Figure 5. A depleted pool of EMT-derived mesenchyme in endocardial Brg1-deficient semilunar valves is replaced by increased proliferation of other mesenchymal lineage(s).

(A–H) Sections of Nfatc1^Cre;Brg1^F/+;R26R^mTmG and Nfatc1^Cre;Brg1^F/F;R26R^mTmG embryos (pulmonic valve except mitral valve in (G, H); genotype and age as indicated) immunostained for GFP (Nfatc1^Cre-lineage derived cells, green) and nuclei (Hoechst-stained, purple). Arrowheads indicate EMT-derived mesenchymal cells at the pulmonic valve cusp base near the cushion-myocardium boundary. (I, J) Quantification of the relative contribution of Nfatc1^Cre-lineage pulmonic valve mesenchyme in control (Brg1^F/+) and endocardial Brg1-deficient embryos (Brg1^F/F) at E14.5 (I) and E16.5 (J). Left (L-PV) and right (R-PV) cusps are scored separately. The percentile fraction of Nfatc1^Cre-lineage mesenchyme for each embryo is normalized to the mean of corresponding littermate wildtype samples. The mean absolute percentage of labeled and unlabeled cells is shown within individual bars of the chart (n=5). (K–L) E14.5 wildtype (Nfatc1^Cre;Brg1^F/+;R26R^mTmG) (K) and Nfatc1^Cre;Brg1^F/F;R26R^mTmG (L) pulmonic valve sections stained with GFP (Nfatc1^Cre-lineage, green), EdU-incorporating proliferating cells (red), and Hoechst (nuclei, blue). White arrows denote EdU⁺, non-Nfatc1^Cre-lineage mesenchymal cells. (M) Quantitation of the proliferation rate of Nfatc1^Cre- and other-lineage pulmonic valve mesenchyme in E14.5 control and Nfatc1^Cre;Brg1^F/F embryos. The mean absolute fraction of EdU-incorporated cells for each condition is shown. For all graphs, error bars show one standard deviation of the mean. Asterisks indicate a significant difference (P < 0.05, two-tailed Student’s t-tests). NS: not significant; GFP⁺Nfatc1^Cre-lineage labeled mesenchyme; Non-LL: non-Nfatc1^Cre-lineage labeled mesenchyme. Scale bars: 100 µm.