This result was confirmed by staining for platelet/endothelial cell adhesion molecule-1 (PECAM-1) and -smooth muscle actin (-SMA), markers for endothelial and smooth muscle cells respectively (Fig. cells In the developing heart, very little is known about the molecular and cellular mechanisms controlling epicardial EMT and the formation of cardiovascular progenitor cells1.Wt1encodes a zinc finger protein, which plays a critical role in the normal development of several organs Rabbit polyclonal to AMAC1 such as kidney, gonads, spleen and heart2. Coronary vascular defects inWt1mutant mice are conjectured to arise through defective EMT3. However, until now it has not been possible to test if Wt1 in the epicardium is directly involved in EMT or whether EMT is essential for the formation of cardiovascular progenitor cells. To investigate the role of Wt1 in the epicardium we generatedWt1conditional knockout mice (Supplementary Fig. 1) that were crossed with Wt1-GFP knockin mice4and transgenicGata5-Cre5mice (See Online Methods for details of breeding). The resultingWt1mutants die between embryonic day 16.5 (E16.5) and embryonic day 18.5 (E18.5) due to cardiovascular failure. Embryos at E16.5 display edema and accumulation of blood in the systemic veins (Fig. 1a, b). Efficient deletion ofWt1in epicardial cells was confirmed using immunohistochemistry on heart sections and real time-PCR analysis on FACS-sorted GFP+epicardial cells isolated fromGata5-Cre+/Wt1loxP/gfp(Cre+) mice (Supplementary Fig. 2a-c). The gross morphology ofCre+embryos is normal. However the right BMS-687453 ventricle (RV) of some mutant embryos (Fig. 1d) showed a thinner free wall as compared to the control (Fig. 1c) (arrows), while the left ventricle (LV) was apparently normal. Mutant embryos also display pericardial haemorrhaging (* inFig. 1d). == Figure 1. == BMS-687453 Heart defects in epicardial-specific Wt1 mutant embryos. (b)Gata5-Cre+/Wt1loxP/gfp(Cre+) embryos display edema and accumulation of blood in the systemic veins; a littermate control (Cre) is shown in panel (a). Scale bars represent 100 m. (c, d) Haematoxylin and eosin-stained sections ofCreandCre+E16.5 embryos. The right ventricle (RV) of some the mutant embryos (d) shows a thinner wall (arrows) as compared to the control (c), while the left ventricle (LV) is apparently normal. Mutant embryos show pericardial haemorrhage (* in d). Scale bars represent 50 m. (e, f) OPT image of control and mutant hearts at E16.5. Scale bars represent 50 m. (g, h ) Immunofluorescence staining for the indicated blood vessel markers. Only control embryos show arteries with a well differentiated smooth muscle layer (g). Analysis of EMT markers with antibodies against: Snail (i, j), E-cadherin (k, l), Vimentin and Cytokeratin (m, n). Abnormal E-cadherin (l) and decreased Snail (j) and Vimentin (n) expression is observed in epicardial cells fromCre+embryos. Scale bars represent 50 m. We confirmed the embryonic expression of theGata5-Cretransgene in the heart. At E10.5 we detected cells expressingCrein the epicardium, while at E12.5Gata5-Crederived cells were abundant within the heart (Supplementary Fig. 3and data not shown)5. Optical projection tomography (OPT) showed that the coronary arteries failed to form in Wt1 mutant mice BMS-687453 (Fig. 1e, fandSupplementary movies 1, 2). This result was confirmed by staining for platelet/endothelial cell adhesion molecule-1 (PECAM-1) and -smooth muscle actin (-SMA), markers for endothelial and smooth muscle cells respectively (Fig. 1g, h). The presence of GFP+epicardial cells covering the surface of the myocardium inCre+mice demonstrated the integrity of this structure in mutant mice (Supplementary Fig. 4b). Since epicardial EMT plays BMS-687453 a very important role in the formation of coronary vascular precursor cells, we studied the expression patterns of the major markers of EMT in theWt1mutant epicardium. We found that E-cadherin and Cytokeratin (CK), epithelial markers, were upregulated in mutant epicardial cells in comparison with control hearts (Fig. 1k-n). Conversely, the expression of mesenchymal markers, Snail (Fig. 1i, j) and Vimentin was reduced (Fig. 1m, n). We also compared the levels ofSlug(Snai2) inCre+andCreFACS-sorted epicardial cells by real time-PCR and confirmed that similar toSnail(Snai1),Snai2levels were reduced by 70% inCre+in comparison withCrecells (data not shown). To determine whether Wt1 plays a direct and cell autonomous role in epicardial EMT we generated tamoxifen inducibleWt1KO immortalised epicardial cells (Cre+CoMEEC)Fig. 2c-f(See Online Methods). Loss of Wt1 after tamoxifen treatment leads to a robust increase in E-cadherin expression in a dose dependent manner (Fig. 2g) which correlates with a downregulation in the levels of N-cadherin and -SMA (Fig 2g). RT-PCR analysis also showed that there was a striking downregulation ofSnai1expression afterWt1deletion.