrows;
panels C–F and panels G–J are from two different pairs of Msx1/2 double mutants and littermate controls) and disrupts conotruncal alignment and organization. In
panel H, the excessive accumulation of mesenchymal cells surrounding the pulmonary OFT will likely obstruct the connection between the right ventricle and the
pulmonary OFTand lead to pulmonary stenosis. Ao, ascending aorta; AoS, aortic sac; CNC, cardiac neural crest; DA, ductus arteriosus; FG, foregut; LA and LV, left
atriumandventricle;OFT, outflowtract; PT,pulmonarytrunk;RA andRV, right atriumandventricle.Scale bars: 0.2mm in panelA(for A,B); 0.4mm in panelsC (for
C–F) and G (for G–J).
429Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
Fig. 6. Increased post-migratory cell proliferation and unaltered cell survival in the Msx1/2 double mutant OFT during aorticopulmonary septation. All nuclei were visualized by DAPI counter-staining (blue
fluorescence). Panels M–O are statistical charts of proliferation indices obtained from data analyses in panels A–L. (A–F) BrdU (red Rhodamine fluorescence) and TUNEL (green FITC fluorescence) double labeling to
localize both proliferating and apoptotic cells in the transverse sections of control and Msx1/2 mutant OFTs between E10.0 and E12.0. TUNEL-positive signals are sparse in both control and Msx1/2 mutant sections,
indicating low levels of cell apoptosis during these developmental stages. Compared with control OFTs, however, there is a dramatic increase in the number of BrdU-positive cells in Msx1/2 mutant OFTs (white arrows)
at both E10.0 (A, B) and E11.0 (C, D), while the increase at E12.0 (E, F) is milder but still reaches statistical significance (panel M; n=4, Pb0.02 at E10.0, Pb0.01 at E11.0 and Pb0.05 at E12.0, Student's t test; see
also Table 1). (G–L) BrdU (red Rhodaminesignals) andβ-galactosidase (green FITC signals) double immunostaining to localize proliferating cardiac NC cells(yellow mergedsignals). The percentages of BrdU-positive
NC cells (yellow) and BrdU-positive non-NC cells (red) are both significantly higher in Msx1/2 mutant OFTs compared with control OFTs at E10.0 (G, H) and E11.0 (I, J) (white arrows), whereas at E12.0 (K, L), only
the percentage of BrdU-positive NC cells is significantly higher in the Msx1/2 mutant OFT (panels N and O; n=4, Pb0.05 for NC cells at all developmental stages, whereas Pb0.05 for non-NC cells at E10.0 and E11.0
but not E12.0, Student's t test; see also Table 1). Notched arrowheads in panels A–L indicate comparable levels of cell proliferation in the pharyngeal, splanchnic and somatic mesoderm between controls and Msx1/2
double mutants. AoS, aortic sac; CNC, cardiac neural crest; LA and LV, left atrium and ventricle; RA and RV, right atrium and ventricle. Scale bars in panels B (for A, B), D (for C, D), F (for E, F), H (for G, H), J (for I, J)
and L (for K, L) are 0.1 mm.
Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
Our findings demonstrate that loss of Msx1 and Msx2 results
in premature down-regulation of p27KIP1levels in the septating
OFT before E12.0. This is correlated with significantly elevated
cell proliferation in the Msx1/2 mutant OFT between E10.0 and
E11.0, which could cause excessive accumulation of cells in the
conotruncal cushions by E12.0.
We did not detect any significant difference in p27KIP1levels
between Msx1/2 null mutants and their littermate controls in the
SHF and 4–6th pharyngeal arches (Figs. 8G–L and data not
shown), consistent with our failure to detect a difference in
proliferation rates between Msx1/2 mutants and controls in the
SHF and pharyngeal arches (Figs. 3I, J and 6A–F, and data not
shown). We also noticed a relatively low number of p27KIP1-
expressing cells in the 4–6th pharyngeal arches between E10.0
and E12.0 (Figs. 8G–L and data not shown), which suggests
that p27KIP1functions primarily within the OFT during
Because no cardiovascular defects have been reported in
p27KIP1null mutant mice (Fero et al., 1996; Kiyokawa et al.,
1996; Nakayama et al., 1996), we reasoned that othergenes may
act together with p27KIP1to regulate post-migratory cell
proliferation in the OFT. Bone morphogenetic proteins (Bmps)
are potential candidates since they have been implicated in OFT
morphogenesis (Cai et al., 2003; Delot et al., 2003; Kaartinen et
al., 2004; Kim et al., 2001; Liu et al., 2004; Stottmann et al.,
2004), and Bmp2, Bmp4 and Bmp7 have all been described as
Ishii et al., 2005; Wu et al., 2003; Zhang et al., 2002, 2003).
Immunostaining for Bmp2/4 and phosphorylated Smad1/5/8
revealed a statistically significant elevation of Bmp signaling in
Msx1−/−; Msx2−/− mutant OFTs compared with control OFTs
at both E10.5 and E11.5 (n=3, Pb0.05, Student's t test; Figs.
9A–J). As Bmp4 is known to play a role in promoting local
proliferation of NC cells after they reached the OFT (Liu et al.,
2004), it is possible that upregulation of Bmp2/4 signaling and
down-regulation of p27KIP1work together to facilitate excessive
Here we show that Msx1 and Msx2 exhibit overlapping
functions in both early and late stages of the morphogenesis of
the cardiac outflow tract. Msx1 and Msx2 seem to display
complete functional redundancy in cardiogenesis as evidenced
by the sufficiency of a single Msx allele for normal heart
development. In addition, Msx1 and Msx2 exert different effects
(inhibit apoptosis and proliferation) on distinct cell populations
(SHF precursors and cardiac NC cells) during early and late
OFT morphogenesis. Our findings demonstrate requirements
for Msx1 and Msx2 in two major aspects of OFT development,
Summary of total cell numbers and proliferation indices in control and Msx1−/−; Msx2−/− double mutant outflow tracts between E10.0 and E12.0a
Controls Msx1−/−; Msx2−/− double mutants
E10.0 All cells
aAll P values were calculated using Student's t test. Significant P values (Pb0.05) are in bold face.
bNon-NC cells include myocardial and endocardial cells in the outflow tract.
Fig. 7. Normal differentiation of cardiac NC cells into smooth muscle cells in
Msx1/2 double mutant hearts and vasculatures. (A–D) Immunostaining for α-
smooth muscle actin (α-SMA) reveals that, at both E11.5 (A, B) and E12.5
(C, D), numbers of α-SMA-positive cells are comparable between Msx1/2
mutants and littermate controls surrounding the vasculature (the left and right
fourth arch arteries at E11.5, and the ductus arteriosus, aorta and pulmonary
trunkat E12.5,as indicatedby notchedarrowheads), at the distal endof the OFT,
where the aorticopulmonary septum lies (indicated by triangle arrowheads), as
well as the proximal conotruncal cushions, where the OFT valves and the
intervalvular fibrous connection will form (indicated by black arrows). Ao,
aorta; APS, aorticopulmonary septum; DA, ductus arteriosus; dAo, dorsal aorta;
L4, left 4th arch artery; LA and LV, left atrium and ventricle; OFT, outflow tract;
R4, right 4th arch artery; RA and RV, right atrium and ventricle. Scale bars in
panels B (for A, B) and D (for C, D) are 0.2 mm.
431 Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
Fig. 8. Premature down-regulation of the CDK inhibitor p27KIP1in the Msx1/2 mutant OFT during aorticopulmonary septation. (A–F) Immunostaining reveals comparable levels of the CDK inhibitor p21CIP1in the
septating OFT between Msx1/2 double mutants and littermate controls at all developmental stages examined: E10.0 (A, B), E11.0 (C, D) and E12.0 (E, F). (G–L) Double immunostaining to colocalize p27KIP1-positive
cells (red Rhodamine fluorescence) and LacZ-expressing cardiac NC cells (green FITC fluorescence) demonstrates greatly reduced numbers of p27KIP1-positive NC cells (yellow merged signals) in the Msx1/2 double
mutant OFTcushions compared with the control at both E10.0 (G, H) and E11.0 (I, J) (indicated by white arrows; n=4, Pb0.01, Student's t test). Numbers of p27KIP1-positive non-NC cells also significantly decreased in
Msx1/2 mutant OFTs at both E10.0 (G, H) and E11.0 (I, J) (n=4, 0.01bPb0.05, Student's t test). On the other hand, fewer p27KIP1-positive cells are present in pharyngeal and somatic mesoderm in both controls and
Msx1/2 null mutants (indicated by triangle arrowheads). (K, L) At E12.0, the number of p27KIP1-positive cells dramatically reduced in the control OFT (in both NC and non-NC cells) and led to comparable levels of
p27KIP1between the control OFT and Msx1/2 mutant OFT (indicated by notched arrowheads). (M, N) Statistical charts showing average percentages of p27KIP1-positive cells in cardiac NC and non-NC cells in the
CNC, cardiac neural crest; FG, foregut; LV, left ventricle; RA and RV, right atrium and ventricle. Scale bars in panels B (for A, B), D (for C, D), F (for E, F), H (for G, H), J (for I, J) and L (for K, L) are 0.1 mm.
Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
one in survival of SHF precursor cells during cardiac looping
and the other in proliferation of OFTcushion mesenchymal cells
during aorticopulmonary septation (Fig. 10).
Roles of Msx1 and Msx2 in the survival and differentiation of
secondary heart field precursors and in the elongation of the
All Msx1−/−; Msx2−/− double mutant mice that we
examined had a malaligned OFT (Table 1), consistent with
defects in the elongation or spiral rotation of the OFT (Bajolle
et al., 2006; Yelbuz et al., 2002). In Msx1/2 null mutants,
increased apoptosis in the SHF was associated with the
reduced accretion of SHF cells into the lengthening OFT,
similar to the consequence of ablating SHF precursors in
chick embryos, which resulted in a shortened OFT and thus
led to dextroposed aorta or pulmonary atresia/stenosis (Ward
et al., 2005). The presence of Pitx2-expressing cells in the
right ventricle and conal region but not the truncal region of
Msx1/2 mutant hearts at E10.5 (Figs. 3M, N) suggests that
Fig. 9. Elevated Bmp2/4 signaling in the Msx1/2 double mutant OFT during aorticopulmonary septation. Immunostaining for Bmp2/4 (green FITC fluorescence)
(A–D) and phosphorylated Smad1/5/8 (F–I) reveals significantly increased numbers of Bmp2/4-positive and phospho-Smad1/5/8-positive cells in Msx1/2 null mutant
OFTs compared with control OFTs at both E10.5 (A, B, F, G) and E11.5 (C, D, H, I), with a much greater difference at E11.5 (for Bmp2/4: n=3, 0.01bPb0.05 at
E10.5, Pb0.01 at E11.5; for phospho-Smad1/5/8: n=3, 0.005bPb0.01 at E10.5, Pb0.005 at E11.5; Student's t test). White arrows indicate increased numbers of
Bmp2/4-positive and phospho-Smad1/5/8-positive cells in the Msx1/2 mutant conotruncal cushions (B, D, G, I). Notched arrowheads indicate increased Bmp2/4-
positive signals in the double mutant somatic mesoderm at E10.5 (B) as well as increased Bmp2/4-positive and phospho-Smad1/5/8-positive signals in the pharyngeal
mesoderm, splanchnic mesoderm and myocardium of the OFT and right ventricle in the Msx1/2 mutant at E11.5 (D, I). (E, J) Statistical charts displaying average
percentages of Bmp2/4-positive and phospho-Smad1/5/8-positive cells in the control and Msx1/2 mutant OFTs at E10.5 and E11.5. AoS, aortic sac; FG, foregut; LA
andLV, leftatrium andventricle;OFT,outflowtract;RA andRV,right atriumandventricle;SoM,somaticmesoderm;SpM,splanchnicmesoderm.Scale barsinpanels
B (for A, B), D (for C, D), G (for F, G) and I (for H, I) are 0.1 mm.
433 Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
one function of Msx1 and Msx2 in the SHF is to maintain the
survival of a subset of SHF precursors that become
incorporated into the distal (truncal) OFT late in cardiac
looping. A flow of migratory SHF precursors along the path
from splanchnic mesoderm adjacent to pharyngeal endoderm
and mesoderm through the aortic sac gives rise to distinct
regions of the OFT at different times of development (Cai et
al., 2003; Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et
Isl1 expression is required to maintain the survival of a
subset of SHF cells that become incorporated into the OFTearly
in its development (Cai et al., 2003). Our data indicate that
Msx1 and Msx2 are necessary for the survival of another
distinct subset of SHF cells that are incorporated into the OFTat
a later stage. These two subpopulations of SHF precursors are
localized in the medial and lateral splanchnic mesoderm, as
shown by labeling of apoptotic cells (Figs. 3A, B, E, F, I, J and
Cai et al., 2003). It is noteworthy, however, that not all SHF
cells within the expression domain of Isl1 or Msx1/2 underwent
ectopic apoptosis. Why this specific subset of SHF cells is more
sensitive to Msx function than other SHF cells is unclear, but is
likely a result of the combinatorial action of a group of genes
within this subset of SHF cells. Our findings raise the intriguing
possibility that Hand1/2 genes may be a part of this group:
differences between Msx1/2 mutant SHF subpopulations in
their susceptibility to apoptosis appear to be associated with
differential down-regulation of Hand1 and Hand2 in distinct
regions of the Msx1/2 mutant SHF.
Hand1/2 may also function together with Msx1/2 in later
stages of OFT morphogenesis. Inactivation of Hand1 in
Nkx2.5-expressing cells, which constitute most of the primary
heart field and a subset of SHF cells (Waldo et al., 2001), results
in OFT malalignment defects (McFadden et al., 2005). Since
Hand1 and Hand2 play pivotal roles in the differentiation of
myocardial precursors and cardiomyocytes (Risebro et al.,
2006; Trinh et al., 2005), reduced Hand1/2 expression in Msx1/
2 mutant hearts may also perturb the differentiation of SHF
precursors and myocardial cells in the OFT, disrupting OFT
morphogenesis. Against this possibility is our finding that the
expression of the smooth muscle marker, α-SMA, was not
detectably altered in Msx1/2 mutant OFTs (Fig 7). Despite this
result, further studies analyzing the expression patterns and
localization of adherens junctional proteins will be required to
determine whether formation of epithelia during myocardial
differentiation is affected by the loss of Msx1 and Msx2
function (Trinh et al., 2005).
Roles of Msx1 and Msx2 in left–right patterning and rotation of
the outflow myocardium
That left–right patterning is important for the correct
rotation and alignment of the OFT myocardium follows from
findings (i) that deficiencies in laterality genes such as Pitx2c
impair the rotation of the OFT (Bajolle et al., 2006; Gaio
et al., 1999; Liu et al., 2002; Oh and Li, 1997), and (ii) that
disruptions in the incorporation of right SHF precursors into
the left side of preseptation OFT myocardium result in
pulmonary stenosis/atresia (Ward et al., 2005). Our results
show that in Msx1/2 mutants, a reduction in the number of
Pitx2-expressing cells in left splanchnic mesoderm (due to
apoptosis) resulted in accretion of fewer Pitx2-expressing cells
to the left side of the distal OFT myocardium by E10.5 (Fig.
3). The dramatically reduced number of Pitx2-expressing cells
on the left side of the Msx1/2 mutant OFT myocardium would
likely diminish left side-specific, Pitx2-mediated signaling
(such as Nodal-Cfc signaling (Bamforth et al., 2004; Linask et
Fig. 10. Summary of the roles of Msx1 and Msx2 in OFT morphogenesis.
Dashed arrows and lines indicate upregulation or down-regulation of gene
expression or protein levels. Previous studies have shown that: (1) Bmp4 down-
regulates Pitx2 expression in the early lateral plate mesoderm, and Bmp4 and
Pitx2 act antagonistically in the pharyngeal arch mesoderm (Brand, 2003;
Branford et al., 2000; Liu et al., 2003; St Amand et al., 2000); (2) Pitx2 specifies
a left cardiac lineage of cells (Campione et al., 2001; Franco and Campione,
2003); (3) Hand genes are required for survival, differentiation and migration of
myocardial precursors (Han et al., 2006; Trinh et al., 2005; Yelon et al., 2000);
(4) Bmp4 is required for Pitx2 expression in the OFT (Liu et al., 2004); (5) Pitx2
promotes cell type-specific proliferation (Kioussi et al., 2002); (6) p27KIP1
inhibits cell proliferation and p27KIP1-deficient mice exhibit hyperplasia of
multiple organs and tissues, including the heart (Nakayama et al., 1996;
Poolman et al., 1999).
434 Y.-H. Chen et al. / Developmental Biology 308 (2007) 421–437
al., 2003; Shen et al., 1997)). This may result in insufficient
left–right signaling for the complete spiral rotation of the OFT.
Angular analyses of the aortic-to-pulmonary valve axes have
attributed the malaligned OFT in DORVand tetralogy of Fallot
to arrests in normal OFT rotation at certain stages of
embryogenesis (Bostrom and Hutchins, 1988; Lomonico et
al., 1988). Similarly, a low level of Pitx2-mediated left–right
signaling in the Msx1/2 mutant OFT may only be sufficient to
support normal rotation of the septating OFT to a specific
developmental stage, leaving the OFT in an intermediate
Msx1 and Msx2 regulate post-migratory expansion of cardiac
neural crest cells as well as endothelial and myocardial cells in
the outflow tract during aorticopulmonary septation
Although in previous work, we observed a delay in NC
migration into the Msx1/2 mutant pharyngeal region at E9.5
(Ishii et al., 2005), our findings in this study indicate that normal
numbers of cardiac NC cells colonize OFT cushions of Msx1/2
double mutants at E10.5 (Figs. 5A, B). Moreover, our findings
reveal a substantial excess of cardiac NC cells in E12.5 Msx1/2
mutant conotruncal cushions (Figs. 5C–J). An increase in the
abundance of cardiac NC cells in the conotruncal region has
been described in mice overexpressing Connexin 43. These
mice displayed obstructions of pulmonary outflow tracts similar
to the phenotype of pulmonary stenosis or atresia in Msx1−/−;
Msx2−/− double mutants (Huang et al., 1998b; Lo et al., 1999;
Lo and Wessels, 1998). However, accumulation of excessive
cardiac NC cells in the OFTs of Connexin 43 overexpressing
mice resulted not from increased proliferation as occurs in
Msx1/2 mutants, but from increased rates of NC migration
(Huang et al., 1998a).
The previous finding of increased cell proliferation in the
ventricular myocardium of Connexin 43 overexpressing mice
suggested that changes in the abundance or functional activity
of cardiac NC cells might perturb the balance of cytokines that
act on cardiomyocyte proliferation (Huang et al., 1998a; Lo et
al., 1999). Similarly, the increased proliferation rates in Msx1/2
mutant cardiomyocytes in the OFT may be secondary to the
increase in cardiac NC cell proliferation. Potential candidates
for such local cytokines include Bmp2 and Bmp4, which
exhibited elevated levels of signaling in Msx1/2 mutant OFTs,
but unaltered patterns of spatial expression (Fig. 9 and data not
shown). A role for Bmp4 in regulating local cell proliferation in
the septating OFT has been demonstrated in mutant mice
deficient for Bmp4 in the myocardium (Nkx2.5cre; Bmp4 n/f).
These mice displayed significantly reduced cell proliferation in
the conotruncal cushions at E11.5 (Liu et al., 2004). We propose
that increased Bmp2/4 signaling may collaborate with reduced
p27KIP1levels to promote local cell proliferation in Msx1/2
mutant OFTs. Furthermore, the resultant excessive accumula-
tion of mesenchymal cells in Msx1/2 mutant conotruncal
cushions may disrupt the spiral configuration required to form
the spiral aorticopulmonary septum (Fananapazir and Kaufman,
1988) and may thus contribute to OFT malalignment and
This work was supported by NIH grants DE12941 and
DE12450 to RM. We would like to thank Bibha Choudhary for
her technical assistance and thank Dr. Chrissa Kioussi for the
kind gift of anti-Pitx2 antibody. We are also grateful to the
following people for cRNA probes: Drs. Sylvia Evans, James F.
Martin, Eric N. Olson and Jeffrey L. Wrana.
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