Download Sox4-Deficiency Syndrome in Mice Is an Animal Model for Common

Sox4-Deficiency Syndrome in Mice Is an Animal Model
for Common Trunk
Jing Ya, Marco W. Schilham, Piet A.J. de Boer, Antoon F.M. Moorman,
Hans Clevers, Wouter H. Lamers
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Abstract—Embryonic mice lacking functional Sox4 transcription factor die from cardiac failure at embryonic day (ED) 14.
Heart morphogenesis in these embryos was analyzed in hematoxylin-azophlochsin or immunohistochemically stained,
3-dimensionally reconstructed serial sections between ED12 and ED14. Although Sox4 is expressed in the endocardially
derived tissue of both the outflow tract and atrioventricular canal, Sox4-deficient hearts only suffer from defective
transformation of the endocardial ridges into semilunar valves and from lack of fusion of these ridges, usually resulting
in common trunk, although the least affected hearts should be classified as having a large infundibular septal defect. The
more serious cases are, in addition, characterized by an abnormal number and position of the semilunar valve-leaflet
anlagen, a configuration of the ridges typical for transposition of the great arteries (with linear rather than spiral course
of both ridges and posterior position of the pulmonary trunk at the level of the valve), and variable size of the aorta
relative to the pulmonary trunk. The coronary arteries always originated from the aorta, irrespective of its position
relative to the pulmonary trunk. The restriction of the malformations to the arterial pole implies that the interaction
between the endocardially derived tissue of the outflow tract and the neural crest– derived myofibroblasts determines
proper development of the arterial pole. (Circ Res. 1998;83:986-994.)
Key Words: mouse n semilunar valve n transposition n common trunk n outflow tract
development of the semilunar valves.4 Homozygous Sox4deficient embryos could not be distinguished externally from
their littermates until the onset of valvular morphogenesis in
the arterial pole of the heart (ED12 to ED13). Then, they
rapidly developed generalized edema to die at ED14. Dying
mutant embryos typically displayed an oscillatory, nearly
futile movement of blood in the arterial pole of the heart,
showing that the valvular function of the outflow tract was
affected. A preliminary histological examination revealed
that the embryos suffered from common arterial trunk and
defective semilunar valve development.4 We now show that
Sox4 deficiency causes a syndrome with, in addition to
impaired semilunar valve development, malformations ranging from infundibular septum defect to complete transposition of the great arteries.
Common trunk is a still poorly understood malformation of
the ventriculo-arterial junction. Due to the lack of an established concept of ventriculo-arterial development and malformations, some opt for purely descriptive definitions and use
the term “common trunk” to mean “single arterial trunk that
leaves the heart by way of a single arterial valve and gives
rise directly to the coronary, systemic, and one or both
pulmonary arteries.”8,9 Others take a more ontogenetic view
and define common trunk as the most severe form of a
C
ongenital malformations of the heart are the most common birth defects in man, accounting for 0.5% to 1% of
live births, 10% of stillbirths, and possibly up to 20% of
spontaneous abortions.1–3 Thus far, the systematic analysis
of these defects has rested almost entirely on findings in
human neonates with these structural anomalies. As a consequence, little direct information is available about the etiology and pathogenesis of the malformations. In particular, it is
usually not known to what extent a particular malformation
can be traced to a single morphogenetic event or process.
However, recent progress in molecular and developmental
biology has changed the general approach for the study of
cardiovascular diseases profoundly. In particular, the technique to genetically modify the expression of genes in vivo
has proven to be a powerful approach to identify genes that
are involved in the development of specific cardiac malformations. A case in point is the Sox4-null mutant.4 The Sox4
gene is a member of the Sox (for Sry-box) family of
transcription factors, of which more than a dozen members
have been identified in the mouse.5,6 The Sox4 protein, which
binds to the sequence motif AACAAAG,7 has been shown to
be a transcriptional activator in immature B- and
T-lymphocytes in adult mice.7 However, gene disruption in
mice resulted in cardiac failure because of the defective
Received June 1, 1998; accepted August 20, 1998.
From the Department of Anatomy and Embryology (J.Y., P.A.J.d.B., A.F.M.M., W.H.L.), Academic Medical Center, University of Amsterdam, and
the Department of Immunology (M.W.S., H.C.), University of Utrecht, The Netherlands.
Correspondence to Wouter H. Lamers, Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Meibergdreef
15, 1105 AZ, Amsterdam, The Netherlands. E-mail [email protected]
© 1998 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
986
Ya et al
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“trunco-conal septal defect,”10 whereas others have been
intrigued by its similarity with tetralogy of Fallot and claim
that pulmonary atresia plus partial or complete absence of the
aorticopulmonary septum are its landmark features.11,12 Irrespective of all these uncertainties, common trunk in genetically defined mouse models becomes manifest after
ED12,4,13,14 that is, at the onset of the period in which the
structural transformation of the embryonic outflow tract into
the ventriculo-arterial junction occurs. The outflow tract of
the preseptation embryonic heart is a long, slowly conducting
myocardial structure that functions as a sphincter at the
arterial pole of the heart prior to the emergence of the 1-way
semilunar valves.15,16 Extensive remodeling of the tissues of
the outflow tract, as well as immigrating cells of the neural
crest are responsible for connecting the right ventricle with
the pulmonary trunk and the left ventricle with the aorta, that
is, for the developmental formation of the definitive
ventriculo-arterial junction. The precise mechanisms underlying this important architectural adaptation are still debated.17–23 Based on our findings in the Sox4-deficient mouse
model and reports in the literature, we will argue that the
tissue interactions between the endocardially derived cells of
the outflow tract and cells derived from the neural crest
determine the developmental fate of the embryonic outflow
tract.
Materials and Methods
Animals
Details concerning the method used to disrupt the Sox4 gene have
been described elsewhere.4 Homozygous Sox4-disrupted embryos
were identified by Southern blot analysis and accounted for 18% to
20% of the litter at embryonic day (ED) 12, 13, and 14 (not shown).
Embryos were fixed either in a mixture of ice-cold methanol:
acetone:water (2:2:1) or in 4% formaldehyde diluted in PBS
(150 mmol/L NaCl; 10 mmol/L Na-phosphate; pH 7.4). After
overnight fixation, the embryos were dehydrated in a graded series of
ethanol, embedded in Paraplast plus, serially sectioned (7 mm thick),
and mounted on poly-L-lysine– or 3-aminopropyltriethoxysilane–
coated slides for immunohistochemistry or in situ hybridization
staining, respectively. The sections were stored at 4°C until use.
Immunohistochemistry
The detailed procedure for the histological processing of the tissues
has been described.24 Briefly, the serial sections were incubated
overnight with alternately monoclonal antibodies against a-myosin
heavy chain (a-MHC; 1:1025), b-myosin heavy chain (b–MHC;
1:1026), a-smooth-muscle actin (a-SMA; 1:1000; Sigma IMMH-2),
desmin (1:50; Monosan, Mon 3001, clone 33), and a polyclonal
antibody against fibronectin (1:6000; AB 1942, Brunschwig Chemie). After incubation with the primary antibodies, the sections
incubated with monoclonal antibodies were stained with rabbit
anti-mouse IgG (1:7500; noncommercial) followed by incubation
with goat anti-rabbit IgG (1:250; noncommercial), whereas the
sections incubated with the polyclonal antifibronectin antiserum
were stained directly with goat anti-rabbit IgG. Antibody binding
was demonstrated with rabbit peroxidase-antiperoxidase complex
(1:750; Nordic, Tilburg, The Netherlands) and 3,3-diaminobenzidinetetrahydrochloride and H2O2 as substrates.
In Situ Hybridization
In situ hybridization was performed on formaldehyde-fixed sections
to visualize the expression of Sox4, Nfatc, and SERCA 2 (sarcoendoplasmic reticulum calcium ATPase 2) as described in detail
previously.27 The cRNA probes were transcribed from linearized
November 16, 1998
987
Figure 1. The arterial pole of the heart in ED 12 and 13 mouse
embryos that are heterozygous for Sox4 deficiency (A through
G), homozygous (H), or wild-type (I through L). Panels A and C
are stained immunohistochemically for the presence of desmin,
and panels B and D are stained for the presence of fibronectin,
whereas panels F, J, and K are stained by in situ hybridization
for the presence of Sox4 mRNA, panels E, I, and K for the presence of SERCA2, and panels G and H for the presence of
Nfatc. SERCA 2 mRNA is expressed in the cardiomyocytes,
whereas Sox4 mRNA is expressed exclusively in the endocardially derived tissue of the outflow tract and the atrioventricular
canal. Expression of Sox4 is also found in the mesenchyme surrounding the branchial arch arteries, trachea, and bronchi. Nfatc
expression is concentrated in the endocardium overlying the
endocardial ridges and cushions. ao indicates aorta; la, left
atrium; lv, left ventricle; os, outlet septum; pt, pulmonary trunk;
pv, pulmonary valve; ra, right atrium; rv, right ventricle; tr, trachea. Bar5100 mm.
cDNA in pBluescript using T3 RNA polymerase and 35S-UTP. The
Sox4 probe (EcoRI-Sacl fragment; 300 bp4) contained unique,
untranslated sequences from the 59 end of the mRNA. The Nfatc3
probe (SacII-XhoI fragment28) was provided kindly by Dr G.R.
Crabtree (Stanford, Calif). Selected hearts of Sox4-mutant mice were
reconstructed 3-dimensionally as described29 and rendered with the
help of a medical artist.
Results
Development of the Arterial Pole in Wild-Type
and Heterozygous Littermates of
Sox4-Deficient Mice
No differences in morphological development were observed
between wild-type and heterozygous littermates of Sox4deficient mice (Figure 1A through 1D). In ED12 mice, the
aorta and pulmonary trunk have become separate vessels at
and above the distal rim of the myocardium of the outflow
tract. The fusion of the ridges continues toward the ventricle
during ED13. Before fusion, the distal ends of the parietal and
septal endocardial ridges each contain a central rod of
condensed, a-SMA–positive mesenchymal cells of neural
crest origin. When both ridges fuse, their central rods of
condensed mesenchyme do so, too, by forming a central mass
988
Sox4-Deficiency Syndrome in Mice
of a-SMA–positive cells. At ED14, the a-SMA–positive
mesenchyme is still recognizable at the level of the valves.
Further upstream, however, the presence of many apoptotic
cells reveals that, soon after fusion, it is being eliminated and
replaced by myocardial cells that can be seen to migrate into
the outlet septum; that is, the septum forms between the
subaortic and subpulmonary outlets on fusion of the septal
and parietal endocardial ridges. The scallops of the semilunar
valve leaflets in the aorta and pulmonary trunk, and the
coronary arteries become discernible at ED13. The normal
morphogenesis of the arterial pole therefore is virtually
complete at ED14, which is comparable to 7 weeks of
development (Carnegie stage 19 to 20) in man.
Expression of Sox4 mRNA in Wild-Type and
Heterozygous Littermates of Sox4-Deficient Mice
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Sox4 mRNA is expressed abundantly in the nervous system,
thymus, mesenchyme surrounding the branchial arch arteries,
trachea, and esophagus, but not in the tissue of the aorticopulmonary septum (Figure 1E, 1F, and 1I through 1L). Sox4
expression levels in the endocardially derived tissues of the
outflow tract and atrioventricular canal are similar.
Expression of a-MHC, b-MHC, a-SMA, Desmin,
Fibronectin, and Nfatc in Heterozygous and
Homozygous Sox4-Deficient Embryos
We used a- and b-MHC expression as a parameter to monitor
myocardial development, fibronectin expression to selectively delineate the endocardially derived tissues, and, finally,
a-SMA and desmin expression to follow both the maturation
of the myocardium and the population of the endocardial
tissue– derived cells with neural crest– derived cells.24,30 We
observed no conspicuous differences in the spatio-temporal
pattern of expression of these genes between deficient embryos and their wild-type or hemizygous littermates, suggesting that Sox4-deficiency does not primarily affect myocardial
or endocardial development. Because Sox4-deficient mice
resemble Nfatc-deficient mice with respect to the (failing)
development of the semilunar valves, Nfatc expression in the
endocardium was investigated and found to be normal (cf
Figure 1G and 1H).
Malformations of the Arterial Pole of the Heart in
Sox4-Deficient Mice
A total of 21 Sox4-deficient embryos were studied histologically, 4 of which were from ED12, 8 from ED13, and 9 from
ED14. Cardiac morphogenesis was found to become progressively retarded in these embryos but did not stop. For
example, in only 25% of Sox4-deficient ED13 hearts, coronary arteries were seen to develop, whereas this was the case
in all hearts of wild-type littermates and in all ED14 Sox4deficient embryos. Furthermore, 2 dorsal aortae were still
present in 7 of 8 ED13 Sox4-mutant embryos, whereas the
right dorsal aorta had disappeared in all wild-type littermates.
In affected ED14 embryos, the right dorsal aorta, if present,
had diminished greatly in diameter.
At ED12, nearly normal amounts of endocardially derived
tissue was found in the outflow tract of Sox4-deficient hearts
(Figure 2), suggesting that the morphogenesis of the endocardial
Figure 2. Embryos Nos. 92 and 13. ED12 embryo No. 92
(A through C) belongs to the least-affected Sox4-deficient
embryos in the series, whereas ED12 embryo No. 13 (D through F)
is among the most seriously affected specimens. The embryos are
stained immunohistochemically for the presence of b-MHC
(A and D) and a-SMA (B, C, E, and F). Note that in the leastaffected embryo, the developing aorticopulmonary septum and
the distal portion of the endocardial ridges are positioned
between the lumen of the aortic and subpulmonary outlets
(A and B), whereas in the seriously affected specimen, the endocardial ridges and the rudimentary aorticopulmonary septum are
positioned anteriorly to the lumen of the outflow tract (D and E).
As a consequence, the subpulmonary (o) and aortic outlets
occupy anterior and right-lateral positions in embryo 92 (A),
whereas the single arterial outlet (o) occupies the right lateral
position in embryo 13 (D). Note also that there is no myocardium covering the aortic outlet at the level of the origin of the 6th
arch arteries in the least affected specimen (A), whereas this
area contains myocardium in the seriously affected specimen
(D). Further upstream, both endocardial ridges occupy more lateral positions in the outflow tract in both embryos (C and F).
Panel F further shows that these ridges contain, in comparison
with the less-affected specimen (C), only few a-SMA–positive
cells. A reconstruction of the heart of embryo No. 13 is shown
in Figure 5D. ao indicates aorta; aps, aorticopulmonary septum;
pt, pulmonary trunk; and r, endocardial ridge. *Condensed mesenchyme. Bar5100 mm.
ridges rather than the formation of endocardially derived tissue
is primarily affected. In ED13 and ED14 hearts, the developing
septal and parietal endocardial ridges could be distinguished
from the so-called intercalated endocardial ridges18 by the
presence of a rod of condensed, a-SMA–positive mesenchyme
(Figures 3 and 4). Except in the most seriously affected embryos,
the 2 endocardial ridges were always present, but the number
and position of intercalated ridges between them differed considerably. Although we noted no correlation between the number
of intercalated ridges and the severity of the malformation, a
normal position of the intercalated ridges was seen only in the
least affected hearts.
Sox4-Deficiency Syndrome
In the Table, the embryos are arranged in order of increasing
severity of their malformation at the arterial pole. Features
Ya et al
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Figure 3. Embryos No. 324 (A through C) and 101 (D through
F). These ED13 embryos are stained with hematoxylinazophlochsin (A through C) or immunohistochemically for the
presence of a-SMA (D and E) or b–MHC (F). Both embryos
demonstrate typical malformations in the arterial pole of Sox4deficient embryos, viz anterior position of the aorta (A and D;
note origin of coronary arteries), 1 (E) or 2 (B) intercalated ridges
that are positioned anterior to the adjacent septal and parietal
endocardial ridges. The septal and parietal ridges, which contain
rods of condensed mesenchyme, gradually converge onto the
inner curvature (C and F). A reconstruction of the heart of
embryo No. 101 is shown in Figure 5C. ao indicates aorta; aps,
aorticopulmonary septum; ca, coronary artery; cm, condensed
mesenchyme; ct, common trunk; is, intercalated ridge; la, left
atrium; pt, pulmonary trunk; r, ridge; ra, right atrium; and rv,
right ventricle. Bar550 mm.
associated with increasing severity of the malformation were
fusion of the endocardial ridges on the posterior wall of the
outflow tract near the lesser curvature, fusion of the condensed mesenchymal rods on the posterior wall of the
outflow tract, side-by-side or posterior position of the pulmonary trunk, difference in size of the pulmonary trunk and
aorta, abnormal number or position of the intercalated ridges,
and, finally, the presence of esophago-tracheal fistula. Based
on the position of the ascending aorta relative to the pulmonary trunk, we distinguished 4 groups.
The least severe grade of malformation (Figures 2A
through 2C, 5A, and 6; group 1 of the Table; '25% of cases)
was characterized by a complete separation of aorta and
pulmonary trunk, 2 separate semilunar valve anlagen, and a
remnant of the developing outlet septum below the valvular
rings. Usually, all 6 endocardial swellings could be identified
at the level of the developing valves. Although the endocardial ridges had fused at and just below the valves, they had
failed to do so further toward the embryonic right ventricle.
As a result, a large infundibular ventricular septal defect
existed in all cases. The aortic arch and arterial duct were
always on the left side. Furthermore, the diameter of the
aortic and pulmonary roots were usually comparable, with the
pulmonary root located anterior to that of the aorta.
In more serious malformations (Figures 5B and 7; group 2
of the Table; '25% of cases), the ascending aorta and
November 16, 1998
989
Figure 4. Embryo No. 114. This ED14 Sox4-deficient embryo is
stained immunohistochemically for the presence of a-SMA (A
through C), and b-MHC (D). The pulmonary and ascending aortic trunks are partially and unequally separated by an underdeveloped aorticopulmonary septum (A and B), whereas the outflow tract is a single tube directly connected to the right
ventricle (C and D). The pulmonary arteries arise from the posteriorly located large pulmonary trunk (A). Note the a-SMA–positive condensed mesenchyme (C) in the underdeveloped outflow
tract ridges, which converge onto the inner curvature at the ventricular end of the outflow tract (D). ao indicates aorta; aps, aorticopulmonary septum; cm, condensed mesenchyme; ct, common trunk; pa, pulmonary artery; pt, pulmonary trunk; and rv,
right ventricle. Bar550 mm.
pulmonary trunk were separate vessels to the extent that the
pulmonary arteries arose from the pulmonary trunk and
coronary arteries arose from the aorta. However, at and below
the level of the semilunar valve anlagen, only a single lumen
was present. Furthermore, the root of the pulmonary trunk
and aorta typically occupied side-by-side positions. Nevertheless, the morphological presentation of the endocardial
ridges was still rather normal with the septal and parietal
ridges each following their characteristic, spiral downstream
course in the outflow tract.
As the severity of the malformation increased further,
posterior position of the pulmonary trunk relative to the aorta
was found invariably (Figures 3, 4, and 5C; group 3 of the
Table; 35% of cases). At the level of the semilunar valve
anlagen, only 3 or 4 endocardial swellings were found. The
swellings containing the condensed mesenchymal rods typically occupied a posterior, adjacent position, that is, without
intercalated swelling between (Figure 3B, 3C, and 3E).
Further toward the ventricle, the septal and parietal ridges
were fused into a single ridge that was positioned on the
posterior wall of the outflow tract, that is, near the lesser
curvature (Figures 3C, 3F, and 4D). In 4 embryos, this fused
990
Sox4-Deficiency Syndrome in Mice
Features Characterizing the Sox4 Deficiency Syndrome
Post Fusion
Embryo No.
92
Pulmonary Trunk
ED
Ridges
CM
Valve
Position
Size
ETF
12
2
2
ND
Ant
PT5A
2
302
13
2
2
Ant
PT5A
2
18
14
2
2
Ant
PT5A
2
91
14
2
2
N1
14
2
2
92
14
2
2
36
12
2
2
101
14
2
2
ND
ND
Ant
PT5A
2
Ant
PT5A
2
Ant
PT.A
2
Lat?
PT5A
2
Lat
PT5A
2
103
14
1
2
Lat
PT.A
2
331
13
2
2
Lat
PT5A
1
307
13
1
2
Lat
PT5A
2
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321
13
2
2
Post
PT5A
2
313
13
2
2
Post
PT5A
2
N2
14
1
2
324
13
1
1
ND
Post*
PT5A
2
Post
PT,A
2
1
101
13
1
1
Post
PT,A
201
13
1
1
Post
PT,,A
1
114
14
1
1
Post
PT.A
2
93
12
1
Abs
ND
Single vessel
2
13
12
1
Abs
ND
Single vessel
2
72
14
1
Abs
Single vessel
1
The Sox4-deficiency syndrome was subdivided into 4 groups with increasing severity of the
abnormalities. Criteria were whether or not the endocardial ridges had fused, whether or not
condensed mesenchyme (CM) was recognizable in 2 of the ridges, and whether or not these rods
fused on the posterior wall of the outflow tract (Post Fusion). Abs indicates absent. The configuration
of the endocardial ridges at the valve level is described by a pictogram, in which dots represent ridges
containing condensed mesenchyme (Valve). The position of the pulmonary trunk (PT) relative to that
of the aorta (A) is indicated as Ant (PT anterior), Lat (PT and A side-by-side), or Post (PT posterior),
whereas differences in diameter are indicated by ,, 5, or .. The presence of an esophagotracheal
fistula (ETF) as indicated by 1. ND indicates not determined because of age (ED12) or plane of
sectioning (Nos. N1 and N2); and ?, the position of the ascending aorta relative to the pulmonary trunk
cannot be determined with certainty yet. *This case was described by Schilham et al4 as having its
ascending aorta in the anterior position; however, reinspection of the limited number of sections that
were available indicated that the aorta of this specimen also occupied an anterior position.
posterior portion of the ridges occupied more than one third
of the length of the outflow tract so that, in addition, the
pillars of condensed mesenchyme had become fused. Because
the condensed mesenchyme in the endocardial ridges is
continuous with the strongly a-SMA–positive mesenchyme
of the aorticopulmonary septum, a ring of a-SMA–positive
mesenchyme encircles the pulmonary outlet in this group
(Figure 5C). The relative diameters of pulmonary trunk and
aorta varied substantially, the most severe cases presenting
with stenosis of the pulmonary trunk (Figures 3 and 5C).
Narrowing of the pulmonary trunk always was accompanied
by absence of the arterial duct, whereas the aortic arch was
left-sided and normal in diameter. On the other hand, when
the diameter of the aortic root was smaller than that of the
pulmonary trunk (Figure 4), the embryo also had an enlarged
diameter of the arterial duct. In all these cases, both the aortic
arch and the arterial duct were situated on the left side. The
coronary artery anlagen always were associated with the aorta
(Figure 3A and 3D).
A single arterial trunk, which was a direct continuation of
the outflow tract and from which both the coronary arteries
and the pulmonary arteries originated, was found in 15% of
cases (Figures 2D through 2F, 5D, 5E, and 8; group 4 of the
Table). In the ED14 embryo that is illustrated in Figure 8, the
arterial duct was absent, whereas the aortic arch was on
the right side. Tissue with the same staining properties as
those of the endocardial ridges surrounded the lumen of the
outflow tract as a cuff, but rods of condensed mesenchyme
were absent. Two ED12 specimens revealed that this condition arose as a result of merging of the distal portion of the
endocardial ridges on the anterior side of the lumen of the
outflow tract (Figure 2D through 2F). As a result, the aortic
Ya et al
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Figure 5. The spectrum of cardiac malformations in Sox4deficient embryos. Panels A through E show 3– dimensional
reconstructions of hearts of Sox4-deficient embryos with
increasing degrees of severity of the malformation. A, Grade 1
malformation (ED14 embryo No. 18; see also Figure 6). B, Grade
2 malformation (ED14 embryo No. 101; see also Figure 7). C,
Grade 3 embryo (ED13 embryo No. 101; see also Figure 3D
through 3F). D and E, Grade 4 embryos (ED12 embryo No. 13,
see also Figure 2D through 2F; and ED14 embryo No. 72, see
also Figure 8, respectively). For a classification of groups 1 to 4,
see the Table. Dashed lines in panels A and D indicate the
upper boundary of the myocardium. ao indicates aorta; ca, coronary arteries; ct, common trunk; et, endocardially derived tissue; pa, pulmonary artery; pt, pulmonary trunk; r, ridge; rv, right
ventricle; and te, common part of trachea and esophagus.
sac is not divided between ascending aorta and pulmonary
trunk. The ED12 embryos further resembled the ED14
specimen in that relatively few a-SMA–positive cells were
seen in the endocardial tissue and that the right-sided fourth
branchial arch artery was better developed than the left,
whereas both sixth-arch arteries were reduced in size. Two
embryos of this group suffered, in addition, from a hypoplastic left ventricle.
Associated Malformations in Sox4-Deficient Mice
November 16, 1998
991
Figure 6. Embryo No. 18. This ED14 Sox4-deficient embryo is
among the least affected in the series. The embryo is stained
immunohistochemically for the presence of b-MHC (A and D),
a-SMA (B), and fibronectin (C). A, Above the semilunar valves,
the outflow tract is separated into aorta and pulmonary trunk by
the aorticopulmonary septum. However, near the junction with
the right ventricle, the endocardial ridges do not fuse (compare
panels B and C with panel D). Note further that the semilunar
valve leaflets in the pulmonary trunk (A) and aorta (D) are rudimentarily developed. A reconstruction of this heart is shown in
Figure 5A. ao indicates aorta; aps, aorticopulmonary septum;
av, aortic valve; cm, condensed mesenchyme; os, outlet septum; pt, pulmonary trunk; pv, pulmonary valve; r, ridges; and rv,
right ventricle. Bar5100 mm.
pulmonary trunk (type IV according to Angelini and Leachman10), via a common origin of aorta and pulmonary trunk at
the level of the valve anlagen (type 1 according to Collett and
Edwards,31 type A1 according to van Praagh and van
Praagh,11 and type II according to Angelini and Leachman10),
to 2 pulmonary artery orifices in the dorso-caudal truncal wall
(type 2 according to Collett and Edwards,31 type A2 according
to van Praagh and van Praagh,11 and type I according to
Angelini and Leachman10). Additionally, we did observe
embryos in which the diameter of the pulmonary trunk was
much smaller than that of the aorta (van Praagh and van
Praagh’s type A311) and embryos in which the diameter of the
Branchial arch anomalies in ED13 and ED14 embryos, such
as absence (4 cases) or increased size (2 cases) of the arterial
duct, were correlated with the size of the pulmonary trunk
relative to that of the aorta. An esophagotracheal fistula was
found in 4 of the more serious cases (Figure 8). These
malformations probably represent an independent feature of
Sox4 deficiency, because the transcription factor also is
expressed strongly in the mesenchyme of the branchial arches
and on the dorsal surface of the trachea.
Discussion
Even though only a single gene (Sox4) was inactivated, we
observed a range of malformations of the arterial pole of the
heart. The anomalies ranged from a solitary defect in the
proximal (conal) portion of the outlet septum, that is, with
separate valve anlagen supporting the roots of the aorta and
Figure 7. Embryo No. 101. This Sox4-deficient ED14 embryo is
stained immunohistochemically for the presence of a-SMA (A)
and b-MHC (B). At the semilunar valve level, the aorta and pulmonary channels occupy side-by-side positions (A and B). A
reconstruction of this heart is shown in Figure 5B. ao indicates
aorta; cm, condensed mesenchyme; la, left atrium; pa, pulmonary artery; pv, pulmonary valve; r, endocardial ridges; ra, right
atrium; and rv, right ventricle. Bar5100 mm.
992
Sox4-Deficiency Syndrome in Mice
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Figure 8. Embryo No. 72. This ED14 Sox4-deficient embryo is
stained with hematoxylin-azophlochsin. In this embryo, only a
single ascending trunk arises from the embryonic right ventricle
(C), from which the coronary arteries (B) and the pulmonary
arteries (A) arise. No condensed mesenchyme can be seen in
the endocardial tissue. Note the common part of the esophagus
and trachea in panel A. A reconstruction of this heart is shown
in Figure 5E. ca indicates coronary artery; ct, common trunk; et,
endocardially derived tissue; la, left atrium; pa, pulmonary
artery; r, ridge; ra, right atrium; rv, right ventricle; and te, common part of trachea and esophagus. A and B, Bar550 mm. C,
Bar5100 mm.
aorta was much smaller than that of the pulmonary trunk (van
Praagh and van Praagh’s type A411). Such a wide spectrum in
the phenotypic presentation of a single gene deficiency is
probably characteristic of genes that are either components of
a signal-transduction pathway or transcription factors. Most
likely, the downstream members of these signal-transduction
pathways or the target genes of the transcription factors
continue to be active, but as a consequence of a lack of
regulation, the level of these downstream activities is no
longer (tightly) controlled, thus allowing the emergence of
large interindividual differences in phenotype. Our finding
that Nfatc is expressed in Sox4-deficient embryos and vice
versa,32 therefore does not prove the presence of independent
regulatory pathways. On the other hand, accurate descriptions
of single gene– deficiency syndromes are necessary to decide
which congenital malformations may share a common deficiency and to obtain a sufficiently large catalogue of syndromes to deduce which regulatory pathways are involved in
a particular morphogenetic process.
Both Sox4 and Nfatc32,33 deficiency are characterized by
death due to heart failure on day 14 of embryonic development. The observed regurgitation of blood in embryos of this
age shows that the lack of transformation of the distal portion
of the endocardial ridges into one-way semilunar valves is, in
all likelihood, the direct cause of the heart failure in both the
mildly and seriously affected embryos (compare with References 34 to 36). The timing of the onset of this functional
failure strongly supports our hypothesis that it is the remodeling of endocardial tissue into valvular structures rather than
the formation of the endocardial tissue itself that is primarily
affected. Before the development of the semilunar valves on
ED13 and ED14, an adequate amount of endocardially
derived stuffer tissue in the outflow tract suffices to allow the
outflow tract to function as a myocardial sphincter that
prevents regurgitation of blood.16 Although population of the
endocardial cushions of the atrioventricular canal by neural
crest cells is quantitatively insignificant compared with that
of the endocardial ridges of the outflow tract,21,37 Sox4
expression levels in both endocardial tissue– derived structures are comparable (Figure 1F, 1J, and 1L). Sox4 expression therefore is localized almost certainly in endocardially
derived cells. Because the malformations in Sox4-deficient
embryos are largely restricted to the arterial pole, whereas the
development of the atrioventricular cushions is not visibly
affected, our study suggests that it is the interaction between
the neural crest– derived myofibroblasts30 and the endocardially derived original cells of the outflow tract, that determines
proper development of the arterial pole.
The least affected Sox4-deficient hearts, strictly speaking,8,9 do not suffer from common trunk but should be
classified as having a large infundibular ventricular septal
defect. In the more serious cases, the following are seen: a
linear rather than a spiral configuration of the ridges and,
consequently, a posterior position of the pulmonary trunk at
the level of the valve (the configuration present in transposition of the great arteries), an abnormal number and position of
the intercalated ridges relative to the parietal and septal
endocardial ridges, lack of fusion of the ridges, and variable
size of the aorta relative to the pulmonary trunk. In the most
serious cases, even the formation of the endocardial ridges
failed, so that a cuff of endocardial tissue, in which the neural
crest– derived rod(s) of condensed mesenchyme could not
even be identified as such, continued to surround the lumen of
the outflow tract. Because the emergence of spiraling septal
and parietal endocardial ridges coincides temporally with the
ingrowth of a-SMA–positive neural crest cells30 and because
the position of these 2 endocardial ridges greatly differs
between individual affected mice, it appears that the migration of the neural crest cells into the endocardial tissue of the
outflow tract is poorly guided in Sox4-deficient mice. The
association of an abnormal position of the endocardial ridges
and an abnormal number and position of the intercalated
ridges with the presence of a single arterial outlet and often
with a difference in diameter of aorta and pulmonary trunk,
implies a correspondingly abnormal position of the aorticopulmonary septum.
We know of only 2 earlier descriptions of common trunk in
embryos.38,39 For that reason, the morphogenetic events underlying the development of common trunk are deduced
mostly from observations in human neonates. The conventional explanation of common trunk is a complete or partial
failure of down-growth of the aorticopulmonary septum,
Ya et al
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together with incomplete development of the endocardial
ridges of the outflow tract (eg, References 8 to 10, 38, and
40). Some consider the abnormal development of the ridges
to be secondary to faulty development of the aorticopulmonary septum (eg, Reference 9). In support of this view are the
extensive population of the ridges of the outflow tract by
neural crest cells21,37 and the prevalence of malformations of
the arterial pole of the heart in animal models in which genes
that are expressed in neural crest derivatives are disrupted13,14,41– 45 (for a review, see Reference 46). Accordingly,
common trunk, together with tetralogy of Fallot and interrupted aortic arch, is described often as one of the symptoms
identifying a typical neurocristopathy in human neonates,47
but it almost never is associated with a configuration reminiscent of (complete) transposition of the great arteries. In
Sox4-deficient embryos, however, we did not observe tetralogy of Fallot or interrupted aortic arch, whereas we did
observe the configuration of the endocardial ridges that
corresponds with transposition in '50% of cases. These
differences in presentation are suggestive of distinctive roles
for the endocardially derived cells and the neural crest cells in
the regulation of morphogenesis of the outflow tract. In
addition, the lack of differentiation of the semilunar valves
in Sox4- and Nfatc-deficient mice, and the almost normal
development of semilunar valves in common trunk caused by
the dysfunction of neural crest cells (eg, resulting from
deficiency of nuclear retinoic acid receptors13,44 or the Pax3
transcription factor14) also support a distinct morphogenetic
role for endocardially derived and neural crest– derived
tissues.
Conclusion
The Sox4-deficiency syndrome can be ascribed to a defective
function of the endocardial tissue of the outflow tract, leading
to a lack of development and/or fusion of the endocardial
ridges, a lack of development of the semilunar valves, and an
arrangement of the ventriculo-arterial connection corresponding with transposition of the great arteries. The Sox4deficiency syndrome is therefore an animal model for abnormal development of the ventriculo-arterial junction because
of a defective function of the endocardially derived tissue.
The fate of the endocardial tissue– derived structures involved
in the formation of the ventriculo-arterial junction could be
followed, thanks to the presence of the highly characteristic
rods of condensed mesenchyme. The restriction of the malformations to the arterial pole, even though the Sox4 gene is
equally expressed in the endocardially derived tissue of the
atrioventricular canal, implies that the interaction between
the endocardially derived tissue of the outflow tract and the
neural crest– derived myofibroblasts30 determines proper development of the arterial pole. The lack of differentiation of
the semilunar valves in Sox4- and Nfatc-deficient32,33 mice
and their normal development in animal models with neural
crest deficiencies implies that the neural crest does not
regulate the development of the semilunar valves.
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Sox4-Deficiency Syndrome in Mice Is an Animal Model for Common Trunk
Jing Ya, Marco W. Schilham, Piet A. J. de Boer, Antoon F. M. Moorman, Hans Clevers and
Wouter H. Lamers
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Circ Res. 1998;83:986-994
doi: 10.1161/01.RES.83.10.986
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