anti-Wt1 (Santa Cruz; catnr SC-192; 1:200), anti-PDGFRa (Santa Cruz; catnr SC-233; 1:200), anti-CD34 (BD Bioscience; catnr 36105; 1:200), anti-cKit (Southern Biotech; catnr 8380; 1:200), anti-a-sma (Sigma; catnr A2522; 1:1000), anti-BrdU (Becton Dickinson, catnr 347580; 1:600), anti-P-Smad1/5/8 (Cell Signal- ing Technology, catnr 9511; 1:200), anti-P-Erk1/2 (Cell Signaling Technology, catnr 4376; 1:200), anti-PECAM (Santa Cruz; catnr SC-1506; 1:100), and ß-Galactosidase (MP Biomedicals(Cappel), catnr 55976; 1:500). To reduce background signal of the antiserum against ß-Galactosidase, the diluted antiserum was pre-incubated with mouse heart powder before application to the respective section. Mouse heart powder was prepared by homogenizing a wildtype adult mouse heart in PBS and precipitating the proteins with 4 volumes ice cold acetone. The precipitate was collected by centrifugation, the dry pellet was ground in a mortar and stored at 220uC. Alexa488 or Alexa568 conjugated goat-anti-rabbit and goat- anti-mouse antibodies (Molecular Probes; 1:250) were used as secondary antibodies. The signal was amplified using tyramide signal amplification (TSA NEL702, Perkin Elmer). Nuclei were visualized using Topro3 (Molecular Probes; 1:500). Fluorescence was visualized using a Leica SPE confocal laser scanning microscope. Results Expression of embryonic epicardial genes in the adult healthy and infarcted heart To gain insight into the response of the epicardium after a myocardial infarction we investigated the expression of markers for embryonic epicardium in the adult heart. During development, Tbx18, Wt1, Raldh1, and Raldh2 are expressed throughout the epicardial layer and in the adjacent mesenchyme, but all four gene products become down-regulated at the end of gestation [25–29]. Their expression during development is associated with the formation of subepicardial mesenchyme by epithelial to mesen- chymal transition (EMT), as well as with myocardial growth and differentiation [30]. In the normal or diseased adult heart, their patterns of expression have not been described in detail [14–17]. In the adult heart Wt1, Tbx18, Raldh1, and Raldh2 mRNA were found to be expressed in the epicardium covering the atrioven- tricular sulcus (Figure 1 A–E) and the ventricular apex, but not in the epicardium covering the ventricles or in the ventricular wall. Raldh1 mRNA was also found in a subset of mesenchymal cells present in the atrioventriuclar sulcus (Figure 1B), whereas none of the other genes is found to be expressed in this mesenchyme (Figure 1A–E). Raldh1 mRNA was further found to be expressed in the epicardial layer covering the atria (Figure 1G). Although the other genes were also expressed in the epicardial lining of the atria, they were expressed at a low level and in a mosaic pattern (Figure 1H–J). To investigate the changes in the spatio-temporal pattern of expression of these epicardial genes in relation to ischemia, we permanently ligated the LAD. To identify the ischemic region we hybridized an adjacent section with a cTnI probe. Already from the first day post-myocardial infarction (post-MI) the level of cTnI mRNA had dropped below detection in the ischemic myocardium (Figure 1K), allowing the identification of the site and extent of the infarct. At one day post-MI the expression of the embryonic epicardial markers had become more prominent in the regions expressing these markers in controls (eg Figure 1O, P, S), and from three days post-MI onwards their expression was detected in the epicardial cells covering the entire infarcted area as well as the border zone (Figure 1L, N, Q, R T, U). Raldh2 mRNA was, Endogenous Epicardial Regenerative Response however, induced throughout the entire epicardial layer of the heart from the first day after the induction of the ischemia (Figure 1V). Concomitant with the appearance of mesenchymal cells in between the epicardium and myocardium of the border zone, these sub-epicardial mesenchymal cells were found to express the four embryonic epicardial genes as well, albeit at lower levels compared to the overlaying epicardium (Figure 1V). Although the patterns and levels of expression differed, the induction of their expression was transient, being confined to the first two weeks post-MI. At one and three months post-MI, the expression of the four embryonic epicardial genes was only found in the regions that also expressed these genes in healthy controls. To further confirm the transient induction of the expression of these genes, RNA was isolated and the expression levels were determined by quantitative PCR. As expected the level of expression of cTnI mRNA was found to be approximately 100- fold lower in infarcted hearts from the first days after the induction of the ischemia up to the latest time point analyzed, being three months post-MI. The expression levels of Wt1, Tbx18 and Raldh1 mRNA were already found to be up-regulated at 1 day post-MI, showing the highest level of expression at three days post-MI and subsequently decreasing to return to levels similar to control at one and three months post-MI (Figure 1W). Analysis of the Wt1-lineage in response to ischemic injury Patterns of gene expression do not allow an assessment of the fate of the epicardial cells after the myocardial infarction. In order to assess the fate of the epicardial cells, we needed to permanently label the epicardial cells. We have recently described a Wt1Cre mouse line, which permanently labels the proepicardium, the epicardium and its derivatives, and in a limited number of small clusters of cardiomyocytes within the ventricular septum [19]. These observations suggest that this mouse line might be a useful tool to study the epicardial lineage after a myocardial infarction [31,32]. As the Cre/loxP system relies on the similarity of the spatio-temporal pattern of expression of Wt1 and Cre, we compared the expression patterns of Wt1 and Cre mRNA in the healthy and infarcted heart. Like Wt1 mRNA, Cre mRNA was not detected in the epicardium overlaying the left and right ventricular free wall or within the adjacent myocardial wall (Figure 2A–C). This expression pattern was not found to have changed 1 day post-MI (Figure 2D–F). At three days post-MI both Wt1 and Cre mRNA expression were detected in the epicardium overlaying the border zone of the infarct (Figure 2G–I) as well as the infarction itself, albeit at lower levels (Figure 2J, L and N), and was absent from epicardium located remotely of the infarct (Figure 2K, M, and O). At one week post-MI Wt1 and Cre mRNA expression had become more prominent in the area of the infarct, being expressed in the epicardium and immediately adjacent mesenchyme (Figure 2P–U). At two weeks post-MI the expression levels of Wt1 and Cre mRNA had decreased (not shown), and were no longer detectable in the infarcted region at 1 and 3 months post-MI (Figure 1V–X). Although the findings with respect to Wt1 mRNA are in line with a previous report [33], we did not observe expression of Wt1 mRNA in the coronaries in the infarcted region, even if the sections were over-stained (data not shown). Taken together, these observations show that the spatio- temporal pattern of expression of Cre mRNA parallels that of Wt1 mRNA, indicating that this Wt1Cre mouse line, concerning this aspect of the Cre/loxP analysis, can be used to trace the fate of Wt1 expressing cells in the region of the infarction. PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44692