Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin

Abstract

The epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of the gene encoding Wilms' tumor-1 (Wt1) leads to a reduction in mesenchymal progenitor cells and their derivatives. We show that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during embryonic stem cell differentiation through direct transcriptional regulation of the genes encoding Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages do not form in Wt1-null embryoid bodies, but this effect is rescued by the expression of Snai1, underscoring the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell-based therapies.

Figure 1

Heart defects in epicardial-specific Wt1 mutant embryos. (b) Gata5-Cre⁺/Wt1 ^loxP/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 μm. (c, d) Haematoxylin and eosin-stained sections of Cre⁻ and Cre ⁺ 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 μm. (e, f) OPT image of control and mutant hearts at E16.5. Scale bars represent 50 μm. (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 from Cre⁺ embryos. Scale bars represent 50 μm.

Figure 2

Wt1 expression is necessary to maintain a mesenchymal phenotype in immortalised epicardial cells. (a) Heart of Wt1-GFP knockin mouse. Scale bar represents 50 μm. (b) Direct GFP expression on heart cryosection of Wt1-GFP knockin embryos shows GFP expression in epicardial cells. Scale bar represents 50 μm. (c) Phase-contrast micrograph of Wt1 conditional KO immortalised mouse epicardial cells (CoMEEC). Scale bar represents 100 μm. (d-f) GFP, ZO-1 and Wt1 expression in CoMEEC. Scale bars represent 50 μm. CoMEEC displayed a cobblestone monolayer typical of epicardial cells (c, e). (g) Western blot analysis was conducted in Cre⁺ tamoxifen inducible CoMEEC cultured in the presence of different concentrations of tamoxifen. (h) RT-PCR analyses of Snai1 and Cdh1 expression in Cre⁺ CoMEEC in presence of tamoxifen. (i) The migratory behaviour of Cre⁺ CoMEEC in presence of tamoxifen was analyzed in an in vitro wound model. Scale bar represents 100 μm.

Figure 3

Wt1 is an activator of Snai1 and a repressor of Cdh1 in epicardial cells. (a, e) Schematic representation of the putative conserved Wt1 binding sites (, □) in the Snai1 and Cdh1 loci, (functional binding site), □ (putative but non functional binding site) and ■ (exons). (b) Luciferase activity of reporter construct carrying mouse Snai1 promoter in epicardial cells in the presence of indicated amounts of −KTS Wt1 expression vector. (c) Luciferase activity of wild-type (Control) or mutated Snai1 promoters in the presence of -KTS Wt1 isoform. (d, f) Cell extracts from epicardial cells were chromatin immunoprecipitated (ChIP), using antibodies against Wt1, anti-acetyl-histone H3 (AcH3), anti-H3 tri methyl K4 (K4Me) and anti- H3 tri methyl K27 (K27Me) or an irrelevant antibody (IgG). The input was used as a positive control for PCR of the Snai1 (d) and Cdh1 (f) promoters, intronic regions and 3′UTRs. (g, h) Luciferase activity of constructs carrying DNA fragments identified by the Cdh1 ChIP assay in epicardial cells, together with different concentrations of −KTS Wt1 isoform. (i) Luciferase activity of Control or mutated Cdh1 constructs in the presence of -KTS Wt1 isoform (100ng). (j) ChIP assays of Snail and Wt1 at the endogenous Cdh1 promoter in epicardial cells. The graphs represent the mean values ±s.e.m from three independent experiments.

Figure 4

Wt1 is required for EMT in ES cells. (a) Wt1-GFP expression during EB differentiation of Wt1-GFP knockin ES cells. Scale bar represents 50 μm (b) Western Blot (WB) analysis of the endogenous Wt1 protein in EB. (c) WB of epithelial and mesenchymal protein markers in wild type EB (control) and Wt1 KO EB (KO). (d) Phase-contrast microscopy of control and Wt1 KO EB migrating cells. Scale bars represent 50 μm.

Figure 5

Wt1 is required for the formation of some mesodermal lineage in EB differentiation. RT-PCR analysis for expression of mesodermal (a), endodermal and ectodermal markers (b) in wild type EB (control) and Wt1 KO EB (KO). (c) RT-PCR analysis for expression of Snai1 and Cdh1 in Wt1 KO Snai1 expressing clones (snailc1 and snailc2) versus Wt1 KO GFP expressing clones (c1 and c2). (d) RT-PCR analysis for expression of mesodermal markers in Snai1 clones (Snai1c1 and Snai1c2) versus GFP clones (c1 and c2). (e) Quantification of the number of beating cardiomyocytes in control, and Wt1 KO clones (c1, c2 and Snai1c1, Snai1c2). The graph represents the mean values ±s.e.m from three independent experiments.