Extended Data Fig. 4: Temporospatial determination of EndoFb fate in developing heart.

a, Schematic diagram showing the strategy for labelling the EndoFb in different time window. 4-Hydroxytamoxifen (4-OH Tam) was injected at E7.5, E11.5, E12.5, and E16.5, then collected at E17.5. b, Whole-mount and epifluorescence images of E17.5 hearts with Tam induced at different time points. c-f, Immunostaining for PDGFRa and GFP on E17.5 heart sections when 4-OH Tam was injected at E7.5, E11.5, E12.5, and E16.5 respectively. Arrowheads, GFP⁺PDGFRa⁺ EndoFb. g, Quantification of the percentage of GFP⁺ EndoFb expressing PDGFRa in total (left panel), different valves (middle panel), and different regions of ventricles (right panel) in different time window. Data are mean ± s.e.m., n = 5 mice. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to compare among means of multiple groups. In All, E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001. In Valve, PL: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; SL: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; AL: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; ML: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; E11.5 > E17.5 vs. E12.5 > E17.5, *P = 0.0002. In Myocardium, RCM: E7.5 > E17.5 vs. E11.5 > E17.5, *P = 0.0226; RTM: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; LCM: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; LTM: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; VS: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; ns, no significance. PL, parietal leaflet; SL, septal leaflet; AL, aortic valve; ML, mural valve; RCM, right compact myocardium; RTM, right trabecular myocardium; LCM, left compact myocardium; LTM, left trabecular myocardium; VS, ventricular septum. Scale bars: yellow, 200 µm; white, 100 µm. Each image is representative of five individual mouse samples.