Lack of Genetic Interaction between Tbx18 and Tbx2/Tbx20 in Mouse Epicardial Development
Lack of Genetic Interaction between Tbx18 and Tbx2/Tbx20 in Mouse Epicardial Development
Franziska Greulich 0 1
Carsten Rudat 0 1
Henner F. Farin 0 1
Vincent M. Christoffels 1
Andreas Kispert 0 1
0 Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany, 2 Department of Anatomy, Embryology and Physiology, Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands
1 Editor: Robert W Dettman, Northwestern University , UNITED STATES
The epicardium, the outermost layer of the heart, is an essential source of cells and signals for the formation of the cardiac fibrous skeleton and the coronary vasculature, and for the maturation of the myocardium during embryonic development. The molecular factors that control epicardial mobilization and differentiation, and direct the epicardial-myocardial cross-talk are, however, insufficiently understood. The T-box transcription factor gene Tbx18 is specifically expressed in the epicardium of vertebrate embryos. Loss of Tbx18 is dispensable for epicardial development, but may influence coronary vessel maturation. In contrast, over-expression of an activator version of TBX18 severely impairs epicardial development by premature differentiation of epicardial cells into SMCs indicating a potential redundancy of Tbx18 with other repressors of the T-box gene family. Here, we show that Tbx2 and Tbx20 are co-expressed with Tbx18 at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of Tbx2 nor the combined loss of Tbx2 and Tbx18 affects epicardial development. Similarly, we observed that the heterozygous loss of Tbx20 with and without additional loss of Tbx18 does not impact on epicardial integrity and mobilization in mouse embryos. Thus, Tbx18 does not function redundantly with Tbx2 or Tbx20 in epicardial development.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by a grant from
the Hannover Biomedical Research School (HBRS)
to FG, and by grants from the German Research
Foundation (DFG) for the Cluster of Excellence
REBIRTH (From Regenerative Biology to
Reconstructive Therapy) and for the Clinical
Research Group KFO136 at Hannover Medical
School to AK. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
The epicardium is an epithelial monolayer that completely covers the outer surface of the
heart. It protects the underlying myocardium and allows mobility of the heart within the
pericardial cavity. In addition to this structural role in homeostasis, the epicardium has been
recognized as an important source of cells and signals directing and modulating myocardial growth
and vascularization both in development and under injury conditions (for recent reviews see
Epicardial development in the mouse starts at embryonic day (E) 9.5 with the formation of
the proepicardium, a cauliflower-like mesothelial cell aggregate at the venous pole of the heart
Competing Interests: The authors have declared
that no competing interests exist.
]. Cells of the proepicardium delaminate and attach to the adjacent myocardium. At E10.5,
a contiguous epithelial epicardial layer surrounds the heart tube. Between E11.5 and E14.5,
individual epicardial cells undergo an epithelial-mesenchymal transition (EMT), invade the
underlying myocardium and largely differentiate into smooth muscle cells (SMCs) and cardiac
]. Concomitantly, the epicardium acts as a source of signals that nurture the
myocardium and promote the in-growth of the coronary plexus and vascularization of the
cardiac muscle [
]. Intriguingly, it has been uncovered in recent years both in zebrafish and
mouse that the adult epicardium can reactivate an embryonic gene program upon injury
]. As a consequence, the epicardium secretes factors that promote neovascularization
of the myocardium, and provides cells that upon differentiation into fibroblasts and SMCs
contribute to scar formation [
Although several signaling pathways and transcription factors have been implicated in the
distinct subprograms of epicardial development, namely proepicardium formation, epicardial
EMT, fate decision or epicardial-myocardial crosstalk [
], we are far from understanding
the tight regulatory networks orchestrating all of these processes in time and space, and using
them for regenerative purposes.
T-box (Tbx) genes encode a large family of transcription factors that regulate a variety of
developmental processes in both vertebrates and invertebrates. They are characterized by a
common DNA-binding motif, the T-box that recognizes and binds conserved DNA-elements
in the genome to mediate transcriptional activation and/or repression of target genes.
Tbox genes often act in a combinatorial or hierarchical fashion and frequently exhibit an
exquisite dose-sensitivity (for reviews see [
]). In the developing mammalian heart, expression
of six of the 17 murine family members have been detected and related to different
subprograms of myocardial patterning and differentiation (for a review see ). Tbx5 and Tbx20 act
in the early heart tube, and independently activate the chamber myocardial gene program [
] whereas Tbx2 and Tbx3 act together to locally repress this program to favor valvuloseptal
and conduction system development [
]. Tbx1 acts in the pharyngeal mesoderm to
maintain proliferation of mesenchymal precursor cells for formation of a myocardialized and
septated outflow tract [
]. Tbx18 is expressed in the sinus venosus region at the posterior pole of
the heart and is required for myocardialization of the caval veins and formation of a large
portion of the sinoatrial node [
Additional roles of these Tbx genes in epicardial development have been suggested. Tbx5
expression was detected in a heterogenous fashion in the proepicardium at E9.5 and the
nascent epicardium at E10.5. Epicardial expression strongly declined after this stage.
Conditional deletion of Tbx5 from the (pro-)epicardium led to reduced attachment of proepicardial
cells to the myocardium and epicardial blebbing that are probably causative for the reduced
epicardial EMT, fibroblast and SMC formation, and defective myocardial and coronary vessel
Tbx18 is strongly expressed in the proepicardium at E9.5 and is maintained in the
epicardium until birth in all vertebrate models analyzed to date [
]. We have recently reported
that Tbx18-deficient mice that were maintained on an outbred background do not exhibit
epicardial defects whereas mice with epicardial overexpression of an activating form of TBX18
(a VP16-fusion protein) show loss of epicardial EMT due to premature SMC differentiation of
epicardial cells [
]. Since TBX18 possesses transcriptional repressor activity via an eh1-motif
near the N-terminus that recruits Groucho corepressors [
], these findings point to a possible
redundancy with another repressing member of this gene family in maintaining the progenitor
character of epicardial cells. This hypothesis is supported by a recent study in which mice
deficient for another null allele of Tbx18 exhibit epicardial blebbing and coronary defects when
maintained on an inbred background [
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We here aimed to decipher a functional redundancy of Tbx18 with other transcriptional
repressors of the Tbx gene family in epicardial development. We identify Tbx2 and Tbx20 as
being coexpressed with Tbx18 in the developing (pro-)epicardium and subsequently test for
genetic interaction of Tbx18 and Tbx2/Tbx20 in this tissue.
Material and Methods
All animal work conducted for this study was performed according to European and German
legislation. Breeding of mutant mouse lines was approved by the Niedersächsisches Landesamt
für Verbraucherschutz und Lebensmittelsicherheit (Permit Number: AZ33.12-42502-04-13/
Mice and genotyping
For the generation of Tbx18-deficient embryos, males heterozygous for a cre knock-in allele of
Tbx18 (Tbx18tm4(cre)Akis, synonym:Tbx18cre) [
] were mated to female mice heterozygous for
a LacZ knock-in allele of Tbx18 (Tbx18tm1.1Akis, synonym: Tbx18LacZ) [
] or a GFP knock-in
allele of Tbx18 (Tbx18tm2Akis, synonym: Tbx18GFP) [
]. Female mice homozygous for a floxed
allele of Tbx2 (Tbx2tm2.1Vmc, synonym: Tbx2fl) [
] were crossed with Tbx18cre/+;Tbx2fl/+ males
to obtain embryos with epicardium-specific loss of Tbx2. The reporter allele Gt(ROSA)26Sortm4
(ACTB-tdTomato,-EGFP)Luo (synonym: R26mTmG) [
] was combined with the Tbx18cre line and a
Tbx2cre line (Tbx2tm1.1(cre)Vmc) [
] for fate analysis. For the generation of mice compound
mutant for Tbx20, we used the previously described null allele Tbx20tm1Akis (synonym:
]. Mice with epicardial overexpression of Tbx2 derived from matings of
Tbx18cre/+;R26mTmG/+ males with females homozygous for Hprttm2(CAG-TBX2,-EGFP)Akis
(synonym: HprtCAG::TBX2) [
]. All mice were maintained on an outbred (NMRI) background. Mice
were kept with regulated temperature (18–22°C) and humidity (~50%) with a 12 h light/dark
cycle. Vaginal plugs were checked in the morning after mating, for timed pregnancies noon
was taken as E0.5. Female mice were sacrificed by cervical dislocation. Embryos were harvested
in PBS, decapitated, fixed in 4% paraformaldehyde overnight and stored in 100% methanol at
-20°C before further use. Genomic DNA prepared from yolk sacs or tail biopsies was used for
genotyping by PCR.
Epicardial explant cultures
Explant cultures of primary epicardial cells were obtained as described before [
RNA isolation, reverse transcription and PCR analysis
RNA of epicardial explants was obtained using PeqGold RNApure (Peqlab) according to the
manufacturer’s manual and subsequently transcribed using the RevertAid Reverse
Transcriptase (Fermentas). 3 μl of undiluted epicardial cDNA or 1 μl of control cDNA (prepared from
different embryonic tissues) were used in the PCR reaction. Primers, PCR conditions and
controls are depicted in S1 Table.
For histological stainings embryos were fixed in 4% paraformaldehyde overnight, transferred
to PBS, paraffin embedded, and sectioned to 5-μm. Sections were stained with hematoxylin
and eosin following standard procedures.
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In situ hybridization analysis
Nonradioactive in situ hybridization analysis with digoxigenin-labeled antisense riboprobes
was performed on 10 μm sections of paraffin-embedded embryos as described [
Immunofluorescent detection of proteins
For immunofluorescence analysis on 5 μm paraffin sections anti-TBX3 (1:50, Santa Cruz,
sc31656), anti-TBX2 (1:1000, Millipore, 07–318), anti-TBX18 (1:50, Santa Cruz, sc-17869),
antiTAGLN (1:300, Abcam, ab14106-100), anti-NOTCH3 (1:300, Abcam, ab23426),
FITC-conjugated anti-ACTA2 (1:200, Sigma, F3777), anti-EMCN (1:50, obtained from D. Vestweber, MPI
Münster, Germany), anti-POSTN (1:300, Abcam, ab14041), anti-WT1 (1:200, Calbiochem,
CA1026), anti-GFP (1:200, Roche, 11 814 460 001) or rabbit polyclonal antibody anti-COL
type IV (1:200, Millipore, AB756P) were used as primary antibodies. Biotinylated anti-rabbit
IgG (1:200, Dianova), biotinylated anti-rat IgG (1:200, Dianova), Alexa488-conjugated
antimouse IgG (1:200, Invitrogen) and Alexa488-conjugated anti-rabbit IgG (1:200, Invitrogen)
were used as secondary antibodies. The signals of all primary antibodies (except anti-GFP,
anti-COLIV and anti-ACTA2) were amplified using the Tyramide Signal Amplification (TSA)
system from Perkin-Elmer (NEL702001KT, Perkin Elmer). For double staining, the second
antigen was stained for after the staining for the first one was finished. Nuclei were stained
with 4,6-diamidino-2-phenylindole (DAPI) (Roth).
Documentation, Quantification and Statistics
Sections were photographed using a Leica DM5000 microscope with Leica DFC300FX digital
camera. All images were processed in ImageJ and Adobe Photoshop CS4. Statistical analyses
were performed using the 2-tailed Student’s t-test. Data were expressed as mean±SD.
Differences were considered not significant when the P-value was higher than 0.05. At least two
specimens were analyzed per stage, genotype and assay.
Expression of T-box genes during epicardial development
To determine whether the members of the cardiac T-box gene family Tbx1, Tbx2, Tbx3, Tbx5,
Tbx20 are coexpressed with Tbx18 in epicardial development, we performed a comparative
expression analysis in mouse embryos. As a sensitive assay we first performed qualitative
RT-PCR analysis from E11.5 epicardial explant cultures grown for three days under serum-free
conditions. This assay revealed the expression of Tbx18, Tbx20, Tbx2 and Tbx5 in
undifferentiated epicardial cells whereas Tbx3 and Tbx1 were not detected (Fig 1A). To resolve epicardial
expression in time and space we subsequently performed section in situ hybridization of E9.5
to E14.5 hearts (Fig 1B). At E9.5, expression of Tbx18 was strong in the proepicardium.
Expression of Tbx20, Tbx5 and Tbx2 was markedly weaker but still above background levels. From
E10.5 to E14.5, Tbx18 expression was detected in the epicardium but not in subepicardial cells
in the right ventricle. (Due to endogenous expression of Tbx18 in the myocardium of the left
ventricle and the interventricular septum [
], we restricted our analysis on the right ventricle).
Expression of Tbx5, Tbx3 and Tbx1 could not be detected in the epicardium at any of these
stages. In contrast, we found expression of Tbx20 in epicardial cells at E10.5 (Fig 1B, arrow).
Due to the myocardial expression of Tbx20 (Fig 1B, ), Tbx20-expressing epicardial and
epicardium-derived cells could not be distinguished by this method. Tbx2 transcripts were detected
in a subset of epicardial cells (Fig 1B, arrow) at E12.5 and at E14.5, and in coronary arteries at
E14.5 (Fig 1B, black arrowheads).
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Fig 1. T-box gene expression during epicardial development. (A) Qualitative RT-PCR analysis detects expression of
Tbx18, Tbx5, Tbx20 and Tbx2 but not of Tbx3 and Tbx1 in undifferentiated epicardial cells from cardiac explant cultures
(Epi). 32 epicardial explant cultures were pooled and used for qualitative PCR. H2O refers to a negative control without
cDNA, pos to a positive control of a tissue with known expression (see S1 Table). (B) In situ hybridization analysis of
Tbx18, Tbx5, Tbx20, Tbx2, Tbx3 and Tbx1 expression on sagital (E9.5) and transverse (E10.5, E12.5, E14.5) sections
through the heart. Shown are higher magnifications of the proepicardium (E9.5) and of the right ventricle (E10.5 to
E14.5). Black arrows indicate proepicardial and epicardial expression of Tbx18, Tbx5, Tbx20 and Tbx2, asterisks point to
known expression domains of Tbx5 and Tbx20 in the atrium, and of Tbx2 and Tbx3 in the liver primordium at E9.5. Black
arrowheads indicate coronary artery expression of Tbx2 at E14.5. Scale bars are 50 μm. CA, common atrium; CAr,
coronary artery; Epi, epicardium; PE, proepicardium; RV, right ventricle; SV, sinus venosus.
For further clarification, we also analyzed TBX2 and TBX3 protein expression by
immunofluorescence. At E9.5, Wilms tumor 1 (WT1) and TBX18 were found in the entire proepicardium as
previously reported [
] whereas TBX2 protein was confined to the caudal part of this tissue.
Lineage analysis using a Tbx2cre line  and a Rosa26mTmG/+ reporter line [
] showed that the
proepicardium itself was completely derived from cells formerly expressing Tbx2 (Fig 2A and 2B).
At E13.5, a subset of epicardial and subepicardial cells expressed TBX2 protein as shown by double
immunofluorescence against TBX2 and GFP, that in this case marked the epicardial lineage by
epicardium-specific recombination under the control of the Tbx18 promoter [
R26mTmG/+ mice) (Fig 2C). Expression of TBX3 was neither detected in the proepicardium at E9.5
nor in the epicardium at E13.5 (Fig 2B and 2C), confirming as in the case of TBX2 our mRNA
expression analysis. We conclude that Tbx18 is co-expressed with Tbx5, Tbx20 and Tbx2 in a
subset of cells in the proepicardium at E9.5 and in a subset of epicardial cells until E14.5.
Combined loss of Tbx18 and Tbx2 does not affect epicardial
Since Tbx2 is co-expressed with Tbx18 in a subset of proepicardial and epicardial cells and
encodes a transcriptional repressor like Tbx18 [
], we tested for functional redundancy of
the two genes in epicardial development by a conditional gene targeting approach.
(Pro)epicardial deletion of Tbx2 was achieved using a floxed allele of Tbx2 and the Tbx18cre mouse
line that mediates robust recombination in the proepicardium, and in the epicardium and its
]. Absence of TBX18 protein in the epicardium/pericardium of Tbx18-null
embryos, and of TBX2 protein in Tbx18cre/+;Tbx2fl/fl embryos at E10.5 confirmed the suitability
of this genetic approach (S1 Fig).
We focused our analysis on E14.5 embryos to be able to compare our findings with that of a
previous study on mice with epicardial overexpression of an activator version of TBX18 [
Hematoxylin and eosin staining of transverse sections through the heart region did not reveal
any difference in the histological appearance of the cardiac chambers in compound mutants
(Tbx18cre/GFP;Tbx2fl/+ and Tbx18cre/+;Tbx2fl/fl), and double mutants (Tbx18cre/GFP;Tbx2fl/fl)
compared to control embryos (Tbx18cre/+); septa and valves were formed normally and the
ventricular myocardium was of normal thickness. In Tbx18-deficient mice pleuropericardial
membranes were not completely resolved from the body wall but remained laterally attached
in agreement with our previous report on the role of Tbx18 in the development of this tissue
] (Fig 3A, arrows). Higher magnification of the histologically stained right ventricle
demonstrated that the epicardium was correctly attached in double and compound mutant embryos
(Fig 3B). To visualize the epicardium and its descendants on a cellular level, we analyzed GFP
expression from the R26mTmG reporter allele in the different mutant combinations.
GFP-positive cells localized to the subepicardial space and intermingled with cardiomyocytes in double
and compound mutants as in control mice; and visual inspection of transverse sections did not
reveal gross changes in the number of immigrating cells (Fig 3C). Similarly, we did not detect
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Fig 2. TBX2 is expressed in the proepicardium and epicardium. (A) Epifluorescence of a Tbx2cre/+;R26mTmG/+ embryo at E9.5 reveals the contribution of
formerly Tbx2-expressing cells to the atrio-ventricular canal (AVC), the otic vesicle (OV), the eye (E) and the proepicardium (PE) (n = 5). The scale bar is
500 μm. (B) Lineage tracing of Tbx2-expressing cells on sections of E9.5 embryos by immunofluorescent detection of a GFP reporter and/or the epicardial
markers TBX18 and WT1 (left row) confirms the contribution of Tbx2-expressing cells to the proepicardium (n = 3). Double immunofluorescence against
GFP and TBX2 or TBX3, respectively, (right row) shows expression of TBX2 in the caudal part of the proepicardium. Note that the anti-TBX2 antibody
recognizes cells that do not recombine after cre expression from the Tbx2 promoter. The third picture of the lower row shows an in silico overlay of the
expression domains of TBX2 and TBX3 co-stained with the Tbx2-lineage label GFP on neighboring sections. Only TBX2-positive (red), TBX3-negative and
GFP-positive (blue) domains relate to Tbx2 expression domains (white arrowhead). The scale bars are 50 μm. (C) TBX2 but not TBX3 protein was detected
by immunofluorescence against TBX2 and TBX3 in epicardial and subepicardial cells of Tbx18cre/+;R26mTmG/+ embryos at E13.5 (white arrows, n = 2).
Costaining against the Tbx18-lineage label GFP clearly identifies epicardial and epicardium-derived cells. The scale bars are 50 μm. CA, common atrium; Epi,
epicardium; RV, right ventricle; SV, sinus venosus.
differences in the expression of Wilms tumor 1 (WT1), a marker for epicardial and
epicardium-derived cells, and more weakly for endothelial cells [
] in double and compound
mutant embryos compared to the control (Fig 3C). Expression of endomucin (EMCN), a
marker of venous and capillary endothelial cells and of the endocardium , was
indistinguishable in double and compound mutant and control hearts indicating that formation of the
coronary plexus occurred normally (Fig 3C). We conclude that epicardial signaling is
unaffected by loss of Tbx18 and/or Tbx2. To clarify whether loss of Tbx18 and Tbx2 impairs SMC
differentiation, expression of SMC proteins NOTCH3 and Transgelin (TAGLN) was analyzed.
Epicardial cells were delineated by costaining for collagen IV (COLIV) expression in the basal
]. TAGLN protein expression was found in subepicardial cells and cardiomyocytes
but not in epicardial cells in any of the analyzed genotypes at this stage (Fig 3C). NOTCH3
protein was detectable in up to 70% of epicardial cells of Tbx18cre/GFP;Tbx2fl/fl and Tbx18cre/GFP;
Tbx2fl/+ hearts but only in 50% of control or Tbx18cre/+;Tbx2fl/fl epicardial cells indicating that
the loss of Tbx18 accounts for this effect (Fig 3C, S2 Fig).
At E18.5, epicardium-derived cells have fully differentiated into fibroblasts and SMCs,
constituting the cardiac fibrous skeleton and completing the formation of the coronary
vasculature. Hematoxylin and eosin staining of transverse sections through the heart at this stage did
not reveal histological defects in addition to the pericardial changes observed in
Tbx18-deficient embryos at E14.5 (Fig 4A). Coronary arteries developed normally in Tbx18/Tbx2
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Fig 3. Phenotypic analysis of hearts with combined loss of Tbx18 and Tbx2 in the epicardium at E14.5. (A) Histological analysis of one to two embryos
per genotype by hematoxylin and eosin staining of transverse heart sections does not reveal any gross morphological defects in Tbx18cre/GFP;Tbx2fl/fl double
mutant hearts compared to control (Tbx18cre/+) or compound mutant (Tbx18cre/GFP;Tbx2fl/+ or Tbx18cre/+;Tbx2fl/fl) embryos. The scale bars are 500 μm. The
black arrows point to pericardial defects observed in Tbx18cre/GFP;Tbx2fl/fl and Tbx18cre/GFP;Tbx2fl/+ hearts. (B) Higher magnification of the right ventricle
shows a tightly attached epicardium on top of the heart in all genotypes. The scale bars are 20 μm (n = 1). (C) Immunofluorescence analysis of GFP and WT1
expression confirms epicardial integrity and subepicardial as well as myocardial localization of epicardium-derived cells in all genotypes. The scale bars
represent 50 μm. The in-growing vasculature, visualized by EMCN immunofluorescence, has almost reached the apex of the right ventricle (white arrows).
TAGLN is not expressed in the epicardium as emphasized by double immunofluorescence with COLIV (grey arrowheads). In contrast, NOTCH3 expression
is found in the epicardium of Tbx18cre/GFP;Tbx2fl/fl and Tbx18cre/GFP;Tbx2fl/+ mice and occasionally in Tbx18cre/+;Tbx2fl/fl and control hearts (white
arrowheads). Double staining with the Tbx18-lineage marker GFP indicates NOTCH3-positive cells in the epicardium of Tbx18cre/GFP;Tbx2fl/fl and
Tbx18cre/GFP;Tbx2fl/+ mice. Dashed lines mark the border between epicardium and myocardium. Scale bars in NOTCH3 and TAGLN single staining are
50 μm, and 20 μm in the double staining of these markers with GFP or COLIV. Two specimens per genotype and stage were analyzed by immunostaining.
Epi, epicardium; LA, left atrium; LV, left ventricle; Peri, pericardium; RA, right atrium; RV, right ventricle.
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Fig 4. Phenotypic analysis of hearts with combined loss of Tbx18 and Tbx2 in the epicardium at E18.5. (A) Histological
analysis by hematoxylin and eosin staining of transverse sections of Tbx18cre/GFP;Tbx2fl/fl hearts reveals a dilatation of the atria in
comparison to Tbx18cre/GFP;Tbx2fl/+ or Tbx18cre/+;Tbx2fl/fl or control (Tbx18cre/+) hearts. Atrial dilatation is occasionally seen in
Tbx18-deficient mice as well (not shown). The ventricular compartment of Tbx18cre/GFP;Tbx2fl/fl hearts appears indistinguishable
from control hearts (n = 2). The scale bars are 500 μm. (B) Immunofluorescence analysis of ACTA2 and TAGLN expression shows
normal differentiation of coronary SMCs and their localization to coronary arteries (CAr). Capillary density, although not quantified,
appears unaffected by the loss of Tbx18, Tbx2 or both genes in the epicardium as visualized by immunofluorescence against
EMCN. The presence of POSTN in the myocardium confirms the formation of cardiac fibroblasts from epicardial cells in all
mutants. Two specimens per genotype were analyzed. Scale bars are 100 μm. Epi, epicardium; LA, left atrium; LV, left ventricle;
Peri, pericardium; RA, right atrium; RV, right ventricle.
compound and double mutant hearts and were surrounded by SMCs expressing actin, alpha 2,
smooth muscle, aorta (ACTA2) and TAGLN (Fig 4B) and the late SMC differentiation marker
myosin, heavy polypeptide 11, smooth muscle (MYH11) (S3 Fig). EMCN expression in
mutants was indistinguishable from the control indicating the formation of a normal
endothelial network in the coronary vasculature. Finally, the formation and distribution of periostin
(POSTN)-positive fibroblasts in the myocardium was not affected by the individual or
combined loss of Tbx2 and Tbx18 (Fig 4B). We conclude, that Tbx2 and Tbx18 are neither
individually nor combinatorially required in the epicardium.
Misexpression of human TBX2 in epicardial cells and their progeny does
not affect cardiac development
Although the loss of Tbx2 does not effect epicardial development, its localized expression in a
subset of epicardial cells may indicate an involvement of the gene in the specification of distinct
epicardial sublineages. To study the potential effect of Tbx2 on lineage segregation in the
epicardium, we generated mice ectopically expressing human TBX2 in the whole epicardium and its
descendants. For this purpose Tbx18cre/+;R26mTmG/mTmG males were mated to females
homozygous for an integration of a cre-inducible TBX2 expression cassette in the X-chromosomal Hprt
locus (HprtCAG::TBX2/CAG::TBX2) [
]. Since female Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/+ embryos
express the transgene in a mosaic fashion due to X-chromosome inactivation, we subsequently
only analyzed male Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y embryos that express the transgene
homogenously. Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y embryos were present in the expected
Mendelian ratio at E18.5 (n = 10/42). Histological analysis revealed that the pleuropericardial
membranes were not fully detached from the body wall. However, septa and valves were
unaffected and the ventricular walls exhibited normal thickness and trabeculation (Fig 5A).
Immunofluorescence analysis confirmed expression of human TBX2 protein in the epicardium and
epicardium-derived cells at levels similar to that of endogenous mouse TBX2 in coronary SMCs.
Weak ectopic expression of TAGLN was associated with human TBX2 protein in the epicardium
and the myocardium in Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y mice (Fig 5B). Formation of
coronary arteries was unaffected although the surrounding SMC layer appeared thinned in Tbx18cre/+;
R26mTmG/+;HprtCAG::TBX2/y hearts as indicated by staining for the SM proteins ACTA2 and
TAGLN (Fig 5C). However, the contribution of epicardium-derived cells to the SM lineage
surrounding coronary arteries was unaffected in Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y hearts (S4
Fig). In contrast to epicardium-derived cells in control hearts, TAGLN but not ACTA2 was
ectopically expressed in epicardial cells and in a majority of epicardium-derived cells of
Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y hearts at E18.5 (Fig 5A and 5B, S4 Fig: red arrow heads).
EMCN expression in mutants was unchanged from the control indicating the formation of a
normal venous and capillary network in the coronary vasculature. Finally, the formation and
distribution of POSTN-positive fibroblasts in the myocardium was normal in Tbx18cre/+;R26mTmG/+;
HprtCAG::TBX2/y hearts at this stage (Fig 5D, S4 Fig). Together, this suggests that homogenous
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Fig 5. Phenotypic analysis of E18.5 hearts with epicardial misexpression of human TBX2. (A) Histological analysis (n = 2) by hematoxylin and eosin
staining of transverse sections of Tbx18cre/+;HprtCAG::TBX2/y hearts reveals pericardial defects (black arrow) but no further anomalies compared to the control
(Tbx18cre/+). Scale bars are 500 μm. (B-D) Immunofluorescence analysis (n = 4) of indicated proteins on transverse sections of Tbx18cre/+ (control) and
Tbx18cre/+;HprtCAG::TBX2/y hearts. Shown are magnified regions of the right ventricle. Scale bars are 100 μm. (B) Human TBX2 protein and TAGLN is found in
the epicardium and in epicardium-derived cells in Tbx18cre/+;HprtCAG::TBX2/y hearts. Note that the level of human TBX2 expression is similar to the level of
endogenous mouse TBX2 protein. (C) Coronary vessels are surrounded by ACTA2- and TAGLN-expressing SMCs in Tbx18cre/+;HprtCAG::TBX2/y hearts at
E18.5, but additional ACTA2- and TAGLN-positive cells are detected in the myocardium of these hearts. (D) Small EMCN-positive coronary vessels form in
comparable densities in Tbx18cre/+;HprtCAG::TBX2/y and control hearts. Intramyocardial deposition of POSTN was detected in both genotypes in a comparable
fashion. CAr, coronary artery; Epi, epicardium; LV, left ventricle; Peri, pericardium; RA, right atrium; RV, right ventricle.
epicardial misexpression of human TBX2 does not impair formation, migration and
differentiation of the epicardial and epicardium-derived cells. Although ectopic expression of TAGLN in
epicardial and epicardium-derived cells of Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y hearts was
observed, the differentiation potential of those cells was maintained.
Reduction of the Tbx20 gene dosage in combination with the loss of
Tbx18 does not affect epicardial EMT
TBX20 like TBX18 is a member of the TBX1 subfamily, and can act either as a transcriptional
] or as a transcriptional activator [
]. It is therefore possible that a TBX20
repressor function acts redundantly with TBX18 in epicardial development. As Tbx20-deficient
mice die shortly after E9.5 [
], and a floxed allele of Tbx20 was not available to us, we decided
to analyze mice compound mutant for a null allele of Tbx18 (Tbx18cre and Tbx18LacZ) [
a null allele of Tbx20 (Tbx20LacZ) that we previously generated and characterized [
determine the effect of a reduced Tbx20 gene dosage in a Tbx18-deficient background. Notably,
Tbx18cre/cre;Tbx20LacZ/+ mutant embryos were underrepresented in E14.5 litters derived from
matings of Tbx18cre/+;Tbx20LacZ/+;R26mTmG/+ male with Tbx18cre/+;Tbx20LacZ/+;R26mTmG/+
female mice (expected 16.7%, obtained 4%, n = 2/50) indicating an early lethality of these
compound mutants. All other Tbx20LacZ/+ compound mutants (Tbx18cre/+;Tbx20LacZ/+: n = 13/50,
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Tbx18+/+;Tbx20LacZ/+: n = 4/50) were slightly underrepresented as well whereas Tbx18cre/LacZ;
Tbx20+/+ (n = 13/50) mutants and Tbx18cre/+;Tbx20+/+ mutants (n = 14/50) were
Histological stainings of transverse sections of Tbx18cre/cre;Tbx20LacZ/+ hearts revealed
normal chamber architecture and septa formation but pericardial defects similar to Tbx18-null
mice (black arrows in Fig 6A). Higher magnification of the right ventricular epicardium
revealed a monolayer of flattened cells covering the myocardium in all mutants; a thin
subepicardial space was formed in Tbx18cre/cre;Tbx20LacZ/+ hearts as in Tbx18cre/LacZ and control
hearts (Fig 6B). Blood filled vessels visualized by the presence of eosin-positive red blood cells
grew into the subepicardial space in all mutants. Section in situ hybridization of transverse
sections against Aldh1a2 confirmed the structural integrity of the epicardium (Fig 6C), and
immunofluorescence of EMCN expression demonstrated that the coronary plexus was recruited
correctly (Fig 6D). Both findings indicate that the epicardial-myocardial crosstalk is
undisturbed. Immunofluorescence of the lineage label GFP and the epicardial marker WT1 further
proved the formation of a complete epicardium that was tightly attached to the surface of the
heart (Fig 6D). Epicardium-derived cells were present in the subepicardium and myocardium
in mutants as in controls as visualized by expression of GFP and/or WT1. The amount of
GFPand WT1-positive cells entering the myocardium and their distribution between subepicardial
and myocardial compartments was unaltered indicating proper EMT and immigration
patterns in Tbx18cre/cre;Tbx20LacZ/+, Tbx18cre/LacZ, Tbx18cre/+;Tbx20LacZ/+ and control hearts (Fig
6D). Expression of TAGLN was restricted to the subepicardial space in Tbx18cre/cre;Tbx20LacZ/+
hearts (Fig 6D) arguing against premature SMC differentiation of epicardial cells. Expression
of NOTCH3 was observed in some epicardial cells of Tbx18-null mutant embryos but was not
enhanced by the additional loss of Tbx20 (S2 Fig). Hence, the loss of one Tbx20 allele does not
alter epicardial development in wild-type or Tbx18-deficient mice.
Here, we found that Tbx2, Tbx20 and Tbx5 are co-expressed with Tbx18 at different stages of
epicardial development. Our genetic experiments showed that Tbx2 is dispensable for
epicardial development, and that neither Tbx2 nor Tbx20 redundantly interact with Tbx18 in any of
the subprograms important for formation, migration and differentiation of epicardial cells in
Tbx18 and combinatorial interaction with Tbx genes in epicardial
Our previous analysis demonstrated that loss of Tbx18 does not affect epicardial function
whereas epicardial-specific misexpression of an activator version of TBX18 (TBX18VP16) led
to premature SMC differentiation of epicardial cells [
]. This suggested that TBX18 possibly
together with a related transcriptional repressor maintains the precursor character of epicardial
cells by repressing SMC differentiation. Our sensitive PCR and in situ hybridization methods
detected expression of three additional cardiac Tbx genes, Tbx5, Tbx20 and Tbx2 in the
proepicardium and epicardium. Expression of Tbx5 was detected in the E9.5 proepicardium, and in
the E10.5–11.5 epicardium but was subsequently down-regulated. A recent report found a
similar pattern of expression both on the level of Tbx5 mRNA and TBX5 protein, and uncovered
that epicardial-specific loss of Tbx5 is associated with blebbing and reduced EMT but not with
premature SMC differentiation of epicardial cells. Augmented expression of Tbx5 in the
proepicardium led to reduced proepicardial migration and enhanced apoptosis in chick embryos
]. Together, this suggests that precise levels of Tbx5 are important for formation and
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Fig 6. Phenotypic analysis of E14.5 hearts with combined loss of Tbx18 and one Tbx20 allele. (A, B) Histological analysis by hematoxylin and eosin
staining of transverse sections of E14.5 hearts shows that the reduction of the Tbx20 gene dosage in a Tbx18-mutant background does not affect epicardial
and myocardial integrity. Pericardial defects are observed in Tbx18cre/cre;Tbx20LacZ/+ as well as in Tbx18cre/LacZ mice (black arrows in A). The scale bars are
500 μm. (B) The right ventricular epicardium (Epi) shows a cellular monolayer on top of a less dense subepicardial layer (SE) and the myocardium in all
genotypes. The scale bars are 20 μm. (C) Section in situ hybridization against Aldh1a2 confirms epicardial integrity. The scale bars are 50 μm. (D) Epicardial
cells, immunologically stained for the Tbx18-lineage label GFP (from an introduced Rosa26mTmG allele) or the epicardial marker WT1, undergo EMT and
populate the subepicardial space as well as the myocardium in a similar fashion in all mutant and control mice. The coronary plexus forms normally in
Tbx18cre/cre;Tbx20LacZ/+ mice and reaches the right ventricular apex similar to Tbx18cre/LacZ single mutant and control mice as indicated by
immunofluorescence against EMCN. The scale bars are 50 μm. A premature differentiation of epicardial cells into SMCs does not occur in any mutant as
visualized by immunofluorescence against TAGLN. Grey arrowheads indicate TAGLN-negative epicardial cells. TAGLN-expressing cells are found within
the myocardium and subepicardial space as confirmed by double staining with COLIV. Immunofluorescence against NOTCH3 on the other hand
demonstrates the presence of few NOTCH3-positive cells in the epicardium of Tbx18cre/+ controls and an increased number of NOTCH3-positive epicardial
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cells in Tbx18cre/LacZ and Tbx18cre/cre;Tbx20LacZ/+ mutants whereas none are observed in the depicted sections of Tbx20LacZ/+ or Tbx18cre/+;Tbx20LacZ/+
mutants. These results are confirmed by double immunofluorescence against NOTCH3 and COLIV. White arrowheads point toward NOTCH3-positive cells
within the epicardium. Scale bars are 50 μm for TAGLN and NOTCH3 staining and 20 μm for double staining with COLIV. Dashed lines indicate the
epicardial-myocardial border. Two specimens per genotype were analyzed. Epi, epicardium; LA, left atrium; LV, left ventricle; Peri, pericardium; RA, right
atrium; RV, right ventricle.
migration of proepicardial cells. To date, TBX5 has only been characterized as a transcriptional
activator in cardiac development [
] whereas a Groucho-dependent role as a
transcriptional repressor was assigned to TBX18 . Although it cannot be formally excluded that
both factors behave in a biochemically equivalent fashion (as activators or repressors) in
epicardial development, the phenotypic differences between Tbx5- and Tbx18-deficient
embryos argue against a redundant function. Given our earlier finding that TBX18 can
compete with TBX5 for DNA-binding sites in vitro [
], it cannot be excluded, however, that
TBX18 is important to fine-tune the transcriptional responses to TBX5 by selectively
repressing some of the TBX5 target genes. More detailed transcriptional profiling of Tbx5- and
Tbx18-deficient (pro-)epicardial cells may help to test such a scenario.
We also detected expression of Tbx20 in the proepicardium and early epicardium albeit
unambiguous assignment to this tissue was hampered by strong myocardial expression of the
gene. Tbx20-deficient embryos form a proepicardium but an analysis of subsequent epicardial
development is impossible due to the lethality at E9.5 [
]. Mice heterozygous for a Tbx20 null
allele survive into adulthood with diverse cardiac pathologies, including defects of septation and
valvulogenesis and cardiomyopathy that however, do not correspond to an epicardial
requirement of the gene [
]. Here, we have shown that mice compound mutant for Tbx18 and
Tbx20 do not exhibit defects in epicardial development. Despite the fact that TBX20 can act as a
repressor in developing and mature hearts [
22, 51, 52, 55
], these genetic findings argue that
Tbx20 neither on its own nor in combination with Tbx18 plays an essential role in the
development of the epicardium. Since TBX20 can also act as a transcriptional activator [
], it may
cooperate with TBX5 in regulating proepicardial and early epicardial development. Such a possibility
may be addressed by proepicardial-specific deletion of both Tbx5 and Tbx20 in the mouse.
Finally, we detected expression of Tbx2 (but not of the closely related Tbx3 gene) in a subset
of proepicardial and epicardial cells in the developing mouse heart. Since Tbx2 like Tbx18
encodes a strong transcriptional repressor [
], we focused our genetic studies on the role
of Tbx2 and its possible redundancy with Tbx18 in epicardial development. However, neither
embryos with an epicardial deletion of Tbx2 nor with combined deficiency of Tbx18 and Tbx2
exhibited epicardial defects, strongly arguing against an individual or combined role for Tbx2
in epicardial development. Similar to Tbx18 , misexpression of TBX2 in the epicardial
lineage did not affect epicardial function nor did it prevent SMC differentiation of
epicardiumderived cells. Both for TBX2 and TBX18, this may reflect the lack of cofactors necessary to
exert this function. Since TBX2 (and TBX3) have been characterized as strong competitive
inhibitors of TBX5-activated gene programs in the atrioventricular canal and outflow tract [
], the possibility again exists, that TBX2 represses TBX5 target genes in individual
proepicardial and epicardial cells.
While TBX5, TBX20 and TBX2 do not hold promise as cooperation partners of TBX18 in
maintenance of epicardial integrity, complex dose-dependent antagonistic and synergistic
interactions between the different TBX family members may exist that play an important role
in generating a molecular heterogeneity in fate decisions in proepicardial and epicardial cells as
in other developmental contexts [
]. Other T-box proteins that have not yet been associated
with cardiac development might additionally be expressed in the epicardium to feed into this
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TBX18 function in repression of SMC differentiation may depend on additional cofactors
While the identity of a TBX protein acting redundantly with TBX18 in repression of SMC
differentiation of epicardial cells remains enigmatic, the maintenance of epicardial precursor
character in Tbx18-deficient embryos may also reflect lack of an activator of the SMC program
in these cells. Interestingly, Wu and colleagues recently analyzed the potential of Tbx18 to
inhibit a SMC differentiation program in vitro. They used C3H10T1/2 cells, multipotent
mesenchymal progenitors that initiate the SMC pathway when exposed to TGFß1, and showed
that cells transfected with a Tbx18 expression vector exhibit a markedly reduced expression of
SMC markers. This result was not due to increased apoptosis or reduced proliferation
indicating that TBX18 is indeed able to suppress a SMC differentiation pathway. Moreover, they
found in transactivation assays that TBX18 inhibits Serum response factor
(SRF)-CArGbox dependent activation of the promotors of the SMC-specific genes Tagln, Fos and Actg2
]. Lack of SMC differentiation in Tbx18-deficient epicardial cells may therefore simply
reflect the lack of expression of the master activators of the SMC program, Myocardin (Myocd)
and/or Srf [
] in the epicardium. TBX18VP16, an activator version of TBX18, on the other
hand may be able to activate target genes usually repressed by TBX18 independently of
co-factors leading to premature differentiation of the epicardium into SMCs [
It is worth to note that NOTCH3 expression was upregulated in epicardial cells
overexpressing TBX18VP16 as well as in cells of Tbx18-null epicardia. Notch3 contains functional T-sites
in the zebrafish [
] suggesting that Notch3 expression is activated independently from
MYOCD/SRF but is directly repressed by TBX18. Although epicardial overexpression of the
intracellular fragment of NOTCH is sufficient to induce SMC differentiation [
], lack of the
appropriate ligand in Tbx18-deficient epicardial cells will prevent activation of Notch3 and
induction of the SMC pathway.
S1 Fig. Absence of TBX18 and TBX2 protein in mutant embryos. (A) Immunofluorescence
analysis of TBX18 on transverse E10.5 sections through right ventricles of control and
Tbx18GFP/GFP mice. Epicardial and pericardial cells express TBX18 in the control (white
arrows) but not in Tbx18GFP/GFP mice. (B) Immunofluorescence analysis of TBX2 on transverse
E10.5 sections through right ventricles of control and Tbx18cre/+;Tbx2fl/fl;R26mTmG/+ mice.
Pericardial cells are positive for TBX2 in the control but not in Tbx18cre/+;Tbx2fl/fl;R26mTmG/+ mice.
Scale bars are 50 μm. Epi, epicardium; Peri, Pericardium; RA, right atrium; RV, right ventricle.
S2 Fig. Increase of NOTCH3+-epicardial cells in Tbx18-null mice is independent from
Tbx2 or Tbx20. In order to quantify NOTCH3-expressing epicardial cells, immunofluorescent
stainings against NOTCH3 were analyzed. (A) Quantification of NOTCH3-positive epicardial
cells in Tbx18cre/GFP;Tbx2fl/fl (70.0±4.4%), Tbx18cre/GFP;Tbx2fl/+ (67.9±8.2%), Tbx18cre/+;Tbx2fl/fl
(52.2±13.8%) and Tbx18cre/+ hearts. Two specimens of each genotype were analyzed and
the ratio of NOTCH3-positive cells within the right ventricular epicardium was determined
and displayed as percentage. Error bars indicate the standard deviation. (B) Ratio of
NOTCH3-expressing cells within the epicardium of Tbx18cre/cre;Tbx20LacZ/+ (74.3±10.4%,
n = 3), Tbx18cre/LacZ (82.9±9.0%, n = 5), Tbx18cre/+;Tbx20LacZ/+ (61.7±5.3%, n = 2) mutant
hearts and Tbx18cre/+ (51.2±14.2%, n = 6) as well as Tbx20LacZ/+ (55.0±11.8%, n = 2) control
hearts is displayed as percentage. Number of specimens per genotype as indicated and error
bars represent the standard deviation. (C) Direct comparison of the ratio of NOTCH3-positive
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cells in Tbx18cre/LacZ (82.9±9.0%) and Tbx18cre/+ (54.5±13.0%) mutants. Five specimens per
genotype were analyzed and the standard deviation blotted as error bar. Student’s t-test
confirmed the significance of these results.
S3 Fig. Myh11 expression in coronary arteries is unchanged in Tbx18- and Tbx2-deficient
hearts at E18.5. (A) In situ hybridization analysis of Myh11 expression on transverse sections
of hearts of control, Tbx18GFP/GFP and Tbx18cre/+;Tbx2fl/fl;R26mTmG/+ mice at E18.5. As in
control hearts, SMCs of the coronary arteries of both mutant hearts show Myh11 expression. (B)
Shown are higher magnifications of the boxed areas in the right ventricle. Scale bars are as
shown. CAr, coronary artery; LV, left ventricle; Peri, pericardium; RA, right atrium; RV, right
S4 Fig. Normal fate of epicardium-derived cells in Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y
mice. Epicardial cells stained for the lineage label GFP enter the myocardium of Tbx18cre/+;
R26mTmG/+;HprtCAG::TBX2/y hearts as in Tbx18cre/+;R26mTmG/+;Hprt+/+ controls and contribute
to coronary SMCs as indicated by double-immunofluorescence staining against the SMC
marker proteins ACTA2 or TAGLN and GFP. Besides, intermyocardial epicardium-derived
cells express TAGLN but not ACTA2 in Tbx18cre/+;R26mTmG/+;HprtCAG::TBX2/y hearts which
was not observed in control hearts at E18.5 (red arrowheads). As in control hearts,
epicardium-derived cells of mutant hearts contribute partially to cardiac fibroblasts as indicated by
double staining for the epicardial lineage marker GFP and the fibroblast marker protein
POSTN. The arrows point towards interstitial epicardium-derived fibroblasts whereas the
arrowheads mark coronary fibroblasts derived from the epicardium. The number of analyzed
specimen is two and the error bar represents 40 μm. CAr, coronary artery.
S1 Table. RT-PCR primer and conditions. The listed RT-PCR primer and conditions were
used for qualitative RT-PCR experiments.
We thank Dietmar Vestweber for the EMCN antiserum.
Conceived and designed the experiments: FG CR AK. Performed the experiments: FG CR.
Analyzed the data: FG CR AK. Contributed reagents/materials/analysis tools: HF VC. Wrote
the paper: FG CR HF VC AK.
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5. Mikawa T, Gourdie RG. Pericardial mesoderm generates a population of coronary smooth muscle cells
migrating into the heart along with ingrowth of the epicardial organ. Dev Biol. 1996; 174(2):221–32.
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