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Molecular and genetic basis of congenital conotruncal heart
defects
Rana, M.S.
Publication date2014
Link to publication
Citation for published version (APA):Rana, M. S. (2014).
Molecular and genetic basis of congenital conotruncal heart
defects.
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Chapter 5
Identification of a Tbx1/Tbx2/Tbx3 genetic pathway governing pharyngeal and
arterial pole morphogenesis
K. Mesbah*, M.S. Rana*, A. Francou, K. van Duijvenboden, V.E. Papaioannou, A.F.M. Moorman,
R.G. Kelly, V.M. Christoffels
Human Molecular Genetics 2012 Mar 15;21(6):1217‐29
*equal contribution
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Abstract
The 22q11.2 deletion syndrome
(22q11.2DS) is the most
common microdeletion disorder and is
characterized by abnormal development
of the pharyngeal apparatus and
heart. Cardiovascular malformations affecting
the outflow tract (OFT) are
frequently observed
in 22q11.2DS and are among the most commonly occuring heart defects. The gene encoding T‐box
transcription factor 1
(Tbx1) has been
identified as a major candidate
for 22q11.2DS. However, cardiovascular malformations are generally considered to have a multigenic basis and
single gene mutations underlying
these malformations are rare. The
T‐box family members Tbx2 and
Tbx3 are individually required in
regulating aspects of OFT
and pharyngeal development. Here, using expression and 3D‐reconstruction analysis, we
show that Tbx1 and Tbx2/Tbx3 are largely uniquely expressed but overlap in the caudal pharyngeal mesoderm
during OFT development, suggesting
potential combinatorial
requirements. Cross‐regulation between Tbx1
and Tbx2/Tbx3 was analyzed using
mouse genetics
and revealed that Tbx1 deficiency affects Tbx2 and Tbx3 expression
in neural crest‐derived cells and pharyngeal mesoderm, whereas Tbx2 and Tbx3 function redundantly upstream of Tbx1 and Hh
ligand expression
in pharyngeal endoderm and BMP‐ and
FGF‐signaling in
cardiac progenitors. Moreover, in vivo we show that loss of two of the three genes results in severe pharyngeal
hypoplasia and heart tube extension
defects. These findings reveal
an indispensable T‐box gene network governing pharyngeal and OFT development and identify TBX2
and TBX3 as potential modifier
genes of the
cardiopharyngeal phenotypes found
in TBX1 haploinsufficient 22q11.2DS patients.
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Introduction
22q11.2 deletion syndrome (22q11.2DS)
comprises DiGeorge syndrome,
velocardiofacial syndrome and conotruncal
anomaly face syndrome and is
the most common
interstitial microdeletion syndrome with an estimated prevalence of 1 per 4,000 live births1. 22q11.2DS patients exhibit a wide spectrum of developmental anomalies, including craniofacial defects, hypoplasia
of the thymus and parathyroid
glands and conotruncal
cardiovascular malformations (CVMs). CVMs
are the leading cause of birth
defect‐related death in
the western world2,
3. Defects in the formation of the arterial pole of the heart, or outflow tract (OFT), account for up to 30% of CVMs. Formation of the OFT is a complex process, requiring spatial and temporal expression of genes
in multiple
interacting cell types. A population of mesodermal
progenitor cells called the second
heart field (SHF) residing in
splanchnic pharyngeal mesoderm in
the dorsal wall of
the pericardial cavity
is progressively added
to the elongating arterial pole of the embryonic heart tube giving rise to the right ventricle and OFT 4. These SHF cells are closely associated with neural crest (NC)‐derived mesenchyme and pharyngeal endoderm, and complex autocrine and paracrine signaling events
regulate SHF development, including pro‐proliferative fibroblast growth factor (FGF) and Sonic hedgehog (Shh) signals and pro‐differentiation bone morphogenetic protein (BMP) signals5‐7. Direct or indirect perturbation of second heart field development leads to failure of correct alignment between the great arteries and ventricles during cardiac septation resulting
in conotruncal CVMs. However, the
mechanisms coordinating intercellular
signaling events during
SHF deployment remain insufficiently understood.
While single gene mutations or
chromosomal abnormalities cause
certain CVMs or syndromes, the majority of CVMs appear to have a multifactorial basis. Despite this, little is known
about the networks of interacting
genes that control cardiac or
pharyngeal morphogenesis. Several scenarios
involving interactions of multiple
genes with major or minor effects
have been proposed to explain
the complex inheritance and
variability of CVMs8. T‐box
transcription factors play a series
of critical roles in patterning
and morphogenesis of the vertebrate heart6,
9. TBX1 is a major candidate gene in the etiology of 22q11.2DS, contributing to a variable phenotypic spectrum including conotruncal congenital heart
defects10. In mice, haploinsufficiency
of Tbx1 causes fourth pharyngeal
arch artery (PAA) defects, whereas
Tbx1 null mutants display most
of the severe defects found
in 22q11.2DS patients, including
common arterial trunk11‐13. Tbx1 is
required in pharyngeal mesoderm to
regulate proliferation and differentiation
of SHF progenitor cells7, 14‐17.
This function is mediated in part by the regulation of FGF ligand expression and signal response18,
19. Other T‐box proteins known
to impact on OFT development
are the closely
related paralogs Tbx2 and Tbx3. Heterozygous mutations in TBX3 cause Ulnar‐Mammary syndrome, but usually do not
include heart defects20. Nevertheless, mouse
studies have shown that Tbx3
plays important roles in the
development of the cardiac conduction
system21. Moreover, whereas Tbx3 haploinsufficiency does not affect heart development, Tbx3 loss‐of‐function results in OFT alignment defects including double outlet right ventricle22,
23. The role of Tbx3
in OFT development appears to be
indirect as Tbx3
is predominantly expressed
in cardiac NC cells and ventral pharyngeal endoderm, rather than pharyngeal mesoderm22,
23. The paralogous gene Tbx2 is expressed in pharyngeal mesoderm, including the SHF and OFT, and
in NC‐derived cells. Although Tbx2 haploinsufficient mice appear normal, a
fraction of Tbx2 null embryos develop OFT alignment defects24,
25.
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Here we show that Tbx2 and Tbx3 are required redundantly during OFT development to
attenuate Tbx1 expression in the
ventral foregut, but also depend
on Tbx1 for
their correct distribution
in pharyngeal mesoderm, endoderm and NC cells. Furthermore,
loss of function of Tbx1 and either Tbx2 or Tbx3 also causes severe defects during OFT and foregut development,
suggesting that these three genes
(Tbx1/Tbx2/Tbx3) comprise a
crucial regulatory network controlling
the topography of intercellular
signaling during pharyngeal and OFT
development. When disrupted, this
network fails to properly
orchestrate morphogenesis of the
pharyngeal apparatus and cardiovascular
system. Our results underscore the
clinical relevance of this
Tbx1/Tbx2/Tbx3 network which
potentially contributes to the phenotypic variation of congenital cardiopharyngeal malformations in the 22q11.2DS patient population.
Materials and Methods
Transgenic mice
Tbx1tm1Pa (synonym: Tbx1‐; 11‐13), Tbx1tm1Bld (synonyms: Tbx1LacZ , Tbx1‐ 12), Tbx2Δ2 (synonym: Tbx2‐ 48), Tbx3tm1Pa (synonym: Tbx3‐ 49) and Tbx3tm1.1(cre)Vmc (synonyms: Tbx3Cre, Tbx3‐ 50) mice have been described previously. Homozygosity for above‐mentioned alleles is also indicated as
‐/‐. All
strains were maintained on mixed or
outbred (Bl6/CD1 or FVB/N)
backgrounds. Embryos were staged considering noon of the day of the copulation plug as embryonic day (E) 0.5. Embryos were
isolated in
ice‐cold 1x Phosphate‐buffered saline
(PBS), fixed
in 4% paraformaldehyde in PBS for 1‐4 hours, examined and processed for in situ hybridization or immunohistochemistry. Genomic DNA obtained
from yolk sac or tail biopsies was used
for genotyping by PCR, using primers described in the above references.
Immunohistochemistry
Embryos were collected and whole
embryos or trunk regions were
fixed in
4% paraformaldehyde, dehydrated and embedded in paraffin prior to sectioning at 7‐12 μm for immunohistochemistry
(IHC), in situ hybridization (ISH)
and haemotoxylin and eosin
(HE) staining. IHC was performed
on hydrated sections treated for
15 min with
antigen unmasking solution (Vector) or pressure‐cooked for 4 min in Antigen unmasking solution (H‐3300,
Vector Laboratories). The sections
were processed according to the
TSA tetramethylrhodamine system protocol
(NEL702001KT, Perkin Elmer LAS).
Primary antibodies were
incubated overnight. Primary antibody concentrations were: Tbx1
(1/100, Zymed), Tbx3 (1/200, Santa Cruz Biotechnology), Islet‐1 (clone 40.2D6 : 1/100, DSHB), AP‐2α (1/50,
clone 3B5 DSHB), phospho‐ERK (1/100,
phospho‐p44/42 MAPK (Thr202/Tyr204) (20G11)
Cell Signaling) phospho‐SMAD (1/100,
Phospho‐Smad1 (Ser463/465)/
Smad5 (Ser463/465)/ Smad8 (Ser426/428), 9511, Cell Signaling). Secondary antibody concentrations were: anti‐species Alexa 488
(1/500, Jackson), Alexa 680
(1:250, Molecular Probes), Alexa 647
(1:250, Molecular Probes), Alexa 568
(1:250, Molecular Probes) and Cy3
(1/500, Jackson). Sections were
counterstained with Hoechst, Sytox
Green nucleic acid stain
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(1:30,000; Molecular Probes) or Dapi
(1:40,000; Molecular Probes) and observed using an ApoTome microscope (Zeiss).
In situ hybridization
Nonradioactive
ISH on sections was performed as described
51 using mRNA probes for
the detection of Tbx2 52, lacZ,
Tbx3, Tbx1, Fgf8, Bmp4,
Isl1, Mlc2a and Shh 23.
Sections were counterstained with Nuclear Fast Red. For whole‐mount in situ hybridization embryos were fixed in 4% paraformaldehyde and processed as described 27.
Ink injection
Visualization of pharyngeal arch
artery formation at E10.5 by
ink injection into
the embryonic ventricle was carried out as described 53.
3D‐reconstruction of gene expression patterns and morphology
Image acquisition of serial sections stained by ISH or IHC, and processing for subsequent 3D reconstructions
was performed using a previously
described method using Amira
5.2 software
54. The mRNA expression patterns were
independently confirmed in at least
two additional embryos.
Results
Three‐dimensional analysis of Tbx1, Tbx2 and Tbx3 transcript distribution in the cardiopharyngeal region
In order to understand the
multigenic cause of cardiopharyngeal
defects observed
in 22q11.2DS, we first explored whether Tbx1, Tbx2 and Tbx3 could be expected to function co‐operatively
during arterial pole development.
This was achieved by examining
their expression patterns relative to
each other by in situ
hybridization (Figure 1A‐C)
and fluorescent immunohistochemistry (Figure 1D‐F) in wildtype embryonic day (E) 8.5 and E9.5 embryos,
stages at which SHF cells are added
to the elongating heart tube. To
facilitate a more accurate
identification of unique and
overlapping expression sites in
the cardiopharyngeal region, we
generated 3D‐reconstructions of transcript
distributions for these three genes
(Figure 1G‐L; Supplementary Material,
Figure S1;
Supplemental 3D‐PDF file). At E8.5 and E9.5, Tbx1
is expressed in
lateral pharyngeal endoderm (arrows
in Figure 1A, arrowhead in 1F) and surrounding mesoderm, in the SHF and in surface ectoderm (Figure 1D‐F). In pharyngeal endoderm, Tbx3 primarily accumulates ventrally (Figure 1C, arrowhead; 1D‐F), overlapping with Tbx2 in the endoderm underlying the aortic sac (Figure 1K, L, arrow). Tbx2 and Tbx3 are also co‐expressed in NC‐derived mesenchyme (Figure 1B, C, black arrow; 1I,
arrow) and the dorsal and
lateral pericardial wall at E9.5
(Figure 1B, C, green
arrow). Overlap between Tbx1 and
Tbx2 was observed in mesoderm
lateral to and
underlying ventral pharyngeal endoderm (Figure 1A, B, arrowhead), including SHF cells. A small patch of
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overlapping Tbx1 and Tbx3
expression was also observed in
the dorsal pericardial
wall (Supplementary Material, Figure S2; Supplemental 3D‐PDF file). Unlike Tbx1 and Tbx3, Tbx2 is robustly expressed in the myocardial wall of the developing OFT at E9.5 (Figure 1G, I). Tbx2 and
Tbx3 are co‐expressed in distal
OFT mesenchyme at this stage
(Figure 1B, C, K).
In summary, Tbx2 and Tbx3 are extensively co‐expressed, whereas Tbx1 demonstrates a more distinct pattern in pharyngeal mesoderm and endoderm (Figure 1J).
Figure 1. Unique and
overlapping expression patterns of
Tbx1, Tbx2
and Tbx3. In situ hybridization at E9.5 showing Tbx1
(A), Tbx2 (B) and Tbx3
(C) mRNA in pharyngeal endoderm
(A, arrows; C, arrowhead), neural
crest‐derived mesenchyme (B, C, black
arrow) and pharyngeal mesoderm
(A, B,
arrowheads; B, C, green arrows). At E8.5 (D, E) and E9.5 (F), Tbx1 protein (red) was found in lateral pharyngeal endoderm (L‐end, arrowhead), pharyngeal mesoderm
(mes) and
surface ectoderm (se) and Tbx3 protein (green)
in ventral pharyngeal endoderm
(V‐end) and neural crest‐derived mesenchyme (arrow). Three‐dimensional
reconstructions showing unique and
overlapping (black arrow) sites of
expression of Tbx1/Tbx3 (G), Tbx1/Tbx2
(H) and Tbx2/Tbx3 (I).
(J) Summary of expression
sites of Tbx1–2–3 at E8.5–9.5. Note that ventral endodermal Tbx2
expression is restricted to
where endoderm underlies the aortic
sac. Cross sections (K) of
corresponding three‐dimensional models
(G, H, I, dashed line). Note
the overlap between Tbx2 and Tbx3 in
ventral endoderm (L, arrow).
avc, atrioventricular canal; dpw,
dorsal pericardial wall; la, left
atrium; lv, left ventricle; oft,
outflow tract; ov, otic vesicle;
pa1, 2, 1st and 2nd
pharyngeal arch; ph, pharynx; pp1–3, first, second and third pharyngeal pouches; ra, right atrium; rv, right ventricle. Scale bar
in (A) and
(D) is 100 mm and in (E) and (F) is 50 µm.
Loss of Tbx1 affects Tbx2 and Tbx3 expression in the caudal pharynx SHF signaling pathways are affected
in Tbx1 null mutant embryos resulting
in hypoplasia of the distal OFT at midgestation26. To determine whether this OFT phenotype coincides with altered expression of Tbx2 and Tbx3, we examined the expression patterns of all three genes by
in
situ hybridization on whole embryos and
transverse sections
in wildtype and Tbx1‐/‐ embryos. The
level of Tbx2 expression was reduced
in the pharyngeal region
in E9.5 Tbx1‐/‐
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embryos (Figure 2C, D) compared
to controls (Figure 2A, B). In
contrast, the expression domain of
Tbx2 was expanded in the first
arch of Tbx1 null embryos
(Figure
2C, arrowhead)27. Tbx2 was downregulated in the dorsal pericardial wall (Figure 2F, red surface), and the area of co‐expression with Tbx1 in the SHF was thus greatly reduced in size (purple surface
in Figure 2F). Similarly, accumulation of Tbx3 transcripts was reduced
in the caudal pharyngeal region
including the dorsal pericardial wall
(Figure 2I, bracket;
Supplementary Material, Figure S2B, D), and expanded in the first arch of Tbx1‐/‐ embryos at E9.5 (Figure 2I, arrow).
Previous work has documented
reduced NC‐derived cell numbers in
the
caudal pharyngeal region of Tbx1 null mutants, showing that Tbx1‐responsive Gbx2 signaling from pharyngeal ectoderm
is required for proper guidance of
the adjacently located NC cells
to the caudal pharynx, where Tbx2
and Tbx3 are co‐expressed 28,
29. We thus analyzed
and quantified the number
of NC‐derived cells in wildtype
and Tbx1 null mutant embryos
by labeling NC‐derived cells with an Ap2α antibody, and found a 50% reduction in the number of Ap2α‐positive NC‐derived cells and, among
the remaining cells, a 20%
reduction of the fraction of
Tbx3‐positive cells compared to
the wildtype situation (n=3 embryos
of each genotype; Figure 2H,
J, K). Hence
loss of Tbx3 expression
in Tbx1‐/‐ embryos
reflects both reduced numbers of
crest‐derived cells and a reduction
of Tbx2‐ and Tbx3‐positive
crest cells.
In wildtype embryos, Tbx2 and Tbx3 overlap
in ventral pharyngeal endoderm. In
the hypoplastic foregut of Tbx1‐/‐
embryos, we observed that ventral
expression of
Tbx2 was decreased (Figure 2M, arrow), whereas Tbx3 expression was maintained (Figure 2O). Thus, Tbx1‐deficiency
affects Tbx2 and Tbx3 expression
in mesodermal, NC‐derived
and endodermal cells in the caudal pharynx, supporting a requirement for Tbx1 upstream of Tbx2 and Tbx3 during OFT development.
Compound Tbx2‐Tbx3‐deficiency affects
endodermal Tbx1 expression and key
signaling molecules in the SHF
We next assessed whether Tbx1 expression
is affected in
the absence of paralogs Tbx2 or Tbx3
by analyzing E9.5 Tbx2‐/‐ and
Tbx3‐/‐ mouse embryos using in
situ hybridization
on transverse sections. The overall expression pattern of Tbx1 was found to be normal in both Tbx2
and Tbx3 null mutants
(Figure 3, Tbx1). Because Tbx2
and Tbx3 are co‐expressed
in multiple regions and share common targets in the heart, they could function redundantly to regulate
Tbx1 and OFT development 9. We
thus generated mice double heterozygous
for Tbx2 and Tbx3 null alleles. Only a
few such mice were obtained because
this genotype is afflicted with
severe craniofacial defects 30.
Tbx2;Tbx3 double heterozygous mice
were interbred to obtain
Tbx2‐/‐;Tbx3‐/‐ embryos (Table 1).
Tbx2‐/‐;Tbx3‐/‐ embryos could
be recovered up to E9.5
(n=11/150) at mendelian ratios and
displayed impaired
overall embryonic growth, pericardial edema and hypoplasia of the OFT and right ventricle (Figure 3D;
Supplementary Material, Figure S3C,
D). Tbx1 expression is restricted
to lateral pharyngeal endoderm
in wildtype, Tbx2‐/‐ and Tbx3‐/‐
embryos. In Tbx2‐/‐;Tbx3‐/‐
embryos, Tbx1 was upregulated in the subdomain of the ventral endoderm that normally co‐expresses Tbx2
and Tbx3 (Figure 3, Tbx1,
arrowhead; Supplementary Material, Figure
S3L,
arrow; Figure 1L, arrow). In contrast, Tbx1 expression was unchanged in pharyngeal mesoderm and in
lateral pharyngeal endoderm that do
not co‐express these factors. This
observation
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suggests
functional redundancy of Tbx2 and Tbx3
in the suppression of Tbx1 expression
in ventral pharyngeal endoderm.
Figure 2. Tbx2 and Tbx3 are affected by
loss of Tbx1. Tbx2 and Tbx3 expression
is broader in the
first arch (arrowhead in C and I) and reduced in the caudal pharynx (brackets in C and I; arrows in D and J) of E9.5 Tbx1 null embryos. Three‐dimensional reconstructions of
the dorsal pericardial wall showed a smaller overlapping region of Tbx1/Tbx2 (compare purple region in E with F) and reduced detection of Tbx2 mRNA (D, arrowheads, blue region absent in F) in the dorsal pericardial wall of Tbx1‐/‐ embryos. Immunofluorescence and histograms (K)
showing a reduction in
Tbx3‐positive mesenchymal cells (J,
white arrows), Ap2a‐positive neural
crest‐derived cells (K) and
Tbx3‐positive/Ap2a‐positive cells (compare
yellow staining in H with J;
K) in Tbx1‐/‐
embryos. 3D‐reconstructions reveal loss
of Tbx2 expression in ventral
pharyngeal endoderm (M,
arrow), whereas expression of Tbx3
is maintained
in pharyngeal endoderm of Tbx1‐/‐ embryos (O, yellow). Ca, caudal; Cr,
cranial; D, dorsal; dpw, dorsal
pericardial wall; L, left lateral;
la, left atrium; lv, left
ventricle; pa1,
first pharyngeal arch; ph, pharynx; pp1–3; first, second and third pharyngeal pouch; R, right lateral; ra, right atrium; V, ventral. Scale bar is 100 µm.
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The LIM homeodomain transcription
factor Isl1 is expressed in
undifferentiated cardiac progenitors in
the SHF and in the distal
OFT. In Tbx2‐/‐;Tbx3‐/‐ embryos,
Isl1 transcripts could
still be detected
in SHF cells and the
shortened OFT, suggesting that
the deployment of SHF cells is
compromised downstream of Isl1
expression (Supplementary Material, Figure
S3D, arrow). We therefore examined
the expression patterns of
known regulators of SHF development. Shh has been implicated in proliferation and deployment of cardiac
progenitor cells and in signaling
events required for cardiac NC
survival from an expression site
in ventral pharyngeal endoderm
31. Moreover, Shh has been
reported
to regulate Tbx1 in pharyngeal endoderm and arches 32,
33. Shh expression in ventral pharyngeal endoderm was
decreased, concomitant with the
increased expression of Tbx1, in
Tbx2‐/‐
;Tbx3‐/‐ embryos, but not in Tbx2 or Tbx3 single mutants (Figure 3, Shh, arrowhead).
We subsequently investigated FGF
signaling, also known to play a
critical role
in regulating SHF progenitor cell proliferation
in pharyngeal mesoderm distal to the OFT. Fgf8 appears
to be the major FGF
ligand driving heart
tube elongation and Fgf8 and Tbx1 have been linked genetically during vascular morphogenesis and to the etiology of 22q11.2DS 6,18,
34,35. At E9.5 Fgf8 is
co‐expressed with Tbx1 in lateral
pharyngeal endoderm,
overlying surface ectoderm and, at low level, in pharyngeal mesoderm lateral to the pharynx including SHF progenitor cells 15,
36,
37. We found elevated Fgf8 transcript accumulation in the SHF and OFT of
compound Tbx2‐/‐;Tbx3‐/‐ embryos
(Figure 3, Fgf8, red arrowheads,
Supplementary Material, Figure S3K, arrowhead).
BMP signaling induces cardiac
differentiation and antagonizes FGF
signaling proximally to the OFT during heart tube extension 7. We therefore included Bmp4, known to be a crucial BMP ligand during OFT development, in our expression pattern analyses 38. We observed a decrease in Bmp4 transcript levels in ventral pharyngeal endoderm, where Tbx2 and Tbx3 are normally co‐expressed, as well as
in pharyngeal mesoderm
including the SHF and distal OFT of Tbx2‐/‐;Tbx3‐/‐ embryos
(Figure 3, Bmp4, arrowheads). Bmp4 and Nkx2‐5 expression
in the dorsal pericardial wall
has been reported to depend on
GATA family member Gata6, where it
is required for proper maturation of cardiomyocyte precursors 39. In Tbx2‐/‐;Tbx3‐/‐ embryos,
in situ hybridization
revealed a decrease
in Gata6 expression
in the dorsal pericardial wall, presumably affecting BMP‐signaling in the SHF (data not shown). Albeit
slightly less severe, a
shortened OFT, hypoplastic
right ventricle and modulations
in FGF and BMP ligand and
Shh expression were also observed
in Tbx2‐/‐;Tbx3+/‐ and Tbx2+/‐
;Tbx3‐/‐ compound mutants (Figure
3E, I; n=21 and n=15,
respectively, Table 1; n=3
as examined by in situ hybridization). However, endodermal expression of Tbx1 was unaffected in
these compound mutant embryos
(Figure 3E, Tbx1). These results
indicate gene dosage effects and functional redundancy between Tbx2 and Tbx3, as a single allele of either gene is insufficient to activate normal FGF and BMP and Hh signaling pathways, yet
is sufficient to repress Tbx1 in ventral endoderm.
Taken together, our findings indicate that endodermal Tbx1 and the abovementioned signaling molecules
are potential downstream effectors of
the redundant actions of
Tbx2 and Tbx3, impacting on both pharyngeal and OFT development.
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Figure 3. Morphological, Tbx1 expression and
signaling component analysis
in Tbx2;Tbx3
compound mutant embryos. Severe hypoplasia of
the outflow tract and
right ventricle, ectopic distribution of Tbx1 mRNA and decreased expression of Bmp4 and Shh
in the ventral endoderm
(black arrowheads) were observed
in E9.5 Tbx2‐/‐;Tbx3‐/‐ embryos
compared with wild‐type, Tbx2‐/‐ and
Tbx3‐/‐ embryos (A–D). Elevated Fgf8
and decreased Bmp4 expression was observed in splanchnic mesoderm (red arrowheads). Tbx2+/‐;Tbx3‐/‐ and Tbx2‐/‐
;Tbx3+/‐ embryos display similar aberrations in transcript levels of Shh, Fgf8 and Bmp4, but no ventral expansion of endodermal Tbx1 (E). Composite Tbx2;Tbx3 mutant embryos with pharyngeal arch, OFT and RV hypoplasia (G–I). Morphology
and three‐dimensional reconstruction of
the pharyngeal endoderm showing
patterning defects of interpouch
endoderm in Tbx2‐/‐;Tbx3‐/‐ embryos
(G′, red arrow) and hypoplasia
of the first
and second pharyngeal pouches (G′‐I′). dpw, dorsal pericardial wall; lv, left ventricle; pa1–3, first, second and third pharyngeal
arch; oft, outflow
tract; ph, pharynx; pp1–3; first,
second and third pharyngeal pouch;
ra,
right atrium; rv, right ventricle. Scale bar in (A) is 100 µm. Pharyngeal segmentation defects in Tbx2‐/‐;Tbx3‐/‐ embryos In
vertebrates, the pharyngeal apparatus
is a transient structure and
contributes to the thymus, thyroid
and parathyroid, structures frequently
affected in 22q11.2DS.
Upon macroscopic examination of Tbx2‐/‐;Tbx3‐/‐, Tbx2‐/‐;Tbx3+/‐ and Tbx2+/‐;Tbx3‐/‐ mutants at E9.5,
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the pharyngeal region was observed
to be severely affected (Table
1) revealing an underdeveloped first
pharyngeal arch, while the more
caudal arches were
hypoplastic compared
to wildtype controls
(Figure 3G‐I). The
foregut of E9.5 control embryos
showed three pharyngeal pouches and
two interpouch regions
(Figure 3F, F’, pp1‐3). Tbx2‐/‐;Tbx3‐/‐ embryos displayed some pharyngeal expansion, as the foregut did not acquire the tube‐like shape observed
in Tbx1‐/‐ embryos
(compare Figure 3G’ with 2M, O). However,
interpouch endoderm could not be distinguished (Figure 3G, red arrow). Pharyngeal segmentation was also, albeit less severely, affected in embryos lacking three of the four alleles (Tbx2+/‐;Tbx3‐/‐ and Tbx2‐/‐;Tbx3+/‐),
indicating that a developmental delay
is not the primary cause of
this phenotype (Figure 3H, I).
Unfortunately, all compound Tbx2;Tbx3
mutants die by mid‐gestation, making
it
impossible to study the development and differentiation of pharyngeal precursors of
the (para)thyroid and
thymus. Nevertheless, our results
indicate
that proper segmentation of the pharyngeal region requires all four alleles of Tbx2 and Tbx3, suggesting roles during development of the pharyngeal apparatus and possibly its derivatives.
Severe arterial pole defects and altered FGF‐ and BMP‐signaling in Tbx1‐/‐;Tbx2‐/‐ and Tbx1‐/‐;Tbx3‐/‐ embryos Since Tbx1 is required for correct pharyngeal expression of Tbx2 and Tbx3, which in turn are redundantly
required to attenuate expression of
Tbx1 in the ventral endoderm,
we hypothesized that these three
genes could constitute a regulatory
T‐box network. We therefore
investigated the combinatorial in vivo
requirements
for Tbx1 and either Tbx2 or Tbx3 by interbreeding Tbx1;Tbx2 or Tbx1;Tbx3 double heterozygous mice and analyzing the respective
compound mutant embryos. Tbx1+/‐;Tbx2+/‐
and Tbx1+/‐;Tbx3+/‐ mice
were obtained at
the expected mendelian
ratio and are viable and
fertile. As Tbx3‐deficiency
is reported to delay aortic arch artery formation 23, we investigated whether heterozygosity for Tbx3
influences the incidence or severity
of pharyngeal arch artery defects
in Tbx1+/‐
embryos. We did not find
significant changes at either E10.5
or E15.5
(Supplementary Material, Figure S4D). Analysis of E9.5 and E10.5 embryos obtained by interbreeding Tbx1+/‐
;Tbx2+/‐ or Tbx1+/‐;Tbx3+/‐ mice
revealed
that Tbx1‐/‐;Tbx2‐/‐ embryos were not
recovered at the expected mendelian
ratio (n=4/134, expected n=8.4/134), in
contrast
to Tbx1‐/‐;Tbx3‐/‐ embryos (12/156). Importantly, 4/4 of Tbx1‐/‐;Tbx2‐/‐ embryos and 9/12 (75%) of Tbx1‐/‐;Tbx3‐/‐
embryos displayed impaired overall
embryonic growth with development
apparently blocked at E8.5 (Figure
4D, J). These embryos all
exhibited pericardial edema.
Severe hypoplasia of the pharyngeal region was also observed, including underdevelopment of the first arch (Figure 4D, J), which forms relatively normally in Tbx1‐/‐ embryos 11. Expansion and segmentation
of the pharyngeal
endoderm was mildly affected in
Tbx1+/‐;Tbx2‐/‐
embryos compared to wildtype and Tbx1‐/‐ embryos (Supplementary Material, Figure S5C). We could not
perform transcript detection or
immunohistochemical analyses on
Tbx1‐/‐;Tbx2‐/‐
embryos, since 3 out of 4 embryos were necrotic and thus unfit for molecular analysis. We compared
cardiac morphology between E9.5
wildtype and Tbx1‐/‐;Tbx2‐/‐ embryos
using histological and 3D analyses and found a severely shortened OFT connected to an embryonic ventricle, whereas morphological signs of a developing
right ventricle were lacking
(Figure 4D, F, n=3). Moreover,
the OFT lumen of the mutant
heart was extremely narrow
or completely obstructed by myocardium.
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128
Figure 4. Heart
tube elongation is severely compromised
by loss of Tbx1 and either
Tbx2 or Tbx3. Right
lateral views at E9.5 showing
pharyngeal and cardiac hypoplasia
in Tbx1‐/‐;Tbx2‐/‐
(D) and Tbx1‐/‐;Tbx3‐/‐ embryos (J).
3D reconstruction of a Tbx1‐/‐
;Tbx2‐/‐ heart revealing
a shortened outflow
tract with a constricted lumen (F, arrowhead).
Transverse sections showing pharyngeal
hypoplasia (ph) and a persistent
dorsal mesocardium in Tbx1‐/‐
;Tbx3‐/‐ embryos (N, arrow). Milder
pharyngeal hypoplasia is observed
in Tbx1‐/‐ embryos (L). In
situ hybidization showing reduced
Tbx5‐negative right ventricular and
OFT myocardium in Tbx1‐/‐;Tbx3‐/‐
embryos (R). lv, left ventricle;
la, left atrium, pa1, 1st
pharyngeal arch; oft, outflow tract;
ph, pharynx; ra, right
atrium; rv, right ventricle. Scale bar is 100 μm.
As we could not
isolate a sufficient number of Tbx1‐/‐;Tbx2‐/‐ embryos, we examined transverse
sections of Tbx1‐/‐;Tbx3‐/‐ embryos,
and found that pharyngeal
endoderm was severely reduced, and
that the dorsal mesocardium failed
to close under the
hypoplastic pharynx (Figure 4N,
arrow). Gene expression analysis
revealed that Tbx1‐/‐;Tbx3‐/‐
hearts were almost entirely positive
for Tbx5 transcripts, normally
restricted to the
left ventricle and
inflow regions of the heart
(Figure 4O‐R). In addition, very
few
Isl1‐positive cells were observed at the arterial pole compared to the situation
in Tbx1‐/‐ or the majority of Tbx3‐/‐ hearts (Supplementary Material, Figure S6A‐D). The observation that Tbx2 and Tbx3 are co‐operatively
required for Hh, FGF and BMP
ligand expression, key regulators of
cardiac progenitor cell proliferation
and differentiation, led us to
investigate the extent
to which these signaling pathways
were altered in Tbx1‐/‐;Tbx3‐/‐
embryos compared to
wildtype, Tbx1‐/‐ and Tbx3‐/‐ embryos. Using in situ hybridization on transverse sections, Shh expression was found to be expressed at a lower level in ventral pharyngeal endoderm of Tbx1‐/‐;Tbx3‐/‐ (Figure
5C) compared to Tbx1‐/‐ (Figure
5B) and Tbx3‐/‐ embryos (Figure
3, Shh). Fgf8 expression was
reduced in lateral pharyngeal
endoderm of Tbx1‐/‐ embryos at
E9.5, as
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129
previously documented (Figure 5E,
arrow) 28. In contrast, in
Tbx1‐/‐;Tbx3‐/‐ compound mutants, Fgf8
transcript accumulation was elevated
in the SHF and OFT (Figure
5F, arrowhead). Consistent with elevated ventral and
loss of lateral Fgf8 expression,
increased p‐ERK was observed
in pharyngeal mesoderm underlying the pharynx and
in the distal OFT of
Tbx1‐/‐;Tbx3‐/‐ embryos (Figure 5M,
white arrow), compared to wildtype
and
Tbx1‐/‐ embryos (Figure 5K, L). Bmp4 expression was unchanged in Tbx1‐/‐ embryos (compare Figure 5G with
5H), but decreased transcript
levels were observed in
the distal OFT and
SHF of Tbx1‐/‐;Tbx3‐/‐ embryos (Figure
5I). Consistent with altered Bmp4
expression, reduced P‐Smad
labeling was observed in the
distal OFT of Tbx1‐/‐;Tbx3‐/‐ (Figure
5Q, white
arrow) compared to wildtype and Tbx1‐/‐ embryos (Figure 5O, P).
Figure 5. Altered signaling pathway components in Tbx1;Tbx3 composite mutant embryos. In situ hybridization showing that endodermal Shh expression was reduced in E9.5 Tbx1‐/‐;Tbx3‐/‐ compared to wildtype and Tbx1‐/‐
embryos (A‐C, arrowhead). Fgf8 expression was reduced
in
lateral endoderm of Tbx1‐/‐ (E, black arrows) and Tbx1‐/‐;Tbx3‐/‐
(F, black arrows) embryos and
elevated in ventral pharyngeal
mesoderm of Tbx1‐/‐;Tbx3‐/‐
embryos (F, arrowhead). Bmp4 transcript detection was decreased
in the distal OFT (I, arrowhead) of Tbx1‐/‐
;Tbx3‐/‐ embryos. Immunofluorescence of wildtype P‐Erk (J) and P‐Smad (N) staining with higher magnifications in wildtype (K, O), Tbx1‐/‐ (L, P) and Tbx1‐/‐;Tbx3‐/‐ embryos (M, Q) of the boxed regions
in J and N. P‐Erk was increased
(M, white arrow) and P‐Smad was
reduced (Q, white arrow) compared
to the situation in
atrial endocardium (Q, yellow arrow)
in the distal OFT of
Tbx1‐/‐;Tbx3‐/‐ embryos. ph, pharynx;
l‐end, lateral pharyngeal endoderm; lv,
left ventricle; oft, outflow tract;
v‐end, ventral pharyngeal endoderm; se,
surface ectoderm. Scale bar in Panels A, J, N is 100 μm and in Panels K‐M, O‐Q 25 μm.
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It has been shown
that Tbx1 deficiency affects proliferation and differentiation of
cardiac progenitor cells 17. Our
results demonstrate that combinatorial
loss of regulators of proliferation
and differentiation of cardiac
precursors in Tbx1‐/‐;Tbx2‐/‐ and
Tbx1‐/‐;Tbx3‐/‐ embryos results in a
severe failure of heart
tube elongation. These changes are
similar to those observed in
Tbx2‐/‐;Tbx3‐/‐ embryos. Together these
data uncover impaired intercellular
signaling in cardiac precursors and
surrounding tissues in
composite Tbx1/Tbx2/Tbx3 mutant embryos
during OFT formation and pharyngeal
development, structures contributing to the pathogenesis of 22q11.2DS.
Discussion
Tbx1, Tbx2, and Tbx3 constitute
a T‐box regulatory network that
controls OFT
and pharyngeal development
Variability in clinical features
between 22q11.2DS patients suggests
that disruptions of multiple genes
or pathways are likely to
contribute to the cardiopharyngeal
phenotypes found in 22q11.2DS. The 22q11.2DS gene Tbx1 is an important regulator of pharyngeal and OFT development, as Tbx1 deficiency affects SHF proliferation and differentiation leading to hypoplasia of the distal OFT and a common ventricular outlet, as well as patterning defects of the pharyngeal apparatus 11‐13. As T‐box factors comprise a family of related proteins that recognize a similar DNA binding site and interact with the common co‐factors 40, mutations in other T‐box factor genes may contribute to phenotypic variability. Loss of function mouse models of
two other T‐box transcription
factors, Tbx2 and Tbx3, revealed
important roles during arterial pole
development. Tbx2 and Tbx3 are
closely related transcriptional repressors
and are required for proper
deployment of SHF cells and
ventriculoarterial alignment 22‐24, 41. Tbx2
and Tbx3 are thus excellent
candidate modifier genes of
the cardiopharyngeal phenotypes resulting from Tbx1 disruption.
Our study indicates that Tbx1, Tbx2, and Tbx3 constitute a T‐box regulatory network that controls OFT and pharyngeal development. Firstly, Tbx1 is required for the expression of Tbx2 and Tbx3
in pharyngeal and NC‐derived mesenchyme. As Tbx1
is not expressed
in NC cells the effect on Tbx2 and Tbx3 in this cell type is likely to be indirect, mediated by altered intercellular
signaling between Tbx1 expressing mesoderm or pharyngeal epithelia and NC cells.
Tbx2 and Tbx3 are also
potential direct targets of Tbx1,
as these genes are co‐expressed
in pharyngeal mesoderm, including SHF
cells in the dorsal pericardial
wall. Ongoing analysis of Tbx2 and Tbx3 cis‐regulatory elements will provide insight into the Tbx1 dependent signaling pathways, and potential direct role of Tbx1 in pharyngeal mesoderm, in the transcriptional regulation of these genes.
Secondly, we demonstrate that Tbx2
and Tbx3 are co‐expressed in
multiple cell populations and that
loss of function of both Tbx2
and Tbx3 causes overall
growth impairment, disturbed patterning of endodermal pouches
(Figure 3) and hypoplasia of
the OFT and right ventricle
suggesting defective SHF deployment.
Tbx2 and Tbx3 overlap
in function
to pattern pharyngeal endoderm, since
Tbx1 expression was observed in
ventral pharyngeal endoderm of Tbx2‐/‐;Tbx3‐/‐ embryos. This
localized up‐regulation of Tbx1
in the
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131
ventral endoderm of Tbx2;Tbx3 compound mutants, coinciding with the site where Tbx2 and Tbx3 are normally co‐expressed, suggests that Tbx2 and Tbx3 directly suppress Tbx1
in this domain of the
foregut. However, putative T‐box
binding sites regulating Tbx1
expression have not yet been identified. In addition, downregulation of Shh expression was observed in ventral
pharyngeal endoderm where Tbx2 and
Tbx3 are co‐expressed. Hh‐signaling
is required during SHF proliferation and migration and, together with downregulation of Bmp4 and upregulation of Fgf8 in the SHF and distal outflow tract, potentially underlies perturbed SHF deployment in Tbx2;Tbx3 compound mutants.
Thirdly, we provide evidence for
the functional importance of a
cross‐regulating Tbx1/Tbx2/Tbx3 network, as embryos lacking Tbx1/Tbx2 or Tbx1/Tbx3 present similar severe pharyngeal and heart tube elongation phenotypes, suggesting overlapping roles for Tbx2 and Tbx3 in conferring network robustness. This phenotype is characterized by a C‐looped heart tube that fails to close dorsally, cardiac dilation and OFT obstruction and pericardial edema, associated with reduced addition of SHF derived progenitor cells to the arterial pole of the heart
as revealed by Tbx5 and Isl1
expression. This severe phenotype is
associated with overall growth
impairment and developmental delay, potentially resulting from the cardiac defects.
These results demonstrate that the
Tbx1/Tbx2/Tbx3 network identified here
is robust, requiring loss of function of two of the three components for network collapse and a severe phenotype to emerge. Tbx1 and Tbx2 are co‐localized in a broader domain of the SHF than
Tbx1 and Tbx3 potentially explaining
the low numbers of Tbx1‐/‐;Tbx2‐/‐
embryos recovered at E9.5. Moreover, downregulation of Tbx2 and Tbx3 in Tbx1‐deficient endoderm and splanchnic mesoderm could further disrupt SHF development in Tbx1‐/‐;Tbx2‐/‐ and Tbx1‐/‐;Tbx3‐/‐ embryos.
Disruption of SHF signaling in compound T‐box factor mutants
Recent work has defined the
importance of intercellular signals
in controlling
SHF deployment during heart tube elongation, including FGF, BMP, Shh, Notch, retinoic acid and Wnt signaling pathways 5,
6. In particular, the balance between FGF signaling, predominant in the
lateral pharynx where it promotes
progenitor cell proliferation, and
BMP signaling, predominant in the
ventral pharynx and distal OFT
where it drives
myocardial differentiation, has emerged as a central axis controlling progressive addition of SHF cells to the OFT. Antagonism between
these signaling pathways has been dissected
revealing that BMP
signaling downregulates FGF signaling
through BMP target genes in NC
cells 7, 42. In addition, BMP
regulates microRNA function to
silence the SHF progenitor cell
genetic program 43. We have investigated the expression of Fgf8 and Bmp4, thought to be the major ligands mediating
this signal exchange during OFT morphogenesis
in the mouse, and p‐Erk and
P‐Smad, intracellular readouts of FGF
and BMP signaling, in embryos
lacking combinations of Tbx1/Tbx2/Tbx3. Our results reveal that in compound mutant embryos, but not in single mutant embryos, combinatorial defects are observed such that proximal to the elongating heart tube FGF signaling is elevated and BMP signaling downregulated (Figure 6). The topography of signaling events
in the caudal pharynx of compound mutant embryos
is thus altered and is likely to contribute significantly to generating the observed phenotypes.
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132
Alterations in FGF and BMP
signaling pathways in Tbx2‐/‐;Tbx3+/‐
and Tbx2+/‐;Tbx3‐/‐ embryos
indicate that more than one functional allele of either Tbx2 or Tbx3
is required to correctly regulate
these signaling pathway components.
The elevated Fgf8 expression
in pharyngeal mesoderm of
Tbx1/Tbx2/Tbx3 compound mutants potentially
results from disrupted BMP signaling
and decreased antagonism between
these pathways (Figure 6). BMP
signaling is likely to be
tightly regulated during normal OFT
development, because expanded BMP
signaling in Nkx2.5 mutant embryos
also leads to failure of heart
tube extension 38.
A small percentage of
Tbx3‐/‐ embryos display a severe
phenotype on a mixed Bl6/CD1
genetic background (Table 1)23,
affecting endodermal Hh‐signaling and
the expression of Bmp4 and Fgf8
in the SHF and distal OFT,
similar to that the
expression changes observed in
Tbx2‐/‐;Tbx3‐/‐, Tbx2+/‐;Tbx3‐/‐ and
Tbx2‐/‐;Tbx3+/‐ embryos on a
FVB/N background. Tbx1/Tbx2/Tbx3 is
thus likely to be part of a
larger network comprising other genetic
factors that differ between different
genetic backgrounds.
Furthermore, downregulation of the BMP
target genes Msx1 and Msx2 is
observed in the pharyngeal region
of severely affected Tbx3 mutant
embryos, supporting a role for
this gene in the regulation of
BMP signaling during pharyngeal
development (Supplementary
Material, Figure S6F, H).
Figure 6. Altered FGF and BMP signaling in Tbx1/Tbx2/Tbx3
composite mutant embryos. Cartoons
showing the distribution of Tbx1,
Tbx2 and Tbx3
and the topography of BMP and FGF signaling in pharyngeal mesenchyme (comprised of mesodermal
cells, dark grey, and
neural crest‐derived cells,
light grey), pharyngeal endoderm, dorsal pericardial wall and the outflow
tract of wildtype (A), Tbx1‐/‐
(C) and compound mutant embryos (D, E). (B) Schematic summary of FGF‐BMP signaling axis
regulation by Tbx1 and by
the redundant action of Tbx2/Tbx3
during pharyngeal and arterial
pole morphogenesis. Red arrow
in C,
reduced expression of Tbx2 and Tbx3; green arrow in D, elevated expression of Tbx1. See text for details.
TBX2 and TBX3 are candidate modifier genes of the cardiopharyngeal phenotypes in TBX1 haploinsufficient 22q11.2DS patients
Tbx1
regulates proliferation and differentiation of
cardiac progenitor cells in
the SHF, and our data demonstrate
that Tbx2/Tbx3 function redundantly
to assist in coordinating
these crucial developmental
processes. While the
detailed mechanisms linking
Tbx1/Tbx2/Tbx3 function to the regulation of this signaling axis remain to be dissected, our study has shown that
in the absence of two components
the Tbx1/Tbx2/Tbx3
regulatory network collapses,
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133
severely compromising crucial intercellular Hh, BMP and FGF signaling events during arterial pole morphogenesis (Figure 6). Interestingly, recent work has shown that DNA variations in TBX1 are unable to fully explain the variable cardiovascular phenotypes in 22q11DS patients, highlighting the importance of genetic modifiers of the 22q11DS phenotype 44. Using mouse models, genetic interactions have already been identified between Tbx1 and the genes Crkl, Fgf8, Pitx2, Gbx2, Chd7, Six1/Eya1 and Hes1 29,
45‐47. Here, we show that Tbx2 and Tbx3 can be
added to this list. Our
findings provide new insights into
the mechanisms by which polygenic
mutations compromise the robustness
of cardiac morphogenesis and lead
to CVMs. In conclusion, this work highlights the central roles of Tbx1/Tbx2/Tbx3 in conotruncal morphogenesis and
identifies TBX2 and TBX3 as potential candidate modifier genes of
the cardiopharyngeal phenotypes in TBX1 haploinsufficient 22q11.2DS patients.
Acknowledgements We thank Jaco Hagoort for his help preparing the interactive 3D‐PDF file and Edouard Saint‐Michel and Laure Lo‐Ré for technical assistance.
FUNDING This work was supported
by the European Commission under
the FP7 Integrated
Project CardioGeNet (HEALTH‐2007‐B‐223463), the Netherlands Organization for Scientific Research (Vidi grant 864.05.006 to V.M.C. and Mosaic grant 017.004.040 to M.S.R.) and the Fondation pour la Récherche Médicale and Agence National pour la Recherche (to R.G.K.).
Table 1. Phenotypic severity of
embryos resulting from Tbx2+/‐;Tbx3+/‐,
Tbx1+/‐;Tbx2+/‐ or Tbx1+/‐;Tbx3+/‐
intercrosses.
Phenotypic severity c
Genotype N (obs) a N (exp) b 1 2
3 4 5
Tbx2+/+;Tbx3+/+ 10 9.4 10 0 0 0 0
Tbx2+/‐;Tbx3+/+ 20 18.8 20 0 0 0 0
Tbx2‐/‐;Tbx3+/+ 7 9.4 6 0 1 0 0
Tbx2+/+;Tbx3+/‐ 20 18.8 20 0 0 0 0
Tbx2+/‐;Tbx3+/‐ 34 37.5 34 0 0 0 0
Tbx2‐/‐;Tbx3+/‐ 21 18.8 0 0 0 19 2
Tbx2+/+;Tbx3‐/‐ 12 9.4 10 0 2 0 0
Tbx2+/‐;Tbx3‐/‐ 15 18.8 0 0 0 10 5
Tbx2‐/‐;Tbx3‐/‐ 11 9.4 0 0 0 0 11
Total 150 100 0 3 27 20
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134
Tbx1+/+;Tbx2+/+ 10 8.4 10 0 0 0 0
Tbx1+/‐;Tbx2+/+ 24 17 24 0 0 0 0
Tbx1‐/‐;Tbx2+/+ 7 8.4 0 7 0 0 0
Tbx1+/+;Tbx2+/‐ 16 17 16 0 0 0 0
Tbx1+/‐;Tbx2+/‐ 36 34 36 0 0 0 0
Tbx1‐/‐;Tbx2+/‐ 13 17 0 13 0 0 0
Tbx1+/+;Tbx2‐/‐ 7 8.4 6 0 1 0 0
Tbx1+/‐;Tbx2‐/‐ 17 17 15 0 2 0 0
Tbx1‐/‐;Tbx2‐/‐ 4d 8.4 0 0 0 0 4
Total 134 107 20 3 0 4
Tbx1+/+;Tbx3+/+ 13 9.75 13 0 0 0 0
Tbx1+/‐;Tbx3+/+ 17 19.5 16 1 0 0 0
Tbx1‐/‐;Tbx3+/+ 7 9.75 0 7 0 0 0
Tbx1+/+;Tbx3+/‐ 19 19.5 17 1 1 0 0
Tbx1+/‐;Tbx3+/‐ 34 39 29 0 5 0 0
Tbx1‐/‐;Tbx3+/‐ 26 19.5 0 23 3 0 0
Tbx1+/+;Tbx3‐/‐ 12 9.75 2 0 8 1 1
Tbx1+/‐;Tbx3‐/‐ 16 19.5 2 0 12 1 1
Tbx1‐/‐;Tbx3‐/‐ 12 9.75 0 1 2 1 8
Total 156 62 65 15 4 10
a Number of embryos collected of each genotype between E9.0 and E10.5
b Number of embryos expected of each genotype
c Phenotypic severity was determined according to the following criteria:
1: wildtype configuration (example panel 3A)
2: arches 2‐6 absent, distal outflow tract hypoplastic (example panel 4B)
3: right ventricle and outflow tract hypoplastic (example panel 3G)
4: pharyngeal hypoplasia and severe right ventricular and outflow tract hypoplasia (example panel 3D)
5: C‐looped dilated heart tube, edema, pharyngeal hypoplasia (including arch 1) (example panel 4D)
d Of which 3 embryos were necrotic
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135
SUPPLEMENTAL FIGURES
Supplemental Figure
S1. 3D‐reconstruction of Tbx1, Tbx2
and Tbx3 transcript distrubution
in wildtype
E9.5 embryos. Expression patterns of Tbx1/Tbx2, Tbx1/Tbx3 and Tbx2/Tbx3
in different embryos were determined using
in situ hybridization and
subsequently used to generate 3D
reconstructions of these
combinations (Supplementary Material, 3D‐PDF file).
Supplemental Figure S2.
Tbx3 expression
in the caudal pharynx
is affected by loss of Tbx1.
In E9.5 Tbx1 null embryos, Tbx3 expression was found to be reduced in pharyngeal mesenchyme (red arrows in B), in the dorsal pericardial wall (black arrows
in B) but not
in ventral endoderm (arrowhead
in B). 3D‐reconstructions of the dorsal pericardial wall showed a smaller overlapping region of Tbx1/Tbx3 (compare orange region in D with C) in Tbx1‐/‐ embryos. dpw, dorsal pericardial wall; la, left atrium; ph, pharynx; ra, right atrium.
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136
Supplemental Figure S3. Transcript distribution of Mlc2a, Isl1, Fgf8 and Tbx1 in Tbx2; Tbx3 compound mutants.
A shortened outflow tract and
right ventricular hypoplasia was observed
in compound mutants (C) compared
to wildtypes (A).
Isl1 was relatively unaffected in
the dorsal pericardial wall and
outflow tract (D). Sagittal section
of an E9.5 wildtype embryo (E,
E’), illustrating the plane
of the sections in F‐I.
In Tbx2‐/‐;Tbx3‐/‐ mutants,
Fgf8 was upregulated in
the dorsal pericardial wall
(K, arrowhead), and Tbx1 was upregulated
in ventral endoderm (L, arrow). dpw, dorsal pericardial wall; la,
left atrium; oft, outflow
tract; ph, pharynx; V‐end, ventral endoderm.
Supplemental Figure S4. Heterozygosity
for Tbx3 does not modify the
frequency or severity of
the Tbx1+/‐ fourth pharyngeal arch
artery (PAA) phenotype. Ink
injection at E10.5 showing the right 3rd, 4th and 6th PAAs of a wildtype embryo (A) and absence
of the right 4th PAA in
Tbx1+/‐ (B) and Tbx1+/‐;Tbx3+/‐ (C)
embryos. (D) Bar graph
showing the incidence of unilateral
(hypoplastic or absent) and bilateral
(hypoplastic, hypoplastic/absent and absent)
4th PAA defects for wildtype,
Tbx3+/‐, Tbx1+/‐ and Tbx1+/‐;Tbx3+/‐
genotypes. Examples
of retro‐oesophageal right subclavian artery
in Tbx1+/‐ (E, arrow) and Tbx1+/‐;Tbx3+/‐ (F, arrow) embryos at E14.5. da, descending aorta.
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137
Supplemental Figure S5. 3D‐reconstructions of pharyngeal endoderm
in wildtype, Tbx1‐/‐, and Tbx1+/‐;Tbx2‐/‐
E9.5 embryos. Proper segmentation and expansion of the foregut
is severely affected
in Tbx1‐/‐ embryos, but also affected in Tbx1+/‐;Tbx2‐/‐ embryos.
Supplemental Figure S6. Expression
of Isl1
in Tbx1;Tbx3 compound nulls and of Msx1 and Msx2 in
Tbx3‐deficient embryos. Expression analysis
of Isl1 revealed no differences
in Tbx1‐/‐ and
Tbx3‐/‐ single mutant embryos but a reduced contribution to
the Tbx1‐/‐;Tbx3‐/‐ compound mutant
outflow tract. BMP target genes
Msx1 and Msx2 were downregulated
in severely affected Tbx3‐/‐ embryos.
dpw, dorsal pericardial wall; lv,
left ventricle; oft, outflow
tract; pa1, pharyngeal arch; ph, pharynx.
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