Figure 3: Wt1 ECRs drive gene expression and BRG1 is required for EPDC activity. | Nature Communications

Figure 3: Wt1 ECRs drive gene expression and BRG1 is required for EPDC activity.

From: BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease

Figure 3

(ai) Wt1 ECRs are sufficient to drive LacZ expression in the epicardium at E11.5. Whole-mount (a,c,e,f,h) and transverse-sectioned hearts (b,d,g,i) are shown. Arrowheads mark epicardium covering the ventricles; arrows mark epicardium covering the atria (a,b) and outflow tract (a). Two (−42 bp), five (+4.0 kb) and six (+5.8 kb) transgenic mice were examined to assess the reproducibility of reporter activity patterns. (js) BRG1 and Wt1 immunostaining of E11.5 hearts documenting Brg1 deletion (asterisks in l,q) and Wt1 reduction (asterisks in m,r) in the epicardium of Brg1 cKOs compared to littermate controls (arrowheads in j,k and o,p). Note reduced BRG1 expression also in the myocardium (arrows; compared j with l versus o with q) of Gata5Cre;Brg1flox/flox, but not Wt1CreERT2;Brg1flox/flox hearts. (n,s) BRG1 ChIP-qPCR data. Three independent ChIP experiments were performed per group (n=10 hearts) and are presented as fold enrichment over IgG. (t,u) Wt1 immunostaining of E16.5 hearts documenting fewer Wt1-positive cells in the epicardium (arrowheads) and EPDCs invading the subepicardial and myocardial layers (arrows) in Brg1 cKOs. Note magnified views of (red) inset boxes in t and u. (v) Quantification of Wt1-expressing cells in E16.5 hearts as %=(total number of Wt1-positive cells per section/total number of DAPI nuclei per section) × 100; co, n=3 hearts; mut, n=4 hearts. (w,x) Endomucin (EMCN) and smooth-muscle myosin heavy chain 11 (SM-MHC) immunostaining of E16.5 hearts documenting a reduction of patent coronary vessels, containing endothelium (arrow) surrounded by smooth muscle (arrowheads), in Brg1 cKOs hearts. Note magnified views of (red) inset boxes in panels w and x. (y) Quantification of patent vessels per field in E16.5 hearts; 6 fields analysed per section per genotype; co, n=3 hearts; mut, n=4 hearts. All error bars are data±s.d. Significant differences (P values) were calculated using two-tailed Student’s t-test (*P≤0.05; **P≤0.01; ***P≤0.001). co, control; ep, epicardium; la, left atrium; lv, left ventricle; mut, mutant; my, myocardium ra, right atrium; rv, right ventricle; v, ventricle. All scale bars 200 μm, except j,o,t,u,w,x 100 μm and magnified views of inset boxes in t,u,w,x 20 μm.

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