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Cellular Biology
Slit–Roundabout Signaling Regulates the Development of the
Cardiac Systemic Venous Return and Pericardium
Mathilda T.M. Mommersteeg, William D. Andrews, Athena R. Ypsilanti, Pavol Zelina, Mason L. Yeh,
Julia Norden, Andreas Kispert, Alain Chédotal, Vincent M. Christoffels, John G. Parnavelas
Rationale: The Slit–Roundabout (Robo) signaling pathway has pleiotropic functions during Drosophila heart
development. However, its role in mammalian heart development is largely unknown.
Objective: To analyze the role of Slit–Robo signaling in the formation of the pericardium and the systemic venous
return in the murine heart.
Methods and Results: Expression of genes encoding Robo1 and Robo2 receptors and their ligands Slit2 and Slit3
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was found in or around the systemic venous return and pericardium during development. Analysis of embryos
lacking Robo1 revealed partial absence of the pericardium, whereas Robo1/2 double mutants additionally showed
severely reduced sinus horn myocardium, hypoplastic caval veins, and a persistent left inferior caval vein. Mice
lacking Slit3 recapitulated the defects in the myocardialization, alignment, and morphology of the caval veins.
Ligand binding assays confirmed Slit3 as the preferred ligand for the Robo1 receptor, whereas Slit2 showed
preference for Robo2. Sinus node development was mostly unaffected in all mutants. In addition, we show absence
of cross-regulation with previously identified regulators Tbx18 and Wt1. We provide evidence that pericardial
defects are created by abnormal localization of the caval veins combined with ectopic pericardial cavity formation.
Local increase in neural crest cell death and impaired neural crest adhesive and migratory properties underlie the
ectopic pericardium formation.
Conclusions: A novel Slit–Robo signaling pathway is involved in the development of the pericardium, the
sinus horn myocardium, and the alignment of the caval veins. Reduced Slit3 binding in the absence of
Robo1, causing impaired cardiac neural crest survival, adhesion, and migration, underlies the pericardial
defects. (Circ Res. 2013;112:465-475.)
Key Words: cardiac neural crest ◼ heart development ◼ pericardium ◼ Slit–Robo signaling
◼ systemic venous return
C
ongenital defects of the arterial side of the heart are frequently life threatening and generally diagnosed immediately after birth. Defects of the systemic venous return to
the heart often only become symptomatic much later in life.
However, these defects can be severe, ranging from congenital
malformations, such as misalignment of the connecting veins,
to atrial arrhythmias like sick sinus syndrome.1,2
Knowledge about the genes involved in the development of
the systemic venous return, the caval veins, and myocardial
sinus horns is limited. The T-box transcription factor Tbx18 is
required for the differentiation of the sinus horn myocardium
from mesenchymal precursors, and its absence causes delayed
formation and malformation of the sinus horns.3,4 Recently, a
role was shown for Wnt/β-catenin signaling in regulating the
differentiation of the sinus horn myocardium.5 Podoplanin,6
Pdgfrα,7 and Wt1, as well as its downstream target Raldh2,8 were
found essential for normal sinus horn formation. Interestingly,
mouse mutants for Tbx18,9 Wt1, and Raldh2/Retinoic acid
signaling8 presented with additional defects in the formation
of the membrane between the pleural and pericardial cavities,
indicating a link between the development of the systemic
venous return and the pericardium. Further data on genetic
pathways involved in pericardium development are lacking.
Absence of part of the pericardium is often symptomless, but
it can reveal itself by sudden acute chest pain or dyspnea.10
Recently, expression of genes of the Slit–Roundabout
(Robo) signaling pathway was detected at the venous pole
of the heart,11 suggesting a possible role in the formation of
Original received July 15, 2012; revision received December 13, 2012; accepted December 19, 2012. In November 2012, the average time from
submission to first decision for all original research papers submitted to Circulation Research was 15.8 days.
From the Department of Cell and Developmental Biology, University College London, London, UK (M.T.M.M., W.D.A., M.L.Y., J.G.P.); Institut
National de la Santé et de la Recherche Médicale, UMR S968, Institut de la Vision, Paris, France (A.R.Y., P.Z., A.C.); Institut für Molekularbiologie,
Medizinische Hochschule Hannover, Hannover, Germany (J.N., A.K.); and Heart Failure Research Center, Department of Anatomy, Embryology and
Physiology, Amsterdam, The Netherlands (V.M.C.).
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.
277426/-/DC1.
Correspondence to John G. Parnavelas, Department of Cell and Developmental Biology, University College London, Gower St, London WC1E 6BT,
United Kingdom. E-mail [email protected]
© 2012 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.112.277426
465
466 Circulation Research February 1, 2013
Nonstandard Abbreviations and Acronyms
E
Robo
Tbx
Wt1
YFP
embryonic day
Roundabout
T-box transcription factor
Wilms tumor 1
yellow fluorescent protein
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this area. Robo receptors are members of the immunoglobulin superfamily of cell adhesion molecules. In mammals, 4
such receptors (Robo1–4) and 3 Slit ligands (Slit1–3) have
been identified.12 In Drosophila and zebrafish, Slit–Robo signaling plays roles in cell adhesion during cardiac cell polarization, morphogenesis, migration, and lumen formation.13–16
However, knowledge about Slit–Robo signaling in the mammalian heart is scant. Slit3 has been shown to be the predominant ligand gene transcribed in the chambers of the early
mouse heart.11 Furthermore, a role for Slit–Robo signaling has
been found during cardiac neural crest migration, where it is
regulated by Tbx1 and gastrulation brain homeobox 2.17
Here, we report a novel role for Slit–Robo signaling in the
development of both the systemic venous return to the heart
and the pericardium. Mice deficient of Robo1 or both Robo1
and Robo2 lack part of the pericardium and show abnormal
caval vein and sinus horn development. The systemic venous
return defects are recapitulated in the absence of Slit3, but not
Slit1 and Slit2. Mainly local Slit3, but also some Slit2, binding
is abolished in Robo1 mutants, whereas both Slit2 and Slit3
binding are absent in Robo1/2 knockouts. Further analysis indicated that abnormal localization of the caval veins combined
with ectopic pericardial cavity formation, caused by increased
cell death and impaired adhesion and migratory responsiveness
of the cardiac neural crest, underlies the pericardial defects.
Methods
Transgenic mice and experimental procedures for in situ hybridization, immunohistochemistry, ligand binding assay, neural crest explants, 3-dimensional reconstruction, and statistical analyses are
provided in the Online Data Supplement. All experimental procedures were performed in accordance with the UK Animals (Scientific
Procedures) Act 1986 and institutional guidelines.
Results
Robo1 Knockout Embryos Lack Part of the
Pericardium and Display Systemic Venous
Return Defects
Sections of embryos lacking the Robo1 receptor showed a
striking defect in the pericardium (n=11/21). Although most of
the pericardium developed normally, the part located between
the superior caval veins, the pleuropericardial membrane, was
missing. As a result, the expanding lungs were not retained
in the pleural cavity, but penetrated into the pericardial cavity and completely enveloped the heart (Figure 1A and 1B).
As a ­result of the absence of the pleuropericardial membrane,
the ­caval veins, which develop in close relation to the pericardium,8 lacked their medial attachment to the mediastinum
(Figure 1A and 1B). Despite the abnormal location of the caval veins, sinus horn myocardium developed relatively normal
(Figure 1A and 1B). The pleuropericardial membrane defect
was predominantly present on the left side (6/11). However, in
several animals, only the right side (2/11) or both sides (3/11)
were affected.
Only after embryonic day (E) 11.5, the pleuropericardial
membranes start to divide the pericardial cavity from the
Figure 1. Pericardial defects in the Robo1
mutants. A–F, In situ hybridization staining for
myocardial marker cTnI on Robo1+/+ (A, C, and D)
and Robo1−/− (B, E, and F) embryos. C–F, Sections
through cranial (C and E) or caudal (D and F)
pericardial cavity in Robo1+/+ and Robo1−/− mice.
Note the normal formation of the pleuropericardial
membrane caudal of the entering caval veins in
the knockout (red arrowheads). G–N, Threedimensional reconstructions of an embryonic day
(E) 12.5 Robo1+/+ and littermate Robo1−/− embryo.
G and H, A ventral view of the heart and of the
dorsal pericardium before and after removal of the
heart. Arrowheads indicate the caval vein entrance.
I and L, Dorsal views of the myocardium of the
heart, sinus horns, and the caval veins. J, K, M,
and N, Arrowheads show where the caval veins
pass the lungs. Ao indicates aorta; LA, left atrium;
LL, left lung; LSCV, left superior caval vein; LSH,
left sinus horn; LV, left ventricle; PC, pericardium;
PT, pulmonary trunk; PV, pulmonary vein; RA, right
atrium; RL, right lung; RSCV, right superior caval
vein; RSH, right sinus horn; and RV, right ventricle.
Scale bars depict 100 μm.
Mommersteeg et al Slit-Robo Signaling in Pericardium Development 467
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pleural cavity.8 In agreement, no morphological defects were
observed in Robo1−/− embryos until after E11.5 (data not
shown). As the caval veins in the wild-type embryos became
progressively located in the pericardial cavity between E11.5
and E12.5, those in the Robo1−/− remained more dorsally inside the body mesenchyme and entered the pericardial cavity
much more caudally (Figure 1C–1H, black arrowheads 1G
and 1H). As a consequence of their more caudal entrance, the
caval veins had to circumvent the developing lungs to gain
access to the heart (Figure 1I–1N). Caudal to their entry into
the heart, pleuropericardial membrane formation was normal
(Figure 1C–1F, red arrowheads).
None of these defects was observed in Robo2−/− embryos,
which were indistinguishable from their wild-type littermates (Figure 2A and 2B). However, the combined absence
of both Robo1 and Robo2 genes increased the incidence of
the pericardial defect (n=5/7; Figure 2C–2F). Furthermore,
Robo1−/−;Robo2−/− embryos showed malformed and very thin
caval veins (Figure 2C–2H). The connection of the left caval
vein to the heart ranged from a small, but relatively normal
connection to the right atrium, to only an entrance into the
coronary circulation (Figure 2G and 2H, red arrowheads;
Online Figure IA–IE). Whereas sinus horn myocardium
formation in the Robo1−/− animals was largely unaffected,
minimal sinus horn myocardium formed around both caval
veins in the double mutants (Figure 2G and 2H) and its size
seemed proportional to the extent of the caval vein connection to the heart (Online Figure IA–IE). Furthermore, some
of the Robo1−/−;Robo2−/− embryos exhibited a persisting left
inferior caval vein, which joined the right inferior caval vein
at the entrance into the liver (Figure 2E–2H, black arrowhead). These defects were confined to the systemic venous
return, as the pulmonary veins developed normally in all mutants (data not shown).
Although Robo1 seems to be the key Robo receptor in the
development of the systemic venous return and pericardium,
the additional defects in the double mutants suggest functional
redundancy with the Robo2 receptor, which is expressed
normally in the absence of Robo1 (Online Figure IF). To
determine which Slit ligands and Robo receptors are involved
in this region, we next analyzed their expression patterns.
Robo1 and Robo2 Receptors and Their Ligands,
Slit2 and Slit3, Are Expressed During Systemic
Venous Return and Pericardium Development
Of all Robo receptor genes, Robo1 was the most prevalent.
Its expression has been described in the outflow tract cushions,11 and we observed additional signal in the myocardium
and cushions of the atrioventricular canal at E9.5 (Figure 3A),
which was maintained at E12.5 and E14.5 (Online Figure IIA
and IIB). Robo1 expression was not observed in the sinus horn
myocardium, but both the pericardium and the mesenchyme
directly surrounding the caval veins showed robust expression (Figure 3A; Online Figure IIA and IIB). Robo2 was less
broadly expressed and, in contrast to earlier observations,11
was never seen in the myocardium at any stage. We detected
Robo2 signal in the outflow tract cushions11 and in the atrioventricular cushions at all time-points analyzed (Figure 3A;
Online Figure IIA and IIB). Although there was no expression
Figure 2. Defects in Robo2 and Robo1/2
mutants. A–D, In situ hybridization for cTnI on
Robo2+/+ (A), Robo2−/− (B), Robo1+/+;Robo2+/+ (C),
and Robo1−/−;Robo2−/− (D) embryos. E–H, Threedimensional reconstructions of Robo1+/+;Robo2+/+
(E and G) and Robo1−/−;Robo2−/− (F and H) embryos.
E and F, Ventral view on the heart and lungs before
and after removal of the heart. G and H, Dorsal
view on the heart and sinus horn myocardium, with
the caval veins enlarged to show the abnormal left
cardiac connection (red arrowhead) in the mutant.
Black arrowheads indicate the persistent inferior
caval vein in Robo1−/−;Robo2−/− embryos (E–H).
Note the small size of the sinus horns in the double
mutant. E indicates embryonic day; ICV, inferior
caval vein; LA, left atrium; LL, left lung; LSCV, left
superior caval vein; LSH, left sinus horn; LV, left
ventricle; RA, right atrium; RL, right lung; RSCV,
right superior caval vein; RSH, right sinus horn; and
RV, right ventricle. Scale bars depict 100 μm.
468 Circulation Research February 1, 2013
Figure 3. Slit3 is required for caval vein alignment
and sinus horn formation. A, In situ hybridization
for Robo1, Robo2, Robo4, Slit1, Slit2, and Slit3 at
embryonic day (E) 9.5. B and C, In situ hybridization
cTnI staining on Slit3+/+ (B) and Slit3−/− (C) embryos.
D and E, Three-dimensional reconstructions
of Slit3+/+ (D) and Slit3−/− (E) embryos. Black
arrowheads Persistent left inferior caval vein in the
mutant; red arrowheads the abnormal left venous
cardiac connection. ICV indicates inferior caval
vein; LA, left atrium; LCV, left caval vein; LL, left
lung; LSCV, left superior caval vein; LSH, left sinus
horn; LV, left ventricle; PV, pulmonary vein; RA, right
atrium; RL, right lung; RSCV, right superior caval
vein; RSH, right sinus horn; and RV, right ventricle.
Scale bars depict 100 μm.
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in the sinus horn myocardium, Robo2 was visible in the mesenchyme surrounding the caval veins and in the body wall at
E9.5 (Figure 3A). Robo3 expression was restricted to the central nervous system as described earlier (data not shown).18
Robo4 expression was found in the vascular endothelium,19
including that of the aorta, pulmonary trunk, and coronary
vasculature, but none was observed in the endocardium. A
small number of Robo4 positive cells were observed in or surrounding the caval veins at E9.5 (Figure 3A, arrowheads), but
not at later stages.
Slit1 was highly expressed in the floor plate of the neural
tube (Figure 3A), but only weakly in the outflow tract vessels at E14.5 (data not shown). In contrast, Slit2 and Slit3
were highly expressed in distinct patterns in and around the
heart, and isolated Slit2 signal in the myocardium was confined to the ventricular trabecules (Figure 3A).11 Furthermore,
Slit2 was expressed in the outflow tract vessels as previously
­observed,11 in the outflow tract endocardium, the epicardium,
and the mesenchyme around the caval veins (Online Figure
IIA and IIB). Slit3 was most broadly expressed in the myocardium, with presence in the outflow tract and atrial myocardium,11 and it was the only gene of the Slit and Robo families
found in the sinus horn myocardium (Online Figure IIA and
IIB, black ­arrowheads). Slit3 was also detected in the epicardium and all tissues connecting the heart to the body. The
high expression of both Slit2 and Slit3 in the systemic venous
­return and pericardium region prompted us to further investigate these ligands.
Slit3–Robo1/2 Interaction Is Required for Caval
Vein Alignment and Sinus Horn Formation
Slit2−/− or even Slit1−/−;Slit2−/− embryos did not show any defects in the systemic venous return and pericardium region
(Online Figure IIC–IIF). However, Slit3−/− mice showed defects partly resembling those observed in Robo1−/−;Robo2−/−
embryos. Slit3−/− caval veins were hypoplastic, although to a
lesser extent than observed in the Robo1−/−;Robo2−/− mutants
(Figure 3B–3E). As in Robo1−/−;Robo2−/− mice, the left superior caval vein persisted into a left inferior caval vein that
connected with the right inferior caval vein at the level of the
liver (Figure 3D and 3E, black arrowheads). The left caval
vein did only connect via a very small long separate vein to
the coronary sinus, and the sinus horn myocardium was very
short and severely hypoplastic (Figure 3D and 3E, red arrowheads). The part of the membrane directly surrounding the
caval veins was much thicker in Slit3−/− embryos, but no missing parts were observed (Figure 3B and 3C). These data suggest that Slit3 acts as the main ligand required by Robo1 and
Robo2 in regulating the formation of the region. However,
the absence of pericardial defects in Slit3 mutants indicates
involvement of additional factors, most likely the other locally present ligand, Slit2.
To determine specificity of Slit3 and Slit2 for Robo1
and Robo2 receptors in the region, we performed a ligand
binding assay on sections. Slit3 binding was observed
precisely between the caval veins and the pericardium in
and adjacent to the Robo1-expressing region in wild-type
embryos (Figure 4A). High-affinity binding of Slit3 was
found for the region between the superior caval veins directly
cranial to the pericardium, which exhibited high Robo1
expression levels (Online Figure IIIA and IIIB). Slit2 also
bound to these regions, albeit at lower levels (Figure 4A and
4B; Online Figure IIIA and IIIB). In contrast to Slit3, affinity
of Slit2 was highest for the locally present Robo2 (Online
Figure IIIA–IIIC). Differential binding of Slit2 and Slit3 was
observed in the outflow tract cushions and surrounding the
trachea and esophagus. In the absence of Robo1, binding
of Slit3 was largely absent, whereas Slit2 binding was
reduced between the caval veins and the pericardium, but
still showed binding just cranial of the pericardium (Figure
4A and 4B; Online Figure IIIA and IIIB). In the absence
of Robo1, Robo2-expressing areas surrounding the trachea
Mommersteeg et al Slit-Robo Signaling in Pericardium Development 469
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Figure 4. Reduced Slit3 and Slit2
binding around the pericardial cavity
and caval veins in Robo1 and Robo1/2
mutants. A and B, Ligand binding assays
on Robo1+/+, Robo1−/− and Robo1−/−;
Robo2−/− embryos. A, Double staining
with Robo1 shows partial overlap of
Slit2 and Slit3 binding with roundabout
(Robo) 1. Note the different binding of
Slit2 and Slit3 in the trachea, esophagus,
and OFT. A and B, White arrowheads
The presence of Slit2 and Slit3 binding
surrounding the pericardial cavity in
the wild-type, which is absent in the
Robo1−/− embryo. Clear arrowheads
Binding of Slit2 and Slit3 in the body
wall, which is reduced in the Robo1−/−.
All binding is lost in the Robo1−/−;Ro
bo2−/− embryos. BW indicates body wall;
Dapi, 4',6-diamidino-2-phenylindole;
E, embryonic day; FG, foregut; LA, left
atrium; LSCV, left superior caval vein; LV,
left ventricle; OFT, outflow tract; RA, right
atrium; RSCV, right superior caval vein;
RV, right ventricle; and Tr, trachea. Scale
bars depict 100 μm.
and oesophagus persisted to bind Slit2 and the ventral body
wall persisted to bind both Slit2 and Slit3 (Figure 4B; Online
Figure IIIC). In the absence of either receptor, binding of
both Slit2 and Slit3 was largely abolished (Figure 4B; Online
Figure IIIB). These data confirm Slit3 as the main regional
ligand for Robo1, showing strong decrease in binding in the
absence of this receptor. Slit2 shows preference for Robo2;
however, the absence of pericardial defects in the Slit3 mutant
and the regional presence and binding of Slit2 indicate likely
functional redundancy of Slit2 and Slit3.
Sinus Node Development and Lack of Crossregulation With Previously Identified Systemic
Venous Return Regulators Tbx18 and Wilms
Tumor 1
At the border of the right sinus horn with the right atrium, the
sinus node develops as part of the sinus horn myocardium.20
As the development of the sinus horns was severely hampered
in Robo1−/−;Robo2−/− and Slit3−/− embryos, we next assessed
if sinus node development was affected. Of the Slit and Robo
genes, only Slit3 was expressed in this structure, identified by
the presence of Hcn4 and the absence of Cx40 (Figure 5A,
arrowheads). Robo1 as well as Slit2 and Slit3 was expressed
in the mesenchyme surrounding the sinus node. Whereas it
normally develops in direct contact with the caval vein, we
noticed separate development of both structures in Robo1
(Figure 5B), Robo1/2 and in Slit3 mutants at E12.5 (data not
shown). The initially malformed sinus node gained a normal
structure during later development in Robo1−/− and Slit3−/−
(Figure 5C), but part of it was still disconnected from the caval
vein in double mutants at E14.5. The size of the sinus node of
all Slit and Robo mutants was indistinguishable from control
littermates at E14.5 (Figure 5C and 5D).
Sinus horn, including the sinus node myocardium, is known
to differentiate from Tbx18-positive mesenchyme into Tbx18positive myocardium.3 Tbx18 was found to be normally expressed
in the sinus horns and sinus node of Robo1−/−, Robo1−/−;Robo2−/−,
and Slit3−/− mice (Figure 5B and data not shown). Furthermore,
the expression of Slit and Robo genes was unaffected in Tbx18
mutants (Figure 5E and data not shown), indicating this
pathway is not downstream of the Tbx18-dependent pathway
for sinus horn development. As Wt1 and Raldh2 are required
for the development of the pleuropericardial membranes and
caval veins,8 we next analyzed the expression of these genes in
the area of pleuropericardial membrane formation in Robo1−/−
embryos. The morphological differences between Robo1−/− and
control embryos made exact regional comparison difficult, but
expression levels and patterns of both genes were normal in
the mesothelium and subcelomic mesenchyme surrounding the
pericardial cavity (Figure 6A; Online Figure IVA). Reciprocal
analysis in Wt1 mutants showed that the expression of Robo1,
470 Circulation Research February 1, 2013
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Figure 5. Sinus node (SN) development
is largely unaffected in Slit and Robo
mutants. A–C and E, In situ hybridization
for the indicated genes. The sinus
node is recognizable by its positivity
for Hcn4 and negativity for Cx40. B,
Arrowheads The developing sinus
node surrounds the caval in the wildtype and is separated from the caval
vein in the Robo1 mutant. C, Note the
partly detached developing sinus node
in the Robo1−/−; Robo2−/− (arrowhead),
compared with the Slit3 mutant and wildtype control. D, Embryonic day (E) 14.5
sinus node volume measurements of
Robo1, Robo1/2, and Slit3 mutants and
control embryos show comparable sizes.
E, Comparable expression of both Robo1
and Slit3 in E12.5 Wt1+/+, Wt1−/−, Tbx18+/+,
and Tbx18−/− embryos. LA indicates left
atrium; LSCV, left superior caval vein;
OFT, outflow tract; RA, right atrium; and
RSCV, right superior caval vein. Scale
bars depict 100 μm.
Robo2, Slit2, and Slit3 was unaffected (Figure 5E and data not
shown), indicating absence of cross-regulation between the
Slit–Robo pathway and Wt1.
Ectopic Celom Formation Underlies the Partial
Absence of the Pericardium
The caval veins were progressively incorporated into the
pericardial cavity in wild types at E12.5,3 but remained
present inside the body mesenchyme in Robo1−/− mice (Figure
1C–1H). Although Wt1 and Raldh2 were normally present
between the caval vein and the pericardial cavity in Robo1−/−
mice, this expression area was much larger near the cranial
pericardium owing to the aberrant location of the veins.
Exactly in the larger area of Wt1- and Raldh2-expressing
mesenchyme, between the caval veins and pericardium,
we observed an area of ectopic celom formation at E12.5
(Figure 6A–A″; Online Figure IVA) that formed directly in
contact with the cranial pericardial cavity and immediately
became occupied by expanding lung tissue (Figure 6A and
6B). In contrast, expansion of the pleural cavity was only
observed caudal to the entrance of the caval veins into the
pericardial cavity in wild-type embryos (Figure 6A–A″, black
arrowheads). As a result of the ectopic celom formation,
the outflow tract connection to the body had to bridge a
significantly increased area inside the pericardial cavity to
reach the heart, which was quantified by measuring the length
of the body connection at outflow tract level, as indicated by
the arrows in Figure 6B (Figure 6C). These results suggest
that abnormal localization of the caval veins in combination
with ectopic celom formation underlies the pericardial defects
observed in the absence of Robo1.
Ectopic Celom Formation Is Caused by Increased
Apoptosis in the Neural Crest
The pleuropericardial membranes are released from the lateral
body wall by apoptosis,8 suggesting ectopic celom formation
may be caused by abnormal cell death. Therefore, we analyzed apoptosis in the region where we observed ectopic cavity formation using the apoptotic marker cleaved Caspase 3. In
wild-type embryos at E11.5, a small cluster of apoptotic cells
was observed in the region between the caval veins and the
cranial pericardial cavity (Figure 6D, arrowhead). However,
the number of apoptotic cells in this area had doubled in
Robo1 mutants, exactly in the area showing ectopic celom
formation 1 day later (Figure 6D, arrowhead). At E12.5, the
Mommersteeg et al Slit-Robo Signaling in Pericardium Development 471
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Figure 6. Ectopic celom formation is caused by
increased apoptosis in the cardiac neural crest.
A and B, In situ hybridization sections stained
for Wt1, level of sectioning as indicated in Online
Figure IVA. A′ and A″ are magnifications of indicated
boxes. Black arrowheads The most cranial area
of pleural cavity formation, which is much more
cranial in the mutant. Clear arrowheads The further
location of the mutant caval vein into the mutant
body. Red arrowheads The location of the wildtype sinus horn inside the pericardial cavity, and
the mutant sinus horn still mostly inside the body
mesenchyme. B, section indicating the location of
length measurements shown in C. D, Robo1+/+ and
Robo1−/−;Wnt1Cre;R26REYFP immunohistochemistry
sections stained for the indicated proteins.
Arrowheads The location of increased apoptosis
in Robo1−/− mice compared with the wild-type
control. Arrows The location of apoptotic cells in
the neural crest. Quantification of cleaved Caspase
3 (CC3)–positive cells shows doubling of the
number of apoptotic cells in the mutant compared
with controls. Note the presence of neural crest
cells between the caval vein and pericardial
cavity at embryonic day (E) 11.5. Dapi indicates
4',6-diamidino-2-phenylindole; LA, left atrium; LL,
left lung; LSCV, left superior caval vein; LSH, left
sinus horn; OFT, outflow tract; RA, right atrium;
RL, right lung; RSCV, right superior caval vein;
RSH, right sinus horn; and YFP, yellow fluorescent
protein. Scale bars depict 100 μm.
amount of cell death was still increased in the entire region
in between the caval veins and the cranial pericardium in
Robo1−/− embryos compared with controls (Figure 6D). At
both E11.5 and E12.5, the region of increased apoptosis was
condensed around the cranial-most part of the pericardial cavity; further caudal, very few apoptotic cells were observed in
both Robo1 mutants and wild-type embryos.
The restricted area of increased apoptosis and its proximity
to the cardiac outflow tract suggested a possible relation with
the cardiac neural crest. Therefore, we analyzed the contribution of neural crest to the region using Robo1;Wnt1cre;R26REYFP
embryos. In addition to the yellow fluorescent protein (YFP)–
positive neural crest–derived cells observed in and around the
outflow tract vessels and innervation, at both E11.5 and E12.5,
we observed a large neural crest contribution exactly to the
area prone to apoptosis in Robo1−/− embryos (Figure 6D, arrows). All clusters of apoptotic cells in the region were located
inside or directly neighboring the YFP-positive neural crest
population (Online Figure VA). These cardiac neural crest
cells expressed high levels of Robo1, whereas Robo2 was only
expressed in a subpopulation (Figure 7A; Online Figure VB).
Interestingly, Robo2 was also expressed exactly in the neural
crest region between the caval veins and the pericardial cavity,
the area of increased apoptosis in Robo1 mutants, explaining
the additional phenotype observed in Robo1−/−;Robo2−/− embryos. Slit2 was not expressed in the neural crest, but was
observed directly surrounding it in the endoderm, ectoderm,
and mesenchyme. Slit3 was expressed at low levels in the pharyngeal neural crest cells, but highly in all tissues, including
the neural crest, immediately dorsal to the heart (Figure 7A;
Online Figures VB and VIA). This local Robo2 and high level
of Slit3 expression, combined with the adjacent expression of
Slit2 in the endoderm, likely accounts for the highly localized increase in apoptosis. Interestingly, we did not observe
increased cell death in the cardiac neural crest or surrounding
the caval veins in Slit3 mutants, explaining the lack of pericardial defects in the absence of Slit3 (Online Figure IVB).
Absent Migratory Inhibition by Slit3 and Impaired
Adhesion of Cardiac Neural Crest Cells in the
Absence of Robo1
Despite the increased apoptosis in the neural crest, the overall
patterning of the neural crest seemed unaltered in Robo1−/−
mice at E11.5 (Figure 7B) and E12.5 (data not shown). In both
Robo1−/− and in wild-type controls, YFP-positive cells were in
the majority ventral and medial of the caval veins at cranial
pericardial cavity level. Below outflow tract level YFP-positive
neural crest–derived cells were only found around the foregut
and in the developing innervation (Figure 7B). At E16.5, neural
crest–derived cells were located in the mesenchyme dorsal to
the cranial pericardium, a subset of YFP-positive cells directly
lined the pericardial epithelium in between the caval veins,
bordering the outflow tract. However, no contribution to the
pericardium itself was observed (Figure 7B). In the absence of
Robo1, fewer cells lined the pericardium, and ectopic cavity
formation was observed in the area dorsal to the pericardium
normally filled with neural crest cells (Figure 7B).
To assess whether the defects observed in ectopic celom
and pericardial cavity formation are owing to altered cardiac
neural crest migration that consequently leads to cell death,
472 Circulation Research February 1, 2013
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Figure 7. No contribution of the neural crest to
the pericardium. A, Double in situ hybridization/
immunohistochemistry for yellow fluorescent
protein (YFP) and the indicated genes, showing
overlapping expression of Robo1 and Slit3 with
YFP. B, Robo1+/+ and Robo1−/−;Wnt1Cre;R26REYFP
YFP immunohistochemistry sections showing
normal neural crest patterning at embryonic day (E)
11.5, but reduced contribution to the mesenchyme
dorsal of the pericardium at E16.5. Note the ectopic
pericardial cavity formation where neural crest
cells are present in the Robo1+/+ (red arrowheads).
The neural crest is located directly adjacent to
the pericardium, but does not contribute to it
(white arrowheads). Ao indicates aorta; Dapi,
4',6-diamidino-2-phenylindole; DM, dorsal
mesocardium; FG, foregut; LA, left atrium; LL, left
lung; LSCV, left superior caval vein; OFT, outflow
tract; RA, right atrium; RL, right lung; RSCV, right
superior caval vein; and Th, thymus. Scale bars
depict 100 μm.
we examined the migratory response of Robo1-deficient and
control neural crest cultures to Slit2- and Slit3-conditioned
media (Figure 8A). Wild-type neural crest cells showed
slightly reduced migration in the presence of Slit3, but not
Slit2 (in agreement with previous findings),21 whereas this
response to Slit3 was absent in Robo1−/− cells. Interestingly,
the presence of Slit2- or Slit3-conditioned media did not affect the number of apoptotic cells in vitro nor did the absence
Figure 8. Reduced migratory response to
Slit3 and adhesion of Robo1−/− neural crest.
A, Migration, apoptosis, and adhesion analysis
on Robo1+/+ and Robo1−/− neural crest explant
cultures cultured for 44 hours. B, First, the caval
veins remain inappropriately localized further into
the body mesenchyme near the cranial pericardial
cavity. Second, as a result, or independently owing
to increased neural crest apoptosis, the cranial
pericardial cavity expands dorsally. Therefore,
the lungs are forced to develop ventral to the
caval veins resulting in defective closure of the
pleuropericardial membranes. CC3 indicates
cleaved Caspase 3; CM, conditioned medium;
E, embryonic day; and GFP, green fluorescent
protein.
Mommersteeg et al Slit-Robo Signaling in Pericardium Development 473
of Robo1 in Robo1−/− explants (Figure 8A), underlining the
requirement for very local Robo2 expression and exposure to
both Slit2 and Slit3. Impaired cell–cell adhesion, as well as
altered migration, has also been shown to cause increased cell
death.22 To assess this, we performed an adhesion assay using
dissociated Robo1-deficient and control neural crest cells and
showed reduced adhesion with Robo1-deficient cells compared with control (Figure 8A). These results indicate that the
increased local neural crest apoptosis that results in ectopic
celom formation is likely owing to impaired cell–cell adhesion, as well as altered neural crest migration.
Discussion
Mice Lacking Slit3 Only Partially Recapitulate the
Defects Observed in Robo1−/−;Robo2−/− Mutants
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Although most phenotypic features of Robo1/2 and Slit3
mutants are similar, the most striking difference is the intact pericardium in the latter. Interestingly, diaphragmatic
hernias have been reported in Slit3−/− mutants,23 providing
evidence that Slit3 does play a role in the division of the celomic cavities. In human, diaphragmatic hernias coexist with
pericardium defects,10 suggesting a related developmental
mechanism or involvement of the same gene pathways. The
absence of pericardial defects in Slit3−/− mice suggests phenotypic rescue by binding of locally present Slit2, similar
to the rescue of systemic venous return defects by Robo1 in
Robo2 mutants, explaining the difference in sinus horn phenotype between the Slit3 and Robo1/2 mutants. Moreover,
the discrepancy in phenotype could be explained by requirement of additional interacting partners for the Slit–Robo
signaling pathway. For example, Robo1 has been shown to
interact with Neuropilin1 to regulate Semaphorin signaling,24
and many of the components of the Semaphorin–Plexin/
Neuropilin signaling pathway are known to be involved in
heart development.25
In patients, syndromes showing coexistence of all or some
of the defects observed in the Robo1/2 and Slit3 mutants have
been described,26,27 but no causative genes have yet been identified. Our data suggest SLIT3, ROBO1, and ROBO2 as candidate genes.
Increased Neural Crest Cell Death Underlies
Ectopic Celom Formation
Knowledge of both the morphological processes and the
molecular pathways underlying pericardial defects is still
limited. Different developmental mechanisms have been put
forward from premature atrophy of the left common cardinal vein to defective release of the membranes from the body
wall.8,10 In Robo1−/− mice, the pericardial defect seems to have
a dual cause. First, the caval veins remain inappropriately localized further into the body mesenchyme near the cranial
pericardial cavity. Second, as a result, or independently owing to neural crest cell death, the cranial pericardial cavity
expands dorsally. Therefore, the lungs are forced to develop
ventral to the caval veins resulting in defective closure of the
pleuropericardial membranes (Figure 8B).
The cardiac neural crest is well known to contribute to
the aorticopulmonary septation complex, the membranous
ventricular septum and innervation.25 However, it has not
been reported that neural crest cells also fill most of the
mesenchyme dorsal to the cranial pericardium, indicating a
broader role for neural crest during heart development than
previously thought. During normal development, limited
controlled cell death is observed in the neural crest contributing to this region, suggesting locally tightly regulated
celom expansion. In the absence of Robo1, the increase in
localized cell death causes ectopic celom formation. During
their migration to the heart, Robo1-positive neural crest
cells only locally express Robo2 and encounter very high
levels of Slit3, and also Slit2 ligand, directly dorsal to the
pericardium, suggesting a vital role, locally, for this signaling pathway. Absent migratory response to Slit3, and significantly reduced adhesive properties in Robo1 mutants,
leads to apoptosis of neural crest that is dependent on the
presence of correct survival signals. Slit–Robo signaling is
a known key player in cell adhesion through regulation of
various cadherins and integrins.12 The significant reduction
in neural crest adhesion in the absence of Robo1 suggests
that the increase in cell death is caused either directly by
loosened cell adhesion or by adhesion-dependent protection
against apoptosis.22
Slit–Robo Signaling Controls Sinus Horn
Development and Acts Independently of Tbx18 and
Wt1 Gene Pathways
Tbx18 and Wilms tumor 1 (Wt1) have been identified as key
transcription factors involved in systemic venous return formation. Therefore, we analyzed the possibility of regulation
of the Slit–Robo signaling pathway by these genes or vice
versa. However, no cross-regulation was observed, indicating
that the Slit–Robo signaling pathway is a novel independent
contributor in the formation of this region. The high expression of Wt1 in the area of ectopic celom formation and previous findings of reduced apoptosis in Wt1 mutants8 indicate
a role for Wt1 in the ectopic celom formation. Interestingly,
as observed in Slit3−/− mice, diaphragmatic hernias were also
found in Wt1 mutants.28
The absence of increased neural crest cell death in Slit3
mutants shows that the caval vein and sinus horn defects
develop independently of the pericardial defect caused by
neural crest cell death. Both the abnormal location and the
hypoplasticity of the caval veins indicate an early role for
Slit–Robo signaling in their formation, either by defective
angioblast migration or by assembly. The initial caval veins
are formed by angioblasts migrating from the somites, which
become ensheathed by smooth muscle cells only much later
in development.29,30 Slit–Robo is expressed in these somites31
and shown to be involved both in angioblast migration and
in lumen formation.13,32 Furthermore, the presence of Robo4,
which is required for angiogenesis19 during early caval vein
development, can explain the discrepancy in phenotype in
the absence of Slit3 in comparison with the Robo1−/−;Robo2−/−
mutants. Furthermore, our data suggest a link between the size
of the caval vein connection to the heart and the size of the
sinus horns.
Like the sinus horn myocardium, the sinus node myocardium forms by differentiation of the surrounding mesenchyme.33
However, the current hypothesis is that the sinus node forms by
474 Circulation Research February 1, 2013
a different mechanism than the sinus horn myocardium, in line
with its distinct gene program and normal sinus node formation in the Wt1 and Raldh2 mutants.8 Our results confirm this
hypothesis, as loss of Slit3 or Robo1 and Robo2 does not affect
the formation of the sinus node, in contrast to the sinus horns.
Knowledge of the molecular mechanisms underlying the
development of the systemic venous return and pericardium
is steadily increasing, and here we found a new signaling
pathway that takes part in the formation of this area. The
Slit–Robo signaling pathway exerts its function through
regulation of a diverse array of processes and, although
we shed light on the molecular mechanism underlying the
pericardial defects, the precise mechanisms involved in the
systemic venous return defects still need to be unraveled.
Furthermore, the extensive expression patterns of the Slit–
Robo signaling pathway genes in and around the heart point
to their likely involvement in other aspects of mammalian
heart development.
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Acknowledgments
We thank Fani Memi and Mary Rahman for technical support and
David Ornitz for Slit3 mutant mice.
Sources of Funding
This work was supported by the Wellcome Trust (Program Grant
089775). M.T.M. Mommersteeg was recipient of an EMBO
Long-Term Fellowship grant (ALTF 441–2010) and Netherlands
Organization for Scientific Research Rubicon grant (825.10.025).
A. Chédotal was supported by a grant from the Fondation pour
la Recherche Médicale (FRM, program équipe). P. Zelina was recipient of a postdoctoral fellowship from the Neuropole Région
ile de France. A.R. Ypsilanti was supported by fellowships from
the Natural Sciences and Engineering Research Council of Canada,
the FRM and the Ecole des Neurosciences de Paris. The laboratory
of A. Kispert was supported by grants from the German Research
Foundation (DFG) for the Cluster of Excellence REBIRTH (From
Regenerative Biology to Reconstructive Therapy) and the Clinical
Research Group KFO136 at Hannover Medical School V.M.
Christoffels was supported by the European Community’s Seventh
Framework Program (CardioGeNet 223463).
None.
Disclosures
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Novelty and Significance
What Is Known?
• Slit–Roundabout (Robo) signaling is important for Drosophila heart
development, but its role in the mammalian heart is largely unknown.
• Congenital malformations of the cardiac venous pole and pericardial
defects often present together and share a common pathogenesis.
What New Information Does This Article Contribute?
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• Defects in Slit–Robo signaling result in pericardial defects, misalignment of the caval veins, and sinus horn defects.
• Local increase in neural crest cell death and impaired neural crest
adhesive and migratory properties underlie the absence of the
pericardium.
• There is no cross-regulation between Slit–Robo signaling and know
regulators of systemic venous return and pericardium development.
Whereas a whole range of genes are known to underlie congenital defects of the arterial pole of the heart, knowledge about the
genetic and cellular mechanisms underlying defects of the venous
pole is still limited. Congenital defects of the systemic venous return to the heart often only become symptomatic after childhood.
However, these defects can be severe, ranging from congenital
malformations, such as misalignment of the connecting veins, to
atrial arrhythmias like sick sinus syndrome. Here, we report a novel role for Slit–Robo signaling in the development of this region.
We show that the absence of components in Slit–Robo signaling
results in misalignment of the caval veins and hypoplastic sinus
horn myocardium. Furthermore, defective Slit–Robo signaling
results in partial absence of the pericardium. These pericardial
defects are caused by increased apoptosis owing to reduced adhesion and migration of the cardiac neural crest. This study is the
first to show defects caused by the Slit–Robo signaling pathway
in the mammalian heart, implicating a role for the cardiac neural
crest in pericardial defects and further elucidating the pathogenesis of congenital defects in this complex region.
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Slit−Roundabout Signaling Regulates the Development of the Cardiac Systemic Venous
Return and Pericardium
Mathilda T.M. Mommersteeg, William D. Andrews, Athena R. Ypsilanti, Pavol Zelina, Mason
L. Yeh, Julia Norden, Andreas Kispert, Alain Chédotal, Vincent M. Christoffels and John G.
Parnavelas
Circ Res. 2013;112:465-475; originally published online December 19, 2012;
doi: 10.1161/CIRCRESAHA.112.277426
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/112/3/465
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2012/12/19/CIRCRESAHA.112.277426.DC1
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CIRCRESAHA/2012/277426/R2
Supplemental Material
Mommersteeg et al.
Detailed Methods
Transgenic mice
All experimental procedures were performed in accordance with the UK Animals (Scientific
Procedures) Act 1986 and institutional guidelines. Robo1+/-,1 Robo2+/-,2 (obtained from
William Andrews, UCL, London) Robo1+/-;Robo2+/-,3 Slit1+/-, Slit2+/-,4 Slit3+/-,5 (obtained from
Alain Chedotal, Institut de la Vision, Paris, France) Wnt1Cre,6 R26REYFP7 (Jackson Lab),
Wt1+/- 8 and Tbx18GFP 9 (obtained from Andreas Kispert, Institut für Molekularbiologie,
Hannover, Germany) mice were described previously. Robo1+/-, Robo2+/-, Slit1+/-, Slit2+/-,
Wnt1Cre and R26REYFP mice were maintained on a C57/bl6J background, and back crossed
>10 crossings. Robo1+/-;Robo2+/- and Slit3+/- mice were maintained on a mixed
C57/bl6J;C3H background (6 back crossings with C57/bl6J) and Wt1+/- and Tbx18GFP mice
were kept on an outbred (NMRI) background. The day the vaginal plug was found was
considered as embryonic day (E) 0.5.
In situ hybridization and immunohistochemistry
Probes and methodology of the non-radioactive in situ hybridization10 and
immunohistochemistry11 were described previously. Embryos were fixed overnight in 4%
paraformaldehyde, embedded in paraffin and transversally sectioned at 7-10 μm for
immunohistochemistry or 12 μm for in situ hybridization. The following primary antibodies
were used: goat polyclonal anti-cardiac Troponin I (cTnI, 1:1000; Hytest Ltd), chicken
polyclonal anti-GFP (1:250; Aves), rabbit polyclonal anti-cleaved caspase-3 (CC3, 1:250;
Cell Signaling Technology), goat polyclonal anti-Robo1 (1:250; R&D systems) and rabbit
polyclonal anti-Robo212 (1:250; gift from J.F. Cloutier). Fluorescent secondary antibodies
used were Alexa 568 donkey anti-goat, 488 and 568 goat anti-rabbit, 488 goat anti-mouse
and goat anti-chicken (1:250; Molecular Probes). Nuclei were counterstained with Dapi
(2.5µg/ml;Sigma).
Apoptosis cell count
Every third 10 μm transverse section was mounted from cranial to the pericardial cavity to
caudal of the outflow tract. 16 sections from each embryo were included in the counts.
Apoptotic cells were labelled by immunohistochemistry for Cleaved Caspase 3 (CC3). As
secondary antibody Alexa 568 donkey anti-rabbit was used. All apoptotic cells in the
mesenchyme dorsal of the pericardial cavity and medial and ventral of the caval veins were
counted using Metamorph imaging software (Universal Imaging Corporation). As CC3 also
non-specifically stained blood cells, sections were counterstained with a 488 goat antimouse monoclonal secondary antibody and any co-labelled cells were exclude from the
counts. For all experiments n≥3.
Three-dimensional reconstructions and volume quantification
Three-dimensional visualization, geometry reconstruction and volume measurements of
protein expression patterns determined by immunohistochemistry were carried out as
described previously using Amira 5.4.1 (Visage Imaging).13 All reconstructions and volume
measurements are based on cTnI expression and tissue morphology. Files with
reconstructions are available upon request.
Section ligand binding assay
Isolated Robo1+/+, Robo1-/-, Robo1+/+;Robo2+/+ and Robo1-/-;Robo2-/- embryos were cryoprotected through a 10-20-30% sucrose gradient (one hour each step) and directly
embedded and frozen in Tissue-Tek OCT (Sakura Finetek). 20 μm sections were kept
1
CIRCRESAHA/2012/277426/R2
frozen until 5 minute fixation in cold 100% Methanol. The sections were washed in PBS,
blocked with Opti-mem (Gibco) and incubated overnight with recombinant His-tagged mouse
Slit2 or Slit3 protein (R&D systems) in Opti-mem (5μg/ml). After washing in PBS the sections
were post-fixed for 10 minutes with 4% paraformaldehyde, treated for 45 minutes with 0.3%
H2O2 in PBS, incubated for 1 hour with biotinylated mouse monoclonal anti-polyHistidine
(R&D systems) and then further processed using the TSA enhancement kit (Perkin Elmer).
Control sections were processed identically, but without Slits in the Opti-mem. For double
staining procedure, sections were subsequently blocked for peroxide activity using 1%
Sodium Azide/0.3% H2O2/PBS and processed using the TSA enhancement kit (for Robo1) or
Alexa 488 goat anti-rabbit (for Robo2). Nuclei were counterstained with Dapi (Sigma).
Neural crest explant cultures
Robo1 embryos were collected at E8.5-9, and the yolk sac from each embryo was harvested
for genotyping by PCR analysis. The method used has been described previously.14 The
neural tube was roughly isolated from most surrounding tissue before treatment with 0.5
mg/ml of collagenase/dispase (Sigma). The parts of the neural tube containing the cardiac
neural crest region (from the otic sulcus to the fourth somite) were cleaned from surrounding
mesenchyme and ectoderm. Isolated neural tube parts were washed in PBS and transferred
to 15mm wells containing fibronectin coated coverslips and Dulbecco’s modified Eagle’s
medium (DMEM) Glutamax (Gibco) with 10% fetal bovine serum and Penicillin/Streptomycin.
To obtain Slit2 and Slit3 conditioned medium, COS cells, transfected with Slit2, Slit3 and
GFP constructs,15 were cultured for 3 days in Dulbecco’s modified Eagle’s medium (DMEM)
with Glutamax (Gibco), 10% fetal bovine serum and Penicillin/Streptomycin, after which the
medium was harvested and filtered. For Slit2, Slit3 and GFP conditioned medium
experiments, 1 part DMEM with Glutamax,10% fetal bovine serum and
Penicillin/Streptomycin to 1 part of conditioned medium was used. The explants were
cultured at 37°C with 5% CO2 for 44h, after which they were fixed for 15 minutes with 4%
paraformaldehyde and processed for apoptosis analysis using CC3 (1:1000) and Alexa 568
goat anti-rabbit (1:500) antibodies. Nuclei were counterstained with DAPI. Metamorph
imaging software (Universal Imaging Corporation) was used for apoptosis counts, whereas
migration analysis was done using Image J (NIH image software). To calculate the
outgrowth area, the area of the dense central neural tube tissue was subtracted from the
total area. To control for differences in migration area resulting from random variations in the
shape of the explant, the outgrowth area (mm2) was divided by the perimeter (mm) of the
explant to calculate the migration index, n≥6 per condition.
Neural crest cell adhesion assay
Cardiac neural crest cells were isolated from Robo1+/+ and Robo1-/- 44-hour explant cultures
cultured in DMEM with Glutamax, 10% fetal bovine serum and Penicillin/Streptomycin. The
central neural tube tissue was removed before cells were washed and harvested after
treatment for 3 minutes at 37°C with 0.25% Trypsin-EDTA (Gibco). The cells were
resuspended in DMEM with Glutamax containing 10% FBS. A sample of the cell suspension
containing about 3,000 cells was deposited on 0.7 cm2 wells culture slides (BD Falcon)
previously coated with 15g/ml fibronectin and blocked for 1 hour with 1% BSA. The slides
were incubated at 37°C for 30 minutes, washed with PBS and fixed with 4%
paraformaldehyde for 15 minutes. Attached cells were visualised with DAPI. Per well 2
pictures were taken (5x magnification) and attached cells were counted.
Statistics
Results are expressed as mean ± SEM. Statistical significance was tested with two-tailed,
unpaired, unequal variance student’s t-test. (* for P < 0.05, ** for P< 0.01 and *** for P<
0.001).
2
CIRCRESAHA/2012/277426/R2
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4
Online Figure I
cTnI
Robo1+/+;Robo2+/+ E15.5
A LSH
B
C
Robo1-/-;Robo2-/LSCV
D
LSCV
LA
F
E
LSH
RA
LV
LV
Robo1+/+
RSCV
LSCV
LSCV
LV
Robo1-/RSCV
LSCV
LA
E12.5
RA
RA
OFT Robo2
RSCV
LA
Robo2
LSCV
LSCV
RSCV
RA
LA
Robo2
LA
RA
Robo2
Normal Robo2 expression in absence of Robo1
A-E, in situ hybridization showing myocardial cTnI expression in Robo1+/+;Robo2+/+ (A-B), Robo1-/;Robo2-/- (C-E) embryos. A-E, the Robo1+/+:Robo2+/+ left sinus horn is myocardial (A) and enters the
right atrium (B). The Robo1-/-;Robo2-/- sinus horn has few myocardial cells (C) and either connects to
the right atrium (D) or the coronary circulation (red arrowhead, E). F, in situ hybridization and
immunohistochemistry for Robo2 on Robo1-/- and Robo+/+ embryos. L/RA, left/right atrium; LSH, left
sinus horn; LV, left ventricle; L/RSCV, left/right superior caval vein. Scale bars depict 100μm.
Online Figure II
Wild-type E12.5
A
PPM RL
LSH
Robo1
Robo2
Slit2
Slit3
C
PV
LSH
E14.5
Slit2+/+ D
LL
RL
cTnI
RV
LV
Robo1
Robo2
Slit2
Slit3
Slit2-/-
RSH
RL
LSH
LA
E
RL RSH
Slit1+/+;Slit2+/+ F
RL
LSH LL
LA
RA
RV
LV
LL
LA
RA
RA
cTnI
LSH
RA
LA
RA
Wild-type E14.5
B
RSH
Slit1-/-;Slit2-/-
LSH
LL
LA
RA
RV
LV
Absence of Slit1 and/or Slit2 does not cause pericardium, caval vein alignment or sinus horn
defects
A-B, in situ hybridization for Robo1, Robo2, Slit2 and Slit3 at E12.5 (A) and E14.5 (B). Black
arrowheads indicate that the sinus horns are negative for all Slit-Robo genes except Slit3. C-F, in situ
hybridization showing myocardial cTnI expression in Slit2+/+ (C), Slit2-/- (D), Slit1+/+;Slit2+/+ (E) and Slit1/-;Slit2-/- (F) embryos. No defects are observed in absence of Slit1 and/or Slit2. PPM, pleuropericardial
membrane. L/RA, left/right atrium; L/RSH, left/right sinus horn; RL, right lung; L/RV, left/right ventricle;
PV, pulmonary vein. Scale bars depict 100μm.
Online Figure III
Robo1+/+
B
Robo1+/+
Slit2
LSCV
Robo1
RSCV
LA
LV
Robo1
FG
LSCV
Tr
LL
Robo1-/-
E12.5
A
Robo2
CPW
FG
Robo1-/-/2-/-
Robo1-/-
Tr
Slit2 Dapi
Robo1-/-
C
RSCV
Slit2
Slit3 Dapi
Slit3
Control
FG
Tr
OFT
Slit2 Robo2 Dapi
Slit2
Robo2
Strongly reduced Slit binding just cranial of the pericardial cavity in Robo mutants
A, immunohistochemistry for Robo1 on Robo1+/+ and Robo2 on Robo1-/- embryos. A-B, the line though
the E12.5 three-dimensional reconstruction indicates the level of section ligand binding assay on
Robo1+/+, Robo1-/- and Robo1-/-;Robo2-/- embryos. Robo1 expression is particularly high immediately
cranial of the pericardial cavity. Most Slit3 binding in this area is abolished in the Robo1-/- ,whereas Slit2
binding is slightly reduced and still present in the Robo2 expressing areas. All binding is abolished in the
double mutants. B-C, white arrowheads point to the Robo2 expressing trachea and oesophagus binding
Slit2 and 3 in the wild-type and Slit2 in the Robo1-/-, showing that remaining Slit2 binding is to the Robo2
receptor in absence of Robo1. CPW, cranial pericardial wall; FG, foregut; Tr, trachea. For other
abbreviations, see the legend of Figure 1. Scale bars depict 100μm.
Online Figure IV
A
E12.5
Robo1+/+
Robo1-/LSCV
LSCV
LA
LA
LL
LV
RSCV
RSCV
LSH
Raldh2
RSCV
LV
LL
LSCV
RSCV
LSCV
LL
RA
myocardium
RA
caval veins
B
lung
500
450
CC3+ cell number
400
350
300
250
200
150
100
50
0
+/+;+/Slit3+/+;+/Slit3
-/Slit3-/Slit3
Abnormal localization of the caval veins and ectopic coelom formation in Robo1-/- embryos
A, the line though the E12.5 three-dimensional reconstruction (lateral view) indicates the level of in situ
hybridization sections stained for Raldh2. Black arrowhead indicates the most cranial area of pleural
cavity formation, which is much more cranial in the mutant. Clear arrowheads point out the further
location of the mutant caval veins into the mutant body. Red arrowhead indicates the location of the wildtype sinus horn inside the pericardial cavity, and the mutant sinus horn still mostly inside the body
mesenchyme B, quantification of CC3-positive cells shows comparable CC3-positive cell numbers in
Slit3-/- and control littermates. L/RA, left/right atrium; LSH, left sinus horn; LL, left lung; L/RSCV, left/right
superior caval vein. Scale bars depict 100μm.
Online Figure V
A
E12.5 Robo1-/-;Wnt1Cre;R26REYFP
CC3 YFP Dapi
B
CC3 YFP
YFP
CC3
E11.5 Wnt1Cre;R26REYFP
RSCV
FG
Robo1 YFP
Robo2 YFP
Slit2 YFP
Slit3 YFP
Cardiac neural crest cells express Robo1 and Slit3
A, confocal imaging of a Robo1-/-;Wnt1Cre;R26REYFP immunohistochemistry section stained for YFP and
CC3. Most apoptotic cells express YFP. B, double in situ hybridization/immunohistochemistry for YFP
and the indicated genes. Magnification of the region between the right caval vein and pericardium shown
in Figure 7A. RSCV, right superior caval vein; FG, foregut; OFT, outflow tract. Scale bars depict 100μm.
Online Figure VI
A
cranial
E11.5 Wnt1Cre;R26REYFP
caudal
PA
YFP
Slit2
Slit3
YFP
Slit2
Slit3
OFT
Slit2 and Slit3 expression is highest immediately dorsal to the heart
A, in situ hybridization for YFP, Slit2 and Slit3 on a Wnt1Cre;R26REYFP embryo. Black arrowheads
indicate higher levels of Slit3 in the neural crest and Slit2 surrounding it immediately dorsal to the heart
compared to the pharyngeal arches. PA, pharyngeal arch; OFT, outflow tract. Scale bars depict 100μm.