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.
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). Co-staining 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.
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.
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.
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.
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 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.