Fig. 8: EndoMT molecular pathways are dysregulated in cilia and ciliary calcium signaling KO hearts.
From: Endocardial primary cilia and blood flow regulate EndoMT during endocardial cushion development
a, Endocardial, EndoMT and mesenchymal gene expression in the WT, cilia heterozygous (Ift20+/−) and cilia KO (Ift20−/−) vEC/EndoMT cluster (1,496 nuclei) (Vwf Cilia WT versus Cilia KO P = 2.5 × 10−11, Cilia Het versus Cilia KO P = 1.9 × 10−11; Twist1 Cilia WT versus Cilia KO P < 2.22 × 10−16, Cilia Het versus Cilia KO P = 3.7 × 10−16; Sox5 Cilia WT versus Cilia KO P < 2.22 × 10−16, Cilia Het versus Cilia KO P < 2.22 × 10−16; Kdr Cilia WT versus Cilia KO P = 2.1 × 10−13, Cilia Het versus Cilia KO P = 3.2 × 10−13; Snai1 Cilia WT versus Cilia KO P = 0.0003, Cilia Het versus Cilia KO P = 0.00075; Vcan Cilia WT versus Cilia KO P < 2.22 × 10−16, Cilia Het versus Cilia KO P < 2.22 × 10−16). b, Gene Ontology biological processes that were lower and higher in cilia KO (Ift20−/−) vEC/EndoMT nuclei compared to WT and cilia heterozygous (Ift20+/−) littermates. Cushion-relevant terms are highlighted (green: up in cilia KO, red: down in cilia KO). c, Immunofluorescence on e9.0 sections of WT and Pkd2−/− hearts for KLF4 (red) and cilia (ARL13B, green) on endocardial cells (CD31, white). Nuclei are shown in blue (Hoechst); the AVC is outlined in white. d, KLF4 protein-positive endocardial cells in the AVC and OFT as a percentage of all endocardial cells in e9.5 WT (blue) and Pkd2−/− (purple) mice (e8.5 (WT n = 5, Pkd2−/− n = 5); e9.0 (WT n = 3, Pkd2−/− n = 3); e9.5 (WT n = 7, Pkd2−/− n = 7)). Cilia KO (green) is provided for comparison (e8.5, n = 4; e9.0, n = 4; e9.5, n = 6). e, CD31+ cells in the cushion as a percentage of total endocardial cells in the AVC of Pkd2−/− hearts (n = 5). WT (blue, n = 4), Dnah11−/− (gray, n = 4) and cilia KO (green, n = 5) are provided for comparison. f, Summary graphic: WT hearts selectively lose cilia in areas of the highest shear stress, leading to KLF4 reduction and subsequent EndoMT/cushion cellularization. Without cilia present, KLF4 is unable to be regionally turned off and EndoMT/cushion cellularization cannot progress. Statistics: NS, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. The statistical test used for a was a two-tailed Student’s t test. Data are represented as mean ± s.e.m. The statistical test used for d and e was a two-sided t test with Welch’s correction. Unless otherwise stated, n is the number of embryos.
