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. Author manuscript; available in PMC: 2014 Nov 15.
Published in final edited form as: Dev Biol. 2013 Sep 7;383(2):307–320. doi: 10.1016/j.ydbio.2013.08.019

Tbx18 Regulates Development of the Epicardium and Coronary Vessels

San-Pin Wu 1,6, Xiu-Rong Dong 2, Jenna N Regan 3, Chang Su 4, Mark W Majesky 2,5,6
PMCID: PMC4172450  NIHMSID: NIHMS523563  PMID: 24016759

Abstract

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18-/- mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22αlacZ/+ activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18-/- hearts at E12.5 reveal altered expression in 79 genes that are associated with development of vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of repressing progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.

Keywords: cardiac development, epicardium, coronary circulation, hedgehog signaling, T-box18

Introduction

Lineage mapping and genetic analysis studies have shown that proepicardial (PE) cells are progenitors for the epicardium, coronary smooth muscle cells (CoSMCs), perivascular and intermyocardial fibroblasts, and a subpopulation of cardiac myocytes (Mikawa and Fishman, 1992, Gittenberger-de Groot et al., 1998). In the mouse, the PE first appears around E8.5 on the surface of septum transversum mesenchyme near the sinoatrial junction (Viragh and Challice, 1981). PE cells reach the heart between E9.5 and E10.5, attach to the myocardium and migrate over it to produce a continuous sheet of mesothelial cells that forms the epicardium (Manner, 1992, Sengbusch et al., 2002). Coronary vessel development begins when epicardial cells undergo an epithelial to mesenchymal transition (EMT), followed by vasculogenesis in the subepicardial space, formation and remodeling of a capillary-like vascular plexus, and eventual recruitment of CoSMCs (Majesky, 2004, Tomanek, 2005). Development of a subepicardial coronary plexus occurs between E11.5 and E13.5 in the mouse, and is followed by formation of left and right coronary stems in the aortic valve sinuses. Unlike the systemic vasculature, which develops gradually along with formation of the heart, when the coronary plexus makes contact with the aortic lumen at E13.5, it establishes continuity with an already functional circulation. Therefore, formation of an optimal network of coronary arteries, veins and capillaries to ensure efficient perfusion in mature hearts requires a preformed vascular plexus in the subepicardium that is already well organized to receive blood flow from the aorta at E13.5.

T-box18 (Tbx18) is a member of the T-box transcription factor family (Kraus et al., 2001, Naiche et al., 2005). Loss of function studies show that Tbx1, Tbx2, Tbx3, Tbx5, and Tbx20 play critical roles in cardiac development (Greulich et al., 2011). Expression of Tbx18 is first detected in presomitic mesoderm, and cranial paraxial mesoderm in E7.75 mouse embryos (Kraus et al., 2001, Bussen et al., 2004). Later, Tbx18 is strongly expressed in the PE, epicardium, epicardial-derived cells (EPDC) in subepicardial mesenchyme, septum transversum, somites, limb buds, aorta-gonad-mesonephros region and a subset of myocardium (Bussen et al., 2004, Airik et al., 2006, Christoffels et al., 2009, Christoffels et al., 2006). Analysis of Tbx18-expressing cell fates in the mouse heart showed that epicardial cells, CoSMCs, atrioventricular valve interstitial cells, and a subpopulation of cardiac myocytes derive from Tbx18-positive progenitor cells (Cai et al., 2008, Christoffels et al., 2009, Mommersteeg et al., 2010). Loss of Tbx18 function causes perinatal lethality with defects in skeletal, urogenital systems and pleuropericardial membranes, and with concomitant disruptions of Notch, Wnt and Hedgehog signaling pathways (Bussen et al., 2004, Airik et al., 2006, Norden et al., 2012). In the developing heart, Tbx18 deficiency results in dysmorphogenesis of the sinus node (Wiese et al., 2009), while forced expression of Tbx18 reprograms ventricular myocytes to pacemaker cells in rodent (Kapoor et al., 2012). Because Tbx18 is highly expressed in the PE and epicardium, we sought to determine its role in coronary vessel formation during heart development. We found that Tbx18-deficient PE produces an epicardium and coronary vasculature with structural and functional defects, and that remodeling of the disorganized subepicardial plexus in Tbx18-deficient hearts produced a mature coronary artery network with fewer distributing conduit vessels and smaller lumen profiles than that of wild type hearts.

Materials and Methods

Generation of Tbx18 mutant mice

The Tbx18tm1mwm allele was generated by replacing exon 1 sequences encoding the predicted translation initiation site and the N-terminal domain of the protein, and exon 2 sequences encoding the amino-terminal residues of the highly conserved T-box, with an IRES-nlacZ reporter as depicted in Fig. S1. All mice were maintained in a 129/C57BL6/J mixed background.

Histological analysis and immunofluorescence Staining

Histological analysis was performed by using standard procedures (Landerholm et al., 1999).

Skeletal preparation

The skeletal preparation procedure was adopted from McLeod (McLeod, 1980).

Whole mount b-galactosidase activity

Embryos or tissues were whole mount-stained for b-galactosidase activity as previously described (Passman et al., 2008).

Scanning electron microscopy (SEM)

Freshly harvested hearts were fixed in 2% PFA/2.5% glutaraldehyde in 0.15M Karlsson and Schultz solution at 4°C overnight, postfixed in 1% buffered osmium tetroxide, dehydrated, critical point dried, sputter coated with a 60/40 Au/Pd alloy and examined on a Cambridge S-200 scanning electron microscope as described (Dong et al., 2008).

Whole mount immunofluorescence and confocal microscopy

Dissected hearts were placed in ice-cold PBS, fixed and permeabilized in methanol/DMSO (4:1) overnight at 4°C, bleached in methanol/DMSO/H2O2 (4:1:1) for 4 hours at room temperature, and preserved in methanol at −20°C. Tissues were prepared, stained, and imaged by confocal microscopy as previously described (Passman et al., 2008, Dong et al., 2008).

Cell proliferation and apoptosis assays

Cell proliferation was assessed in whole mount or tissue sections using anti-phosphohistone-H3 antibody. Apoptosis was determined by TUNEL assay using the ApopTag Fluoroscein in situ Apoptosis Detection kit (Chemicon, Inc) according to the manufacturer's instructions.

Coronary vascular casts

Vascular casts were prepared as described (Adamson et al., 2002). Coronary casts were mounted on an SEM stub, with the apex of the ventricles oriented upward, and visualized by scanning electron microscopy (SEM).

Microarray analysis

Generation of labeled antisense RNA for microarray: Total RNA was extracted from four pooled Tbx18-null hearts and three pooled wild type E12.5 hearts (ToTally RNA kit, Ambion, Inc.), and underwent one round of linear amplification (Amino Allyl MessageAmpII kit, Ambion, Inc.). The paired antisense RNA samples were hybridized to glass microarrays containing the Operon Mouse Aros 4.0 Oligo library (oligomers spotted by the Microarray Core Facility, Department of Systems Biology & Translational Medicine, Texas A&M University). After imaging, the acquired data were transformed, normalized and filtered using the GeneSpring v7.0 software package (Silicon Genetics, Agilent Technologies). Gene expression significance was assessed using multiple t-tests (p<0.05) and the Benjamini-Hochberg false discovery rate multiple testing correction. Molecular function and pathway analysis was performed via Ingenuity Pathway Analysis.

Real-time quantitative RT-PCR analysis

cDNA was generated by M-MLV Reverse Transcriptase (Invitrogen) followed by Taqman real time PCR analysis on an ABI-7700 thermal cycler according to the manufacturer's instructions. Data were analyzed by the Δ ΔCT method.

Transient Transfection and Reporter Assays

COS7 and C3H10T1/2 cells were cultured in 24-well tissue culture plates at a starting density or 5 × 104 cells/well in DMEM supplemented with 10% fetal bovine serum and transfected with FuGENE6 (Roche) per the manufacturer's instructions. For reporter assays, the amount per well of plasmid DNA was kept constant at 50ng for luciferase constructs, 50ng for SRF and 5ng for myocardin, while titering in Tbx18 (50-200ng) or complimentary amounts of empty vector DNA to reach a constant 300ng total amount of these two plasmid DNAs. Each well was co-transfected with pGL4-SV40-Renilla luciferase-reporter (Promega, with a constant ratio of Firefly luciferase-reporter: Renilla luciferase-reporter = 50:1) for normalization. Luciferase assays were performed using a commercially available substrate kit (Promega).

Materials

Antibodies used in this study include anti-PECAM-1 (BD Pharmingen, #550274, 1:100), anti-phosphohistone H3 (Upstate, 1:100), anti-Wilms' Tumor-1 (Santa Cruz, 1:100), anti-cytokeratin (Dako, 1:100), anti-SMγActin (Seven Hills Bioreagents,1:500), anti-SM22α (Abcam, 1: 500), anti-COUP-TFII antibody (R&D Systems, 1:2000) and anti-V5 (Invitrogen, 1: 800). Nuclei counterstaining was performed with SYTOX Green, TOPRO-3 or Hoechst 33258 (Invitrogen). Tie2GEP/+ reporter mice (Tg(TIE2GFP)287Sato) were obtained from The Jackson Laboratory. SM22αLacZ/+ reporter mice (SM22TMlili) were a generous gift of Dr. Li Li (Wayne State University). The Tbx18 expression vector was generated by cloning the cDNA fragments encoding full-length murine Tbx18 (accession number AF306666) into V5-tagged pcDNA3.1 expression vector (pcDNA3.1/V5-His-TOPO, Invitrogen). Expression plasmid encoding SRF.HA and myocardin cloned into pcDNA3 as well as the -447 bp mouse SM22α promoter-luciferase and the -356 bp c-fos promoter-luciferase reporters were kind gifts from Dr. Da-Zhi Wang (Boston Children's Hospital). Constructs encoding -1224, -176, -65 bp chick smooth muscle γ-Actin (SMγA) promoter cloned in front of a pGL-3 luciferase reporter gene have been previously described (Carson et al., 2000).

Results

Tbx18-deficiency is perinatal lethal

Tbx18+/- mice were viable and fertile, and exhibited no obvious defects in morphology, growth or behavior. Tbx8+/- mice had difficulty breathing and died within 24h after birth. Mendelian ratios of expected genotypes were found in embryos up to E18.5, confirming a perinatal lethal phenotype. Although the Tbx18 targeting construct used by Bussen et al (Bussen et al., 2004) deleted a shorter segment of the Tbx18 locus than our mutant allele (Fig. S1), our mice have essentially identical defects in the axial skeleton (Fig. S2) and kidney-ureter (not shown) as described by their group (Bussen et al., 2004, Airik et al., 2006). In this study, we focus on phenotypes not previously reported for Tbx18-deficient mice.

Expression of Tbx18-nlacZ during development of the epicardium

The nLacZ reporter gene in the targeted allele faithfully reflected expression of the endogenous Tbx18 gene in the developing proepicardium (PE), epicardium, septum transversum and AGM (aorta-gonad-mesonephros) region (Fig. 1A-D) (Kraus et al., 2001). The epicardial layer is uniformly positive as it covers the heart (Fig. 1C-F, S3B). Moreover, LacZ-positive cells can be detected in the subepicardial mesenchyme at E12.5 (arrowheads, Fig. 1D), but much less frequently in deeper layers of the myocardial wall. Expression of Tbx18 in the sinus horns, and caval veins has been described (Christoffels et al., 2006). We failed to detect Tbx18nlacZ/+ expression in coronary endothelium or smooth muscle (Fig 1F, S3B). Other tissues that are marked by the Tbx18nlacZ/+ reporter include limbs, mesothelium and whisker primoridium (Fig. 1C and S3A).

Figure 1. Expression of Tbx18 nLacZ.

Figure 1

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Tbx18 expression was detected by β-galactosidase activity from an nLacZ reporter gene introduced into the Tbx18 locus. (A,B) Tbx18 expression in the proepicardium (pe), septum transversum (stm), and aorta-gonad-mesonephros (agm) region in E9.5 Tbx18-/- embryos. Note that the PE has begun to invest epicardial cells onto the surface of the heart (h) (arrow). (C,D) At E12.5, expression of Tbx18 is found in the epicardial layer (h, panel C), and in the subepicardial mesenchyme (arrowheads, panel D). (E,F) Tbx18 is selectively expressed in the epicardium (epi) at E18.5. a; atrium, v; ventricle, lv; liver, cv; coronary vessel, epi; epicardium, myo; myocardium.

Analysis of coronary vasculature in Tbx18-deficient hearts at E18.5

Histological analysis of Tbx18-deficient hearts at E18.5 revealed fewer large diameter coronary vessels in the subepicardium and ventricular septum compared to wild type hearts (Fig. 2 A,B). To gain insight into the basis for this difference in coronary profiles, we bred the Tbxl8 mutant allele into two different cell type-specific reporter backgrounds. Tie2+/GFP reporter mice express green fluorescent protein (GFP) in an endothelial cell-specific pattern throughout development (Motoike et al., 2000). By confocal microscopy, no obvious differences in number or organization of microvascular endothelial cells within the ventricular walls were found in wild type and Tbx18-deficient hearts (Fig. 2 C,D). Significant differences were found, however, when Tbx18-mutant hearts were examined on an SM22α+/lacZ CoSMC reporter background (Qiu and Li, 2002). For this analysis, hearts were examined between 0-24 hours after birth so as to minimize residual background lacZ signals due to earlier expression of SM22α by immature cardiac myocytes (Li et al., 1996). Both wild type and Tbx18+/- hearts exhibited well-developed subepicardial coronary arteries invested with SM22α-expressing CoSMCs (Fig. 2E). By contrast, coronary arteries in Tbx18-/- hearts displayed reduced smooth muscle content and shorter overall smooth muscle coverage along the length of coronary arteries at birth (Fig. 2E). In addition, a less well-developed branching network of subepicardial conduit vessels in mutant hearts was also apparent in the SM22α+/lacZ background (Fig. 2E). Morphometric analysis of the left coronary artery in the SM22α+/lacZ background showed a reduced overall diameter of the coronary stems in Tbx18-null hearts (wild type: 76 ± 7 mm; Tbx18-/-: 51 ± 2 mm), and a reduced branching index (wild type: 6.5 ± 2 branch vessels/left coronary artery; Tbx18-/-: 3.7 ± 2 branch vessels/left coronary artery). To further investigate this smooth muscle phenotype, we examined endogenous levels of smooth muscle markers by immunostaining. In E18.5 hearts, levels of SM22α are comparable between control and Tbx18-/- mice in the portion of coronary stems that lies between the coronary ostia and the first branch of circumflex artery (Figures S4A and B). This result suggests that, in coronary smooth muscle cells, SM22α expression does not depend on Tbx18. Conversely, in the distal part of coronary arteries, Tbx18-/- displays reduced expression of overall SM22α in comparison to control (Fig. S4 C and D), which is consistent with our observation using the SM22α-LacZ reporter. To determine whether the reduction of smooth muscle coverage is due to differentiation defects or reduced numbers of cells, we examine the presence of differentiating smooth muscle cells by staining of COUP-TFII (You et al., 2005). Our results reveal that, in Tbx18-null mutant, the distal part of coronary arteries is covered with fewer COUP-TFII-positive smooth muscle cells in comparison with control (Figure S4 E and F). Notably, these COUP-TFII-positive smooth muscle cells still express SM22α despite of the reduction in numbers (Figure S4D), suggesting the process of smooth muscle differentiation remains functional in the Tbx18-/- background. In summary, our data suggest that deletion of Tbx18 results in fewer numbers of smooth muscle cells invested on coronary arteries, which causes reduced smooth muscle content and overall smooth muscle coverage along the length of coronary arteries.

Figure 2. Defects in coronary vessel formation in Tbx18-/- hearts.

Figure 2

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(A,B) Histology of the left ventricular free wall at E18.5 (H&E stain). Note large diameter subepicardial coronary vessels are evident in wild type, but not in Tbx18-/- mutant hearts. Also note that the ventricular wall of Tbx18-/- hearts is not abnormally thin. (C,D) The Tbx18tm1mwm allele was crossed into a Tie2-GFP reporter background. Capillary vessel density in Tbx18-/- hearts at E18.5 was not obviously different from wild type. (E) The structure of major coronary conduit arteries was examined by crossing the Tbx18tm/mwm allele into an SM22αLacZ/+ reporter background. Three representative hearts (arranged vertically) from P0 pups of the indicated genotype were stained for b-galactosidase activity and photographed to show the left coronary artery (LCA) and its major branches. Significant reductions in overall length and branching complexity of the LCA were found in Tbx18-/- hearts.

Appearance of the epicardium at E12.5

To gain insight into the developmental basis for the altered coronary network structure in Tbx18-/- hearts described above, we first examined the epicardial layer at E12.5, a time point at which coronary vessels are beginning to form in the subepicardium (Tomanek, 2005). Using low angle illumination, the epicardium of mutant hearts was seen to form a complete, but irregular covering of the heart with frequent areas that were distended from the myocardial layer (Fig. 3B) when compared to wild type hearts (Fig. 3A). Histological analysis confirmed the impression of a continuous, but irregular epicardial surface (Fig. 3 C,D), and also revealed that the subepicardium of Tbx18-/- hearts contained scattered clusters of densely packed nucleated erythroid cells covered by a continuous epicardial layer (Fig. 3G, arrow). Comparable structures were only rarely encountered in wild type hearts (Fig. 3E). Despite occasional mutant hearts in which cystic structures containing erythroid cell pools were found (Fig. 4B, inset), we did not observe pericardial hemorrhage in Tbx18-null embryos. When viewed at the level of the epicardial surface, the spherical clusters caused mound-like protrusions of the epicardium (Fig. 3H). PECAM-1 (CD31) is a cell surface protein expressed by platelets, endothelial cells, and erythroid progenitors (Baldwin et al., 1994, Ema et al., 2006). Quantitation of the number of PECAM-1-positive spherical clusters in different regions of wild type and Tbx18-deficient hearts is shown in Fig. 3I. Large increases in the number of these clusters were confined to the left and right ventricular free walls of mutant hearts (Fig. 3I).

Figure 3. Defects in epicardium at E12.5 in Tbx18-/- hearts.

Figure 3

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(A,B) Gross view of epicardium at E12.5. A translucent epicardial layer (yellow arrowheads) forms a uniform covering in wild type hearts, and a more irregular and blistered surface in Tbx18-/- hearts. (C,D) H&E staining of cross-sections of ventricular free wall at E12.5 shows a smooth surfaced epicardium (red dotted line), and a subepicardial space of uniform thickness in wild type hearts, and an irregular epicardium with a subepicardial space of variable thickness in Tbx18-/- hearts. (E,F) Low magnification images of PECAM-1 whole mount stains reveal numerous ectopic nodules (arrows, panel F) of PECAM-1-positive cells (PECAM1pos, red) in Tbx18-/- ventricles (E12.5) (nuclei stained green). A small number of these PECAM-1pos nodules are normally found in the atrioventricular sulcus of wild type hearts (arrow, panel E). (G) H&E stained section of E12.5 heart showing that subepicardial nodules contain erythroblasts (arrow) or mesenchymal cells (arrowhead) in Tbx18-/- heart. (H) High power view of (F) showing PECAM-1pos clusters (red) in the subepicardium (arrow) (nuclei, green). (I) PECAM-1pos nodules were counted from different regions of wild type (WT, blue cylinders, n=4) and Tbx18-deficient (KO, red cylinders, n=5) hearts at E12.5. vLV and dLV; ventral and dorsal surface of left ventricle, vRV and dRV; ventral and dorsal surface of right ventricle, vIVS and dIVS; ventral and dorsal surface of interventricular sulcus.

Figure 4. Epicardial surface of Tbx18-/- hearts.

Figure 4

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(A-D) Scanning electron microscopy (ScEM) of E12.5 hearts. (A,B) Anterior view of hearts with apex of ventricles positioned upward. In contrast to the smooth appearance of epicardium covering wild type hearts (A), numerous mound-like protrusions and occasional cyst-like structures (collapsed in B) were found in mutant epicardium (B). Inset in (B) shows a blood-filled cyst on right ventricle prior to specimen preparation for ScEM. (C,D) High power view of left ventricle of (A) and (B) respectively. (E,F) Analysis of cell proliferation (E), and cell death (F) in epicardium that covers ventricular free walls (see panels A,B). Mean values ± standard deviations are shown for wild type (blue triangles) and Tbx18-deficient (red circles) epicardial cells. Asterisks indicate significant differences in wild type versus mutant epicardium (p<0.05). (G-J) Stacked Z-plane confocal images of E12.5 hearts stained for PECAM-1 (red) with apex of heart oriented toward top left, and atrioventricular canal at lower right. (G) A representative wild type heart showing a normal subepicardial vascular plexus covering ∼90% of the left ventricle. Dotted line outlines the ventricle, dashed line marks the extent of capillary plexus coverage (arrow indicates blood island-like cluster). (H-J) Tbx18-deficient hearts fail to develop a normal coronary plexus. Note that newly formed vessels are disorganized with fewer interconnections and more variable diameters in mutant hearts. The extent of myocardial coverage (dashed lines) is much less than in wild type hearts, and many more PECAM-1pos nodules (arrows) are found scattered over the ventricular surface. Dense collections of unorganized PECAM-1pos cells (panel J, asterisk) were also occasionally found in Tbx18-/- hearts.

The irregular appearance of the epicardial surface of Tbx18-/- hearts is more apparent by scanning electron microscopy (Fig. 4A-D). The epicardium covering the ventricular free walls of Tbx18-deficient hearts at E12.5 is composed of numerous mound-like structures and occasional cyst-like protrusions (collapsed in preparation for SEM) (Fig. 4B), whereas epicardium of wild type hearts is flattened and more uniform (Fig. 4A). Higher magnification images show an intact epicardium covering the mound-like structures in which the continuity of cell-cell junctions between individual epicardial cells is maintained (Fig. 4C,D). The irregular-appearing epicardium covering the ventricular free walls of Tbx18-deficient hearts exhibited reduced rates of cell proliferation and increased rates of apoptosis when compared to equivalent areas of epicardium in wild-type hearts (Fig. 4E,F). By contrast, morphologically normal appearing epicardium covering the inflow tract, atrium and atrioventricular canal of Tbx18-/- hearts exhibited rates of cell proliferation and cell death that were not significantly different from equivalent areas of wild type epicardium (not shown).

Formation of a subepicardial coronary vascular plexus at E12.5

We used PECAM-1 whole mount immunostaining and confocal microscopy to reconstruct the subepicardial coronary plexus (Fig. 4G-J) by stacking a Z-series of confocal images from the atrioventricular canal (lower right) to the apex of the left ventricle (dotted line, upper left). At E12.5, coronary vasculogenesis in wild type hearts is nearly complete, but the coronary stems have not yet formed connections to the aorta and there is no coronary blood flow. In wild type hearts, the subepicardial coronary plexus covered 80% to 95% of the ventricular surface area with a regular array of interconnected capillary-like vessels of uniform diameter (Fig. 4G). In Tbx18-mutant hearts, the ventricular surface area coverage (50% to 75%) was reduced (dashed line), and an irregular network of variably sized tubular structures that made fewer connections within the network was present (Fig. 4H-J). In addition, occasional nodular clusters of PECAM1-positive cells seen in the wild-type hearts (Fig. 3E and Fig. 4G, arrows) were greatly increased in number and scattered ectopically throughout the subepicardium in Tbx18-/- hearts (Fig. 3F and Fig. 4G-J, arrows). In some cases, loose aggregates of PECAM-1-positive cells that had not incorporated into the tubular network were found among the tightly packed nodular clusters (Fig. 4J, asterisk).

Coronary plexus remodeling after initiation of blood flow at E13.5

A coronary vascular plexus that forms around the aortic root connects with the aortic lumen and establishes directional blood flow through the coronary vasculature at E13.5 in the mouse (Tomanek, 2005). At E14.5, one day after establishment of coronary blood flow, two main coronary stems had formed in the correct locations in both wild type and Tbx18-deficient hearts (Fig. 5A,B). In addition, vascular channels had begun to form in wild-type hearts in positions where the left coronary artery would later be found in mature hearts (Fig. 5C, dashed lines). By contrast, it was difficult to identify early coronary channels in the disorganized vascular plexus that forms in Tbx18-/- hearts at E14.5 (Fig. 5D). Differences in coronary vessel remodeling between wild type and Tbx18-/- hearts continued to be evident in PECAM-1-stained hearts at E16.5 (Fig. 5E,F).

Figure 5. Coronary plexus remodeling dejects in Tbx18-/- hearts.

Figure 5

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(A,B) Stacked Z-plane confocal images of PECAM-1 (red) whole mount staining of E14.5 hearts. Two coronary stems (dashed yellow lines) form normally in Tbx18-/- hearts (B) compared to wild type hearts (A). (C,D) Once coronary blood flow is established at E13.5, vascular remodeling occurs. Continuous, branching channels for blood flow can be identified in the subepicardium of E14.5 wild type hearts (C, yellow dashed lines), whereas similar channels are difficult to find in Tbx18-/- hearts (D). Ventral views of E16.5 (E,F) hearts showing significant alterations in the pattern, and reductions in length and diameter (arrow) of the left descending coronary artery in mutant hearts. Note persistence of PECAM-1pos nodules in mutant hearts.

Analysis of artery-vein identity

To address the possibility that defects in coronary plexus remodeling might result from a loss of arterial-venous identity of coronary endothelium in Tbx18-/- hearts, we examined the expression of COUP-TFII, a key transcriptional regulator of venous identity (You et al., 2005, Chen et al., 2012). When hearts from either wild type or Tbx18+/- embryos at E18.5 were examined for COUP-TFII expression, we found clear differences between arterial (negative) and venous (positive) endothelium consistent with previous reports (You et al., 2005, Van den Akker et al., 2008) (Fig. S5A,B). Upon examination of multiple Tbx18-/- hearts, we found no evidence for misexpression of COUP-TFII in coronary arterial endothelial cells (Fig. S5C-F). We conclude that arterial-venous specification of coronary endothelial cells is not disrupted in Tbx18-deficient hearts.

Analysis of coronary vascular casts by scanning electron microscopy

To directly examine the structure of the coronary vascular network we employed vascular casting on E18.5 embryos. In Tbx18+/+ hearts, the left coronary artery gives off first order branch vessels of more or less uniform diameter at regular intervals that extend long distances along the surface of the heart to efficiently distribute blood flow to all regions of the left ventricular myocardium (Fig. 6A). The casts of these first order-distributing arteries describe a vascular pattern that matches well with the histological cross-sectional profiles of subepicardial vessels (Fig. 2A). In Tbx18-/- hearts, fewer first order vessels are found branching off the main left coronary artery (Fig. 6B-D). Also, the lumen diameter of first order branches in mutant hearts is significantly reduced (Fig. 6E,F). When normalized to the diameter of the left coronary artery directly upstream of the branch point, the lumen diameter ratios of first order distributing arteries are 0.51±0.18 for wild type (n=8 hearts, 74 branch points), and 0.39±0.21 for Tbx18-/- hearts (n=6 hearts, 58 branch points) (p=5.89×10-4) (Fig. 6G). Moreover, the overall length of first order distributing arteries before encountering secondary branches is also reduced in mutant hearts, resulting in fewer large diameter subepicardial distributing vessels appearing in histological sections (Fig. 2B). The average calculated maximal flow rates using Poiseuille's law for the Tbx18-/- left descending coronary artery and its first order branch arteries is only 34% of that for wild type coronary vessels at E18.5. These results confirm and extend the observations of coronary network defects obtained when the hearts of Tbx18 mutant mice were examined histologically (Fig. 2A,B), or in an SM22α+/lacZ reporter background (Fig. 2E).

Figure 6. Coronary vascular casts at E18.5 examined by scanning electron microscopy.

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(A) Wild type heart showing the left coronary artery (LCA) and its first order distributing branch arteries (L1, L2, etc). (B-D) Coronary casts of Tbx18-/- hearts showing a range of deviations from the wild type pattern, particularly in the number and size of branch conduit arteries. (E,F) Coronary casts showing lumen diameters of the LCA (m, m') and first order branch arteries (b, b') from wild type (E) and Tbx18-/- (F) hearts. (G) Morphometric analysis of lumen diameter ratios (b/m) for first order branch arteries from SEM images of coronary casts. Note the significant reductions in lumen diameter ratios in Tbx18-/- hearts (n=6, total branch points analyzed = 69), compared to Tbx18+/+ hearts (n=8, total branch points analyzed=84).

Gene expression profiling in Tbx18-/- hearts

To gain insight into the molecular basis for the coronary developmental defects Tbx18-/- hearts at E12.5, we carried out genome-wide gene expression profiling on whole hearts by microarray analysis. Tbx18 deficiency altered expression of 774 genes with changes greater than 1.5 folds (Table S1). Among them, Ingenuity Pathway Analysis identified 71 genes in association with development of vascular system (Fig. 7A and Table S2). Compared with wild type, Tbx18-/- hearts exhibited reduced expression of multiple genes that have been shown promoting or are essential for endothelial sprouting, tubular branching and capillary formation, including Vegfa (VEGF-A) (Gerhardt et al., 2003), Lama4 (Gonzales et al., 2001), Angptl2 (Kim et al., 1999), Nos3 (Lee et al., 2003), Ctbp2 (Roukens et al., 2010) and Meis1 (Hisa et al., 2004). Differential gene expression profiles were further independently investigated by quantitative real-time RT-PCR (qRT-PCR) using separately prepared pools of E12.5 heart RNA, distinct from those used for the microarray study (Table S3). Analyses from microarray and qRT-PCR together demonstrated that loss of Tbx18 expression in mutant hearts resulted in significantly reduced levels of Vegfa, Angpt1 (Angiopoietin-1), and endoglin transcripts, and significantly increased levels of Vegfd (VEGF-D) and Angpt2 (Angiopoietin-2) gene expression (Fig. 7A and Table S2 and S3). Expression levels of Vegfb (VEGF-B) and Vegfc (VEGF-C) were not significantly different between genotypes. Strong down regulation of critical hedgehog signaling components Ptch1 (patched-1), Smo (smoothened), and gli2 was also found in Tbx18-/- hearts (Table S3 and Fig. 7A). In addition, an expression profile consistent with down regulation of Wnt signaling (reduced levels of Wnt4, Wnt5a, Ccnd1, Ppp2r1b and Cskn1e and increased expression of the soluble inhibitor Sfrp2), and altered TGF-β signaling was also observed (decreased levels of Tgfb2, Tab1, Inhba and Kras) (Table S1, S2 and S3). Taken together, the gene expression profile of Tbx18-/- hearts suggests that Tbx18 deficiency produces an altered molecular microenvironment in which disrupted Hedgehog, VEGF, Angiopoietins, TGF-β and Wnt signaling may cause failure of forming a well developed subepicardial coronary vascular plexus (Fig. 7B,C).

Figure 7. Altered molecular profile of Tbx18-/- hearts.

Figure 7

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(A) Fold changes of genes associated with vascular development in E12.5 mutant hearts compared with control. Color codes representing change of mRNA levels relative to control are listed at the bottom of the panel. Actual numbers can be found in Table S2 and S3. (B and C) A model for roles of Tbx18 in coronary vasculogenesis. Bottom panels: a model whereby loss of Tbx18 expression in the epicardium produces a defective coronary plexus. Disruption of normal epicardial-myocardial signaling interactions (B) in Tbx18-/- hearts (C) leads to reduced levels of hedgehog signaling in the myocardium and lower expression levels of hedgehog target genes including ptch1, Vegfa, and Angpt1. In addition, Wnt and TGF-b pathways are also responsive to loss of Tbx18 function in the heart. The combined result is failure to form a well-ordered, highly branched coronary vascular plexus at E12.5 (D and E). Remodeling of the defective plexus in mutant hearts leads to a mature network of distributing coronary arteries that are fewer in number, shorter in length and smaller in lumen diameter (D and E) with a reduced capacity for coronary blood flow distribution (E18.5 LCA).

The SRF-CArG box dependent repressor activity of Tbx18

Tbx18 possesses strong transcriptional repressor activity via an engrailed homology-1 motif near the N-terminus that interacts with Groucho family corepressor proteins (Farin et al., 2007). To test the possibility that Tbx18 might play a role in maintenance of a coronary smooth muscle progenitor phenotype in EPDC via its intrinsic transcriptional repressor activity, we transfected COS7 cells with smooth muscle promoter-luciferase reporter constructs in the presence and absence of increasing amounts of Tbx18 expression vector. We found that Tbx18 inhibits the activity of smooth muscle cell (SMC) differentiation marker promoter-luciferase reporters in an SRF-CArG box-dependent manner (Fig. 8A-D). SMC differentiation marker expression is greatly increased by interaction of the strong transcriptional coactivator myocardin with SRF (Wang et al., 2001). Tbx18 also strongly represses smooth muscle promoter activity stimulated by a combination of SRF and myocardin (Fig. S6A). Moreover, SRF-CArG box-dependent transcriptional repression by Tbx18 was also observed in two other SRF-CArG box-dependent promoters from the SM22α gene and from the c-fos gene (Fig. S6B,C).

Figure 8. Tbx18 is a potent CArG box-dependent transcriptional repressor for smooth muscle differentiation.

Figure 8

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(A-D) Transcriptional activity of CArG-box (open box) containing SMyA promoter-reporter constructs was measured in COS7 cells by luciferase assay with the indicated combination of expression plasmids encoding SRF, Tbx18, or empty vector. The results of at least three independent experiments performed in triplicate are presented. All values are normalized to cotransfected pSV40-Renilla luciferase activity. Values are presented as fold activation increases in luciferase activity ± standard error of the mean compared to the level of activity with empty expression vector alone. The promoter-reporter constructs used are shown schematically. *, p<0.05 compared with the SRF(-)/Tbx18(0) group. #, p<0.05 compared with the SRF(+)/Tbx18(0) group. (E-G) C3H10T1/2 cells were transfected with or without the V5-tagged Tbx18 expression vector (Tbx18OE and control, respectively) for 24 hours followed by TGF-β1 stimulation for 24 or 48 hours. Then cells were fixed and immunostained for V5 (green) plus SM22α (F, red) or SMγA (G, red). The percentage of SM22α+ or SMγA+ cells was quantified by scoring 3,000 cells in 10 non-overlapping fields each from 3 independent experiments (E) and shown as mean ± standard error of the mean. *, p<0.05 compared with non-transfected control.

To further test the ability of Tbx18 to inhibit SMC differentiation, we turned to a commonly used model for SMC differentiation in vitro. C3H10T1/2 cells are multipotent mesenchymal progenitor cells that will differentiate along a SMC pathway when exposed to TGFβ1 (Hirschi et al., 1998). As shown in Figures S7, addition of TGFβ1 produces significant increases in smooth muscle marker-positive cells at 24 hours that are further increased at 48 hours. However, in cultures that were transfected with Tbx18 expression vector 24 hours before addition of TGFβ1 and examined by immunofluorescence staining at 24 hours or 48 hours after exposure to TGFβ1, the appearance of SMC marker-positive cells was strongly inhibited (Figure 8E). This was not due to an increase in the rate of apoptosis of Tbx18 overexpression cells (TUNEL assay, data not shown), nor was there a significant difference found in the rate of cell proliferation (phospho-Histone H3, data not shown) of Tbx18 overexpression cells compared to cells that did not overexpress Tbx18. Moreover, we did observe expression of SMC markers in the Tbx18 overexpression cells (Fig. 8F,G). Taken together, the data described above strongly suggest that Tbx18 is a transcriptional co-repressor of SRF-CArG box-dependent SMC marker gene expression that can inhibit SMC differentiation from multipotent mesenchymal progenitor cells.

Discussion

Development of the coronary vasculature requires reciprocal signaling interactions between epicardium, myocardium and coronary vessels. We found that Tbx18 is required for proper formation and remodeling of the subepicardial coronary vascular plexus. Since we observed defects in plexus formation that precede the establishment of coronary connections to the aorta at E13.5, the early vascular defects are unlikely to be secondary to alterations in either cardiac output or general systemic circulation in Tbx18-/- embryos. Therefore, we suggest that defects in coronary vasculogenesis in Tbx18-deficient hearts are the consequence of altered epicardial cell signaling or cell fate that results in abnormal differentiation, assembly and remodeling of coronary endothelial and perivascular cells within the subepicardial space. Indeed, gene expression analysis at E12.5 showed reduced levels of VEGF-A, VEGF-B, Endoglin, and Angiopoietin-1 transcripts, increased expression of Angiopoietin-2, and strong down regulation of the hedgehog signaling markers Ptch1, Smo and Gli2. Although each of these factors has been implicated in coronary vessel development (Tomanek, 2005, Lavine et al., 2006, Ward and Dumont, 2004, Perez-Pomares and de la Pompa, 2011), the nature of an epicardial control pathway that regulates their production in the developing heart has not been identified.

Tbx18 and development of the epicardium

Tbx18 is strongly expressed in the PE and septum transversum mesenchyme (Kraus et al., 2001). Despite high levels of Tbx18 expression in PE from wild type hearts, we found no differences in the localization, growth or vesiculation of the PE in Tbx18-/- embryos compared to Tbx18+/+ littermates (not shown). Moreover, our studies of freshly isolated E9.5 PE ex vivo failed to identify significant differences in expression of epicardial markers (WT1, capsulin, a4-integrin) between wild type and Tbx18-deficient cells (not shown). Expression of collagens type I, III, and IV as well as fibronectin and tenascin C in explanted PE also did not significantly differ between Tbx18 genotypes (not shown). Formation of epicardium appeared to initiate properly in Tbx18-mutant mice and a complete epicardial covering of the heart was present at E11.5 and at E18.5 (not shown). Our results agree with the observation reported by Greulich et al. that epicardium of Tbx18-/- hearts is intact at the perinatanl stage (Greulich et al., 2012). However, we identified a previously undescribed epicardial phenotype in which the appearance and organization of Tbx18-/- epicardium showed clear differences from that of wild type at E12.5, the critical time of epicardium-dependent coronary vasculogenesis in the subepicardial space. Epicardium covering the ventricular free walls in Tbx18-/- hearts was irregular with small mound-like distensions and occasional large cyst-like structures deforming the epicardial surface. These protrusions were associated with underlying accumulations of erythroblasts clustered in structures reminiscent of blood islands. While formation of blood islands has been reported during coronary vasculogenesis (Kattan et al., 2004, Tomanek et al., 2006), they are usually few in number and confined to the atrio-ventricular canal. In Tbx18-/- hearts, ectopic blood island-like structures were greatly increased in number and distributed over ventricular free walls. An origin of hematopoietic cells from PE has been previously reported (Kattan et al., 2004). Since we were unable to detect β-galactosidase expression from our mutant allele in these blood island-like structures, their mislocalization and elevated numbers may be an indirect consequence of a defective signaling environment in the subepicardium of Tbx18-/- hearts. Given reports that the PE contains erythroid progenitors (Tomanek et al., 2006, Wilting et al., 2007), that reduction of VEGF signaling with a soluble VEGF-R1/R2 chimera (VEGF trap) produced large increases in subepicardial erythroid cells (Tomanek et al., 2006), together with an absence of coronary blood flow at E12.5, we favor the idea that ectopic blood island-like structures arise de novo from PE-derived cells, yet other possibilities cannot be excluded at this time (Ratajska et al., 2006). Interestingly, this epicardial phenotype was observed in the Tbx18-null mice of the 129/C57BL6/J background but not in that of the NMRI outbred background (Greulich et al., 2012), suggesting a currently unknown genetic modifier that might interact with Tbx18 to regulate epicardial development.

Tbx18 and development of the coronary vasculature

Coronary endothelial cells appear in the subepicardial space between E10.5 and E11.5, and are directed to assemble into a tubular plexus by growth factors, matrix proteins, hypoxia and proteases within the subepicardium (Tomanek, 2005). These vasculogenic factors are produced by both the epicardium and the bordering myocardium. Tbx18-deficient hearts produce a malformed subepicardial plexus characterized by an irregular network of tubular structures of variable sizes that made fewer connections within the plexus. Tube formation in coronary vessels is under control of angiogenic growth factors including members of the VEGF, angiopoietin, Wnt and TGFβ families (Tomanek, 2005, Ward and Dumont, 2004, Holifield et al., 2004, Perez-Pomares and de la Pompa, 2011, Zamora et al., 2007). Each of these growth factor families is misexpressed in Tbx18-/- hearts. Moreover, Vokes et al reported that vascular tube formation in avian embryos requires factors produced by the endoderm, and that a key endoderm-derived factor that promotes tube formation is sonic hedgehog (Shh) (Vokes et al., 2004). Lavine et al reported that myocardial FGF signaling triggers a wave of Shh activation in the myocardium of developing mouse hearts that is required for VEGF-A, -B, and -C expression during coronary development (Lavine et al., 2006). Our gene expression studies indicated that Shh signaling is down-regulated in Tbx18-deficient hearts, pointing to a key potential pathway by which subepicardial plexus formation may be deficient in these hearts (Fig. 7). In the later stage at E18.5, observations from Greulich et al. and us both indicate that the main coronary arteries and capillary vessels are developed in the Tbx18-/- hearts (Fig. 2 C-E and Fig. 6)(Greulich et al., 2012). Our data further demonstrated that Tbx18-/- hearts exhibited simplified branching complexity and reduced lumen diameter ratios of first order distributing arteries in the principle subepicardial distributing vessels at the perinatal stage (Fig. 6). Given that the development of coronary stems appeared unaffected in Tbx18-/- mice (Fig. 5B), it is not surprised to see that hemodynamic effects contribute to vascular remodeling after connecting to systemic circulation (Tomanek, 2005, Lucitti et al., 2007).

Tbx18 and maintaining the progenitor status of epicardial-derived cells

Expression levels of Tbx18 are rapidly downregulated following epithelial to mesenchymal transition of the epicardium to generate EPDC, a critical early step in coronary vessel development. Our data and those of Greulich et al (2012) reach a similar conclusion that Tbx18 may play an important role as a transcriptional corepressor to control the timing of EPDC differentiation during early steps in coronary vessel development. In particular, Tbx18 may prevent premature SMC differentiation in EPDC and thereby ensure timely progression of CoSMCs differentiation and their recruitment onto coronary vessels during heart development. Our in vitro transcription studies suggest that the ability of Tbx18 to inhibit premature smooth muscle differentiation may be due to its repressor activity on SRF/myocardin-CArG-dependent transcription of smooth muscle differentiation marker genes. Conversely, our model also predicts that converting the transcription-repressing Tbx18 into a transcription-activating factor should promote smooth muscle differentiation through driving the expression of smooth muscle genes. Indeed, Greulich and colleagues demonstrate premature adoption of the smooth muscle phenotype by EPDC in a Tbx18VP16 misexpression study. Results of our study are in agreement with their model and further support the notion that Tbx18 maintains the epicardial progenitor status by repressing smooth muscle gene expression.

Tbx18, a novel epicardial mutant phenotype

To date, most genes whose loss of function phenotypes produce epicardial defects exhibit a pronounced thinning of the compact layer myocardium (Sucov et al., 1994, Kwee et al., 1995, Moore et al., 1999, Merki et al., 2005, von Gise et al., 2011, Zamora et al., 2007, Acharya et al., 2012, Lin et al., 2012). These embryos die in utero from cardiac failure. On the other hand, despite defective EMT was observed in mice with ablation of Nf1 or PDGF receptors, no obvious myocardium or coronary vessel development was reported (Baek and Tallquist, 2012, Smith et al., 2011). In contrast, Tbx18-/- hearts exhibited defective coronary vessel patterning from E12.5 and beyond while myocardial wall thickness and heart weight to body weight ratios were not significantly different from wild type at E18.5 (not shown), and an intact epicardium was present from E11.5 to birth. A mendelian ratio of Tbx18 genotypes was found at E18.5, indicating an absence of significant embryonic lethality due to cardiac failure. Death of Tbx18-/- mice within hours after birth precludes a detailed analysis of cardiac function in Tbx18 mutants. Yet a reduction in coronary network structural capacity to conduct blood flow, as described here, may not compromise cardiac development or baseline cardiac function. Such defects may only be evident with high or sustained levels of cardiac activity or in hearts compromised by coronary artery disease.

Supplementary Material

01

Highlights.

  1. Tbx18-null mice exhibit morphological defects in epicardium and coronary vessels.

  2. Mutant epicardial cells show impaired proliferation and increased apoptosis.

  3. Mutant mice display defects in the vascular plexus remodeling process.

  4. Expression of Hedgehog and VEGF signaling components are altered in mutant hearts.

  5. Tbx18 possesses a SRF/CArG box dependent repressor activity.

Acknowledgments

We thank Adriana Gittenberger de-Groot and Rob Poelmann for critical reading of the manuscript, Sophia Tsai and Ming-Jer Tsai for help with COUP-TFII analysis, and Da-Zhi Wang and Frank Conlon for helpful discussions.

This work was supported by NIH grants HL-19242, HL-93594 and HL-07816, by American Heart Association grant AHA-0715320U, and the Seattle Children's Research Institute.

Footnotes

Disclosure: None

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References

  1. Acharya A, Baek ST, Huang G, Eskiocak B, Goetsch S, Sung CY, Banfi S, Sauer MF, Olsen GS, Duffield JS, Olson EN, Tallquist MD. The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors. Development. 2012;139:2139–49. doi: 10.1242/dev.079970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adamson S, Lu Y, Whiteley K, Holmyard D, Hemberger M, Pfarrer C, Cross J. Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta. Dev Biol. 2002;250:358–373. doi: 10.1016/s0012-1606(02)90773-6. [DOI] [PubMed] [Google Scholar]
  3. Airik R, Bussen M, Singh M, Petry M, Kispert A. Tbx18 regulates the development of the ureteral mesenchyme. J Clin Invest. 2006;116:663–674. doi: 10.1172/JCI26027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baek ST, Tallquist MD. Nf1 limits epicardial derivative expansion by regulating epithelial to mesenchymal transition and proliferation. Development. 2012;139:2040–9. doi: 10.1242/dev.074054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baldwin H, Shen H, Yan H, Delisser H, Chung A, Mickanin C, Trask T, Kirschbaum N, Newman P, Albelda S, Al E. Platelet endothelial cell adhesion molecule-1 (PECAM1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development. Development. 1994;120:2539–2553. doi: 10.1242/dev.120.9.2539. [DOI] [PubMed] [Google Scholar]
  6. Bussen M, Petry M, Schuster-Gossler K, Leitges M, Gossler A, Kispert A. The T-box transcription factor Tbx18 maintains the separation of anterior and posterior somite compartments. Genes Dev. 2004;18:1209–1221. doi: 10.1101/gad.300104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cai C, Martin J, Sun Y, Cui L, Wang L, Ouyang K, Yang L, Bu L, Liang X, Zhang X, Stallcup W, Denton C, Mcculloch A, Chen J, Evans S. A myocardial lineage derives from Tbx18 epicardial cells. Nature. 2008;454:104–108. doi: 10.1038/nature06969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carson JA, Fillmore RA, Schwartz RJ, Zimmer WE. The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor. J Biol Chem. 2000;275:39061–72. doi: 10.1074/jbc.M006532200. [DOI] [PubMed] [Google Scholar]
  9. Chen X, Qin J, Cheng CM, Tsai MJ, Tsai SY. COUP-TFII Is a Major Regulator of Cell Cycle and Notch Signaling Pathways. Mol Endocrinol. 2012;26:1268–77. doi: 10.1210/me.2011-1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Christoffels VM, Grieskamp T, Norden J, Mommersteeg MT, Rudat C, Kispert A. Tbx18 and the fate of epicardial progenitors. Nature. 2009;458:E8–9. doi: 10.1038/nature07916. discussion E9-10. [DOI] [PubMed] [Google Scholar]
  11. Christoffels VM, Mommersteeg MT, Trowe MO, Prall OW, De Gier-De Vries C, Soufan AT, Bussen M, Schuster-Gossler K, Harvey RP, Moorman AF, Kispert A. Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18. Circ Res. 2006;98:1555–63. doi: 10.1161/01.RES.0000227571.84189.65. [DOI] [PubMed] [Google Scholar]
  12. Dong X, Maguire C, Wu S, Majesky M. Development of coronary vessels. Meth Enzymol. 2008;445:209–228. doi: 10.1016/S0076-6879(08)03009-7. [DOI] [PubMed] [Google Scholar]
  13. Ema M, Yokomizo T, Wakamatsu A, Terunuma T, Yamamoto M, Takahashi S. Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin, PECAM1, Tie2, endoglin and CD34 in the mouse embryo. Blood. 2006;108:4018–4024. doi: 10.1182/blood-2006-03-012872. [DOI] [PubMed] [Google Scholar]
  14. Farin HF, Bussen M, Schmidt MK, Singh MK, Schuster-Gossler K, Kispert A. Transcriptional repression by the T-box proteins Tbx18 and Tbx15 depends on Groucho corepressors. J Biol Chem. 2007;282:25748–59. doi: 10.1074/jbc.M703724200. [DOI] [PubMed] [Google Scholar]
  15. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 2003;161:1163–77. doi: 10.1083/jcb.200302047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gittenberger-De Groot A, Vrancken Peeters M, Mentink M, Gourdie R, Poelmann R. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res. 1998;82:1043–1052. doi: 10.1161/01.res.82.10.1043. [DOI] [PubMed] [Google Scholar]
  17. Gonzales M, Weksler B, Tsuruta D, Goldman RD, Yoon KJ, Hopkinson SB, Flitney FW, Jones JC. Structure and function of a vimentin-associated matrix adhesion in endothelial cells. Mol Biol Cell. 2001;12:85–100. doi: 10.1091/mbc.12.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Greulich F, Farin HF, Schuster-Gossler K, Kispert A. Tbx18 function in epicardial development. Cardiovasc Res. 2012 doi: 10.1093/cvr/cvs277. [DOI] [PubMed] [Google Scholar]
  19. Greulich F, Rudat C, Kispert A. Mechanisms of T-box gene function in the developing heart. Cardiovasc Res. 2011;91:212–22. doi: 10.1093/cvr/cvr112. [DOI] [PubMed] [Google Scholar]
  20. Hirschi KK, Rohovsky SA, D'AMORE PA. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol. 1998;141:805–14. doi: 10.1083/jcb.141.3.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hisa T, Spence SE, Rachel RA, Fujita M, Nakamura T, Ward JM, Devor-Henneman DE, Saiki Y, Kutsuna H, Tessarollo L, Jenkins NA, Copeland NG. Hematopoietic, angiogenic and eye defects in Meis1 mutant animals. EMBO J. 2004;23:450–9. doi: 10.1038/sj.emboj.7600038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Holifield J, Arlen A, Runyan R, Tomanek R. TGF-beta1, -beta2, and -beta3 cooperate to facilitate tubulogenesis in the explanted quail heart. J Vasc Res. 2004;41:491–498. doi: 10.1159/000081805. [DOI] [PubMed] [Google Scholar]
  23. Kapoor N, Liang W, Marban E, Cho HC. Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol. 2012;31:54–62. doi: 10.1038/nbt.2465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kattan J, Dettman R, Bristow J. Formation and remodeling of the coronary vascular bed in the embryonic avian heart. Dev Dyn. 2004;230:34–43. doi: 10.1002/dvdy.20022. [DOI] [PubMed] [Google Scholar]
  25. Kim I, Moon SO, Koh KN, Kim H, Uhm CS, Kwak HJ, Kim NG, Koh GY. Molecular cloning, expression, and characterization of angiopoietin-related protein. angiopoietin-related protein induces endothelial cell sprouting. J Biol Chem. 1999;274:26523–8. doi: 10.1074/jbc.274.37.26523. [DOI] [PubMed] [Google Scholar]
  26. Kraus F, Haenig B, Kispert A. Cloning and expression analysis of the mouse T-box gene Tbx18. Mech Dev. 2001;100:83–86. doi: 10.1016/s0925-4773(00)00494-9. [DOI] [PubMed] [Google Scholar]
  27. Kwee L, Baldwin H, Shen H, Stewart C, Buck C, Buck C, Labow M. Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice. Development. 1995;121:489–503. doi: 10.1242/dev.121.2.489. [DOI] [PubMed] [Google Scholar]
  28. Landerholm T, Dong X, Lu J, Belaguli N, Schwartz R, Majesky M. A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells. Development. 1999;126:2053–2062. doi: 10.1242/dev.126.10.2053. [DOI] [PubMed] [Google Scholar]
  29. Lavine K, White A, Park C, Smith C, Choi K, Long F, Hui C, Ornitz D. Fibroblast growth factor signals regulate a wave of hedgehog activation that is essential for coronary vascular development. Genes Dev. 2006;20:1651–1666. doi: 10.1101/gad.1411406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee EW, Michalkiewicz M, Kitlinska J, Kalezic I, Switalska H, Yoo P, Sangkharat A, Ji H, Li L, Michalkiewicz T, Ljubisavljevic M, Johansson H, Grant DS, Zukowska Z. Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles. J Clin Invest. 2003;111:1853–62. doi: 10.1172/JCI16929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Li L, Miano J, Cserjesi P, Olson E. SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis. Circ Res. 1996;78:188–195. doi: 10.1161/01.res.78.2.188. [DOI] [PubMed] [Google Scholar]
  32. Lin FJ, You LR, Yu CT, Hsu WH, Tsai MJ, Tsai SY. Endocardial Cushion Morphogenesis and Coronary Vessel Development Require Chicken Ovalbumin Upstream Promoter-Transcription Factor II. Arterioscler Thromb Vasc Biol. 2012 doi: 10.1161/ATVBAHA.112.300255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lucitti JL, Jones EA, Huang C, Chen J, Fraser SE, Dickinson ME. Vascular remodeling of the mouse yolk sac requires hemodynamic force. Development. 2007;134:3317–26. doi: 10.1242/dev.02883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Majesky M. Development of coronary vessels. Curr Top Dev Biol. 2004;62:225–259. doi: 10.1016/S0070-2153(04)62008-4. [DOI] [PubMed] [Google Scholar]
  35. Manner J. The development of pericardial villi in the chick embryo. Anat Embryol. 1992;186:379–385. doi: 10.1007/BF00185988. [DOI] [PubMed] [Google Scholar]
  36. Mcleod M. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology. 1980;22:299–301. doi: 10.1002/tera.1420220306. [DOI] [PubMed] [Google Scholar]
  37. Merki E, Zamora M, Raya A, Kawakami Y, Wang J, Zhang X, Burch J, Kubalak SW, Kaliman P, Izpisua Belmonte JC, Chien KR, Ruiz-Lozano P. Epicardial retinoid × receptor alpha is required for myocardial growth and coronary artery formation. Proc Natl Acad Sci U S A. 2005;102:18455–60. doi: 10.1073/pnas.0504343102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mikawa T, Fishman D. Retroviral analysis of cardiac morphogenesis: Discontinuous formation of coronary vessels. Proc Natl Acad Sci USA. 1992;89:9504–9508. doi: 10.1073/pnas.89.20.9504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mommersteeg MT, Dominguez JN, Wiese C, Norden J, De Gier-De Vries C, Burch JB, Kispert A, Brown NA, Moorman AF, Christoffels VM. The sinus venosus progenitors separate and diversify from the first and second heart fields early in development. Cardiovasc Res. 2010;87:92–101. doi: 10.1093/cvr/cvq033. [DOI] [PubMed] [Google Scholar]
  40. Moore A, Mcinnes L, Kreidberg J, Hastie N, Schedl A. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland, and throughout nephrogenesis. Development. 1999;126:1845–1857. doi: 10.1242/dev.126.9.1845. [DOI] [PubMed] [Google Scholar]
  41. Motoike T, Loughna S, Perens E, Roman B, Liao W, Chau T, Richardson C, Kawate T, Kuno J, Weinstein B, Stainier D, Sato T. Universal GFP reporter for the study of vascular development. Genesis. 2000;28:75–81. doi: 10.1002/1526-968x(200010)28:2<75::aid-gene50>3.0.co;2-s. [DOI] [PubMed] [Google Scholar]
  42. Naiche L, Harrelson Z, Kelly R, Papaioannou V. T-box genes in vertebrate development. Annu Rev Genet. 2005;39:219–239. doi: 10.1146/annurev.genet.39.073003.105925. [DOI] [PubMed] [Google Scholar]
  43. Norden J, Grieskamp T, Christoffels VM, Moorman AF, Kispert A. Partial absence of pleuropericardial membranes in tbx18- and wt1-deficient mice. PLoS One. 2012;7:e45100. doi: 10.1371/journal.pone.0045100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Passman J, Dong X, Wu S, Maguire C, Hogan K, Bautch V, Majesky M. A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1+ smooth muscle progenitor cells. Proc Natl Acad Sci USA. 2008;105:9349–9354. doi: 10.1073/pnas.0711382105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Perez-Pomares JM, De La Pompa JL. Signaling during epicardium and coronary vessel development. Circ Res. 2011;109:1429–42. doi: 10.1161/CIRCRESAHA.111.245589. [DOI] [PubMed] [Google Scholar]
  46. Qiu P, Li L. Histone CREB-binding protein onto SM22 promoter during SM22 gene expression. Circ Res. 2002;90:858–865. doi: 10.1161/01.res.0000016504.08608.b9. [DOI] [PubMed] [Google Scholar]
  47. Ratajska A, Czarnowska E, Kolodzinska A, Kluzek W, Lesniak W. Vasculogenesis of the embryonic heart: Origin of blood island-like structures. Anat Rec Part A. 2006;288:223–232. doi: 10.1002/ar.a.20311. [DOI] [PubMed] [Google Scholar]
  48. Roukens MG, Alloul-Ramdhani M, Baan B, Kobayashi K, Peterson-Maduro J, Van Dam H, Schulte-Merker S, Baker DA. Control of endothelial sprouting by a Tel-CtBP complex. Nat Cell Biol. 2010;12:933–42. doi: 10.1038/ncb2096. [DOI] [PubMed] [Google Scholar]
  49. Sengbusch J, He W, Pinco K, Yang J. Dual functions of a4b1 integrin in epicardial development: initial migration and long-term attachment. J Cell Biol. 2002;157:873–882. doi: 10.1083/jcb.200203075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Smith CL, Baek ST, Sung CY, Tallquist MD. Epicardial- derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling. Circ Res. 2011;108:e15–26. doi: 10.1161/CIRCRESAHA.110.235531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sucov H, Dyson E, Gumeringer C, Price J, Chien K, Evans R. RXRα mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev. 1994;8:1007–1018. doi: 10.1101/gad.8.9.1007. [DOI] [PubMed] [Google Scholar]
  52. Tomanek R. Formation of the coronary vasculature during development. Angiogenesis. 2005;8:273–284. doi: 10.1007/s10456-005-9014-9. [DOI] [PubMed] [Google Scholar]
  53. Tomanek R, Ishii Y, Holifield J, Sjogren C, Hansen H, Mikawa T. VEGF family members regulate myocardial tubulogenesis and coronary artery formation in the embryo. Circ Res. 2006;98:947–953. doi: 10.1161/01.RES.0000216974.75994.da. [DOI] [PubMed] [Google Scholar]
  54. Van Den Akker NM, Winkel LC, Nisancioglu MH, Maas S, Wisse LJ, Armulik A, Poelmann RE, Lie-Venema H, Betsholtz C, Gittenberger-De Groot AC. PDGF-B signaling is important for murine cardiac development: its role in developing atrioventricular valves, coronaries, and cardiac innervation. Dev Dyn. 2008;237:494–503. doi: 10.1002/dvdy.21436. [DOI] [PubMed] [Google Scholar]
  55. Viragh S, Challice C. The origin of the epicardium and the embryonic myocardial circulation in the mouse. Anat Rec. 1981;201:157–168. doi: 10.1002/ar.1092010117. [DOI] [PubMed] [Google Scholar]
  56. Vokes S, Yatskievych T, Heimark R, Mcmahon J, Mcmahon A, Antin P, Krieg P. Hedgehog signaling is essential for endothelial tube formation during vasculogenesis. Development. 2004;131:4371–4380. doi: 10.1242/dev.01304. [DOI] [PubMed] [Google Scholar]
  57. Von Gise A, Zhou B, Honor LB, Ma Q, Petryk A, Pu WT. WT1 regulates epicardial epithelial to mesenchymal transition through beta- catenin and retinoic acid signaling pathways. Dev Biol. 2011;356:421–31. doi: 10.1016/j.ydbio.2011.05.668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang D, Chang PS, Wang Z, Sutherland L, Richardson JA, Small E, Krieg PA, Olson EN. Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell. 2001;105:851–62. doi: 10.1016/s0092-8674(01)00404-4. [DOI] [PubMed] [Google Scholar]
  59. Ward N, Dumont D. Angiopoietin 1 expression levels in the myocardium direct coronary vessel development. Dev Dyn. 2004;229:500–509. doi: 10.1002/dvdy.10479. [DOI] [PubMed] [Google Scholar]
  60. Wiese C, Grieskamp T, Airik R, Mommersteeg MT, Gardiwal A, De Gier-De Vries C, Schuster-Gossler K, Moorman AF, Kispert A, Christoffels VM. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res. 2009;104:388–97. doi: 10.1161/CIRCRESAHA.108.187062. [DOI] [PubMed] [Google Scholar]
  61. Wilting J, Buttler K, Schulte I, Papoutsi M, Schweigerer L, Manner J. The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart. Dev Biol. 2007;305:451–459. doi: 10.1016/j.ydbio.2007.02.026. [DOI] [PubMed] [Google Scholar]
  62. You L, Lin F, Lee C, Demayo F, Tsai M, Tsai S. Suppression of notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature. 2005;435:98–104. doi: 10.1038/nature03511. [DOI] [PubMed] [Google Scholar]
  63. Zamora M, Manner J, Ruiz-Lozano P. Epicardium-derived progenitor cells require beta-catenin for coronary artery formation. Proc Natl Acad Sci U S A. 2007;104:18109–14. doi: 10.1073/pnas.0702415104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhou B, Ma Q, Rajagopal S, WU S, Domian I, Rivera-Feliciano J, Jiang D, Von Gise A, Ikeda S, Chien K, Pu W. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature. 2008;454:109–113. doi: 10.1038/nature07060. [DOI] [PMC free article] [PubMed] [Google Scholar]

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