Extended Data Fig. 1: Spatiotemporally dynamic endocardial ciliation of the ECCs during cushion EndoMT is blood-flow dependent and is conserved in zebrafish.
From: Endocardial primary cilia and blood flow regulate EndoMT during endocardial cushion development
a) Immunofluorescence on mouse embryo sections for cilia (Arl13b, green) on endocardial cells (CD31, white) of the OFT over cushion development (e8.5, e9.5, e10). Nuclei are shown in blue (Hoechst). Close ups with and without CD31 are given; white arrows indicate cilia; luminal endocardial cells outlined with white and whole OFT (including cushion mesenchyme) outlined in red. b) Diagram of an e9.5 mouse heart for cryosection orientation (yellow: mesenchyme, red: myocardium, blue: endocardium, grey: lumen). c) Immunofluorescence on e10 mouse embryo sections for cilia (Arl13b, green) in the AVC. Nuclei are shown in blue (Hoechst). Luminal endocardial cells are outlined with dashed white and the whole AVC (including cushion mesenchyme) outlined in dotted white. d) Number of ciliated mesenchymal cushion cells as a percentage of total mesenchymal cushion cells in the AVC over time (e9.5 (n = 3), e10 (n = 3), e10.5 (n = 3)) (p = 0.0101). e) Still of live 54hpf tg(Arl13b:eGPF;Kdrl (Flk-1):mCherry;Cmlc2:eGFP) zebrafish heart showing cilia and myocardium in green and endocardium in red. Closeup of cilia provided in yellow box. f) Zebrafish endocardial ciliation over time in the atrioventricular canal (n = 3). (48 hpf vs 54 hpf p = 0.0213). g) Ciliated endocardial cells as percentage of all endocardial cells versus distance in the mouse OFT at e8.5 (n = 2) and e9.5 (n = 3). Distance runs left to right, Ventricle to Dorsal Aorta. h) Immunofluorescence on e9.5 wildtype and Ncx1−/− mouse heart sections for TUNEL (red) in endocardial cells (CD31, white). Nuclei are shown in blue (Hoechst). Version without CD31 is provided; endocardium is outlined in white. i) Quantification for TUNEL staining in h) (WT n = 7 sections, Ncx1−/− n = 11 sections). j) Endocardial ciliation in the OFT over time as percentage of all endocardial cells in wildtype mice (blue) and Ncx1−/− mice (red) (e8.5 (WT n = 7, Ncx1−/− n = 7), e9.0 (WT n = 4, Ncx1−/− n = 4), e9.5 (WT n = 7, Ncx1−/− n = 7)) (e8.5 p = 0.3438, e9 p < 0.0001, e9.5 p = 0.0397). Statistics: ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). Statistical test used was two-sided t-test with Welch’s correction. Data are represented as mean ± SEM. Unless otherwise stated, n is number of embryos. Abbreviations: A-Atrium, AVC-Atrioventricular Canal, EC-Endocardial Cell, LV-Left Ventricle, V-Ventricle, OFT-Outflow Tract, RV-Right Ventricle, WT-Wildtype.
