Lack of Genetic Interaction between <i>Tbx18</i> and <i>Tbx2/Tbx20</i> in Mouse Epicardial Development

<div><p>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 <i>Tbx18</i> is specifically expressed in the epicardium of vertebrate embryos. Loss of <i>Tbx18</i> 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 <i>Tbx18</i> with other repressors of the T-box gene family. Here, we show that <i>Tbx2</i> and <i>Tbx20</i> are co-expressed with <i>Tbx18</i> at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of <i>Tbx2</i> nor the combined loss of <i>Tbx2</i> and <i>Tbx18</i> affects epicardial development. Similarly, we observed that the heterozygous loss of <i>Tbx20</i> with and without additional loss of <i>Tbx18</i> does not impact on epicardial integrity and mobilization in mouse embryos. Thus, <i>Tbx18</i> does not function redundantly with <i>Tbx2</i> or <i>Tbx20</i> in epicardial development.</p></div

Phenotypic analysis of hearts with combined loss of T<i>bx18</i> and <i>Tbx2</i> in the epicardium at E18.5.

<p>(A) Histological analysis by hematoxylin and eosin staining of transverse sections of <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> hearts reveals a dilatation of the atria in comparison to <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> or <i>Tbx18</i>^<i>cre/+</i><i>;Tbx2</i>^<i>fl/fl</i> or control (<i>Tbx18</i>^<i>cre/+</i>) hearts. Atrial dilatation is occasionally seen in <i>Tbx18</i>-deficient mice as well (not shown). The ventricular compartment of <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> 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 <i>Tbx18</i>, <i>Tbx2</i> 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.</p

T-box gene expression during epicardial development.

<p>(A) Qualitative RT-PCR analysis detects expression of <i>Tbx18</i>, <i>Tbx5</i>, <i>Tbx20</i> and <i>Tbx2</i> but not of <i>Tbx3</i> and <i>Tbx1</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156787#pone.0156787.s005" target="_blank">S1 Table</a>). (B) <i>In situ</i> hybridization analysis of <i>Tbx18</i>, <i>Tbx5</i>, <i>Tbx20</i>, <i>Tbx2</i>, <i>Tbx3</i> and <i>Tbx1</i> 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 <i>Tbx18</i>, <i>Tbx5</i>, <i>Tbx20</i> and <i>Tbx2</i>, asterisks point to known expression domains of <i>Tbx5</i> and <i>Tbx20</i> in the atrium, and of <i>Tbx2</i> and <i>Tbx3</i> in the liver primordium at E9.5. Black arrowheads indicate coronary artery expression of <i>Tbx2</i> at E14.5. Scale bars are 50 μm. CA, common atrium; CAr, coronary artery; Epi, epicardium; PE, proepicardium; RV, right ventricle; SV, sinus venosus.</p

Phenotypic analysis of E14.5 hearts with combined loss of <i>Tbx18</i> and one <i>Tbx20</i> allele.

<p>(A, B) Histological analysis by hematoxylin and eosin staining of transverse sections of E14.5 hearts shows that the reduction of the <i>Tbx20</i> gene dosage in a <i>Tbx18</i>-mutant background does not affect epicardial and myocardial integrity. Pericardial defects are observed in <i>Tbx18</i>^{<i>cre/cre</i>}<i>;Tbx20</i>^{<i>LacZ/+</i>} as well as in <i>Tbx18</i>^{<i>cre/LacZ</i>} 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 <i>in situ</i> hybridization against <i>Aldh1a2</i> confirms epicardial integrity. The scale bars are 50 μm. (D) Epicardial cells, immunologically stained for the <i>Tbx18</i>-lineage label GFP (from an introduced <i>Rosa26</i>^<i>mTmG</i> 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 <i>Tbx18</i>^{<i>cre/cre</i>}<i>;Tbx20</i>^{<i>LacZ/+</i>} mice and reaches the right ventricular apex similar to <i>Tbx18</i>^{<i>cre/LacZ</i>} 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 <i>Tbx18</i>^<i>cre/+</i> controls and an increased number of NOTCH3-positive epicardial cells in <i>Tbx18</i>^{<i>cre/LacZ</i>} and <i>Tbx18</i>^{<i>cre/cre</i>}<i>;Tbx20</i>^{<i>LacZ/+</i>} mutants whereas none are observed in the depicted sections of <i>Tbx20</i>^{<i>LacZ/+</i>} or <i>Tbx18</i>^<i>cre/+</i><i>;Tbx20</i>^{<i>LacZ/+</i>} 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.</p

Phenotypic analysis of E18.5 hearts with epicardial misexpression of human <i>TBX2</i>.

<p>(A) Histological analysis (n = 2) by hematoxylin and eosin staining of transverse sections of <i>Tbx18</i>^<i>cre/+</i><i>;Hprt</i>^{<i>CAG</i>::<i>TBX2/y</i>} hearts reveals pericardial defects (black arrow) but no further anomalies compared to the control (<i>Tbx18</i>^<i>cre/+</i>). Scale bars are 500 μm. (B-D) Immunofluorescence analysis (n = 4) of indicated proteins on transverse sections of <i>Tbx18</i>^<i>cre/+</i> (control) and <i>Tbx18</i>^<i>cre/+</i><i>;Hprt</i>^{<i>CAG</i>::<i>TBX2/y</i>} 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 <i>Tbx18</i>^<i>cre/+</i><i>;Hprt</i>^{<i>CAG</i>::<i>TBX2/y</i>} 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 <i>Tbx18</i>^<i>cre/+</i><i>;Hprt</i>^{<i>CAG</i>::<i>TBX2/y</i>} 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 <i>Tbx18</i>^<i>cre/+</i><i>;Hprt</i>^{<i>CAG</i>::<i>TBX2/y</i>} 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.</p

Phenotypic analysis of hearts with combined loss of <i>Tbx18</i> and <i>Tbx2</i> in the epicardium at E14.5.

<p>(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 <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> double mutant hearts compared to control (<i>Tbx18</i>^<i>cre/+</i>) or compound mutant (<i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> or <i>Tbx18</i>^<i>cre/+</i><i>;Tbx2</i>^<i>fl/fl</i>) embryos. The scale bars are 500 μm. The black arrows point to pericardial defects observed in <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> and <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> 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 <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> and <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> mice and occasionally in <i>Tbx18</i>^<i>cre/+</i><i>;Tbx2</i>^<i>fl/fl</i> and control hearts (white arrowheads). Double staining with the <i>Tbx18</i>-lineage marker GFP indicates NOTCH3-positive cells in the epicardium of <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> and <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> 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.</p

Decreased proliferation and increased apoptosis contribute to hypoplasia of <i>Tbx2</i>-deficient lungs.

<p>(A,A′,D,D′,G,G′) Analysis of apoptosis by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and (B,B′,E,E′,H,H′) of proliferation by anti-BrdU immunohistochemistry. (C,F,I) Statistical analysis of the BrdU labeling index for the lung mesenchyme, the proximal and distal epithelium, and the diaphragm at different developmental stages. Genotypes and stages are as indicated. Scale bars represent 100 µm in A,A′,D,D′,G,G′, and 50 µm in B,B′,E,E′,H,H′. For statistics see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003189#pgen.1003189.s012" target="_blank">Table S1B</a>.</p

Precocious differentiation in the <i>Tbx2</i>-deficient lung mesenchyme.

<p>Immunohistochemistry for the endothelial marker Emcn and immunofluorescence analysis for smooth muscle cells (Tagln), immature fibroblasts (S100a4) and fibrocyte-secreted extracellular matrix proteins (Fn1, Postn) on frontal sections of wildtype (wt) and <i>Tbx2</i>-deficient (<i>Tbx2^−/−</i>) lungs at E14.5, E16.5 and E18.5 as indicated. S100a4 is lost in <i>Tbx2</i>-deficient lungs whereas deposition of Fn1 and Postn is greatly increased. Scale bars represent 100 µm.</p

Tbx2 Controls Lung Growth by Direct Repression of the Cell Cycle Inhibitor Genes <em>Cdkn1a</em> and <em>Cdkn1b</em>

<div><p>Vertebrate organ development relies on the precise spatiotemporal orchestration of proliferation rates and differentiation patterns in adjacent tissue compartments. The underlying integration of patterning and cell cycle control during organogenesis is insufficiently understood. Here, we have investigated the function of the patterning T-box transcription factor gene <em>Tbx2</em> in lung development. We show that lungs of <em>Tbx2</em>-deficient mice are markedly hypoplastic and exhibit reduced branching morphogenesis. Mesenchymal proliferation was severely decreased, while mesenchymal differentiation into fibrocytes was prematurely induced. In the epithelial compartment, proliferation was reduced and differentiation of alveolar epithelial cells type 1 was compromised. Prior to the observed cellular changes, canonical Wnt signaling was downregulated, and <em>Cdkn1a</em> (<em>p21</em>) and <em>Cdkn1b</em> (<em>p27</em>) (two members of the Cip/Kip family of cell cycle inhibitors) were strongly induced in the <em>Tbx2</em>-deficient lung mesenchyme. Deletion of both <em>Cdkn1a</em> and <em>Cdkn1b</em> rescued, to a large degree, the growth deficits of <em>Tbx2</em>-deficient lungs. Prolongation of <em>Tbx2</em> expression into adulthood led to hyperproliferation and maintenance of mesenchymal progenitor cells, with branching morphogenesis remaining unaffected. Expression of Cdkn1a and Cdkn1b was ablated from the lung mesenchyme in this gain-of-function setting. We further show by ChIP experiments that Tbx2 directly binds to <em>Cdkn1a</em> and <em>Cdkn1b</em> loci <em>in vivo</em>, defining these two genes as direct targets of Tbx2 repressive activity in the lung mesenchyme. We conclude that Tbx2-mediated regulation of <em>Cdkn1a</em> and <em>Cdkn1b</em> represents a crucial node in the network integrating patterning information and cell cycle regulation that underlies growth, differentiation, and branching morphogenesis of this organ.</p> </div

<i>Upk3b</i> expression in adult tissues.

<p><i>In situ</i> hybridization analysis of <i>Upk3b</i> expression on sections of the adult heart (A, D), the urinary bladder (B, E) and the kidney (C, F). (A–C) Overview images of whole organ sections, (D–F) higher magnification images of the regions marked by open rectangles (in A–C). The arrow points to the epicardium. bl, urinary bladder; cv, coronary vessel; la, left atrium; lu, urinary bladder lumen; lv, left ventricle; m, urinary bladder mesothelium; p, renal pelvis; pa, renal papilla; ra, right atrium; rv, right ventricle; sm, smooth muscle layer, u, urothelium.</p