Fig. 5: Endocardial KLF4 expression correlates with ciliation and depends on blood flow.
From: Endocardial primary cilia and blood flow regulate EndoMT during endocardial cushion development
a, A model of mechanical forces in an e9.5 mouse heart. Red arrows indicate blood flow/shear stress; gray arrows indicate contractile stress. b, Immunofluorescence on e10 mouse heart sections for KLF4 protein (red) and cilia (ARL13B, green) on endocardial cells (CD31, white). Nuclei are shown in blue (Hoechst). Close-ups of the AVC (boxed region in the left image) with and without CD31 are provided; white arrows indicate cilia, while the endocardium is outlined in white. Close-ups of endocardial cilia are shown in orange and yellow boxes. c, KLF4 protein-positive endocardial cells (dotted line), ciliated endocardial cells (solid line) and KLF4 protein-positive + ciliated endocardial cells (lighter color line) versus distance in the AVC at e8.5 (n = 3), e9.5 (n = 4) and e10 (n = 3 KLF4, n = 2 KLF4 + cilia). d, xy plots for the correlation of average plot points from c. e, Violin plot for KLF4 protein immunofluorescence signal intensity (corrected total cell fluorescence) in ciliated (n = 62 cells) and nonciliated (n = 160 cells) AVC endocardial cells (P < 0.0001). f, KLF4 protein-positive endocardial cells in the AVC over time as a percentage of all endocardial cells in WT mice (blue) and Ncx1−/− mice (red) (e8.5 (WT n = 10, Ncx1−/− n = 10), e9.0 (WT n = 6, Ncx1−/− n = 6), e9.5 (WT n = 4, Ncx1−/− n = 4)) (e8.5 P = 0.003, e9.0 P = 0.0014, e9.5 P = 0.0236). g, KLF4 protein-positive endocardial cells versus distance in the AVC of Ncx1−/− mice at e9.5 (n = 3). For comparison, the WT line is provided in blue (n = 4). Distance runs from left to right: atrium to left ventricle. h, Immunofluorescence on e9.5 WT and Ncx1−/− mouse heart sections for KLF4 protein (red) and cilia (ARL13B, green) on endocardial cells (CD31, white). Nuclei are shown in blue (Hoechst). A version without CD31 is provided; white arrows indicate cilia, while the endocardium is outlined in white. Close-ups of endocardial cilia are shown in yellow boxes. Statistics: NS, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. The statistical test used was a two-sided t test with Welch’s correction. Data are represented as mean ± s.e.m. Unless otherwise stated, n is the number of embryos. Pos, positive; RV, right ventricle.
