Fig. 4: KLF4 expression negatively correlates with cushion EndoMT progression.
From: Endocardial primary cilia and blood flow regulate EndoMT during endocardial cushion development
a, HCR-FISH on whole-mount mouse embryos at e9.5 showing Klf4 mRNA (red), Vim mRNA (green) and nuclei (Hoechst, blue). The AVC endocardium is outlined in white. b, mRNA expression as measured by the gray value over distance for Kdr (blue, n = 4), Vim (green, n = 4) and Klf4 (red, n = 6) in the e9.5 AVC endocardium. Distance runs from left to right: atrium to left ventricle. Shading indicates ±s.e.m. from the mean (line). c, Immunofluorescence on e9.5 WT mouse heart sections for FN protein (red) in endocardial cells (CD31, white). Nuclei are shown in blue (Hoechst). A version without CD31 is provided; the AVC is outlined in white. d, CD31+ cells in the cushion as a percentage of the total CD31+ endocardial cells in the AVC and OFT over time (e8.5 (AVC n = 3, OFT n = 3), e9.0 (AVC n = 4, OFT n = 4), e9.5 (AVC n = 4, OFT n = 4), e10 (AVC n = 4, OFT n = 5)) (OFT e9.5 versus e10 P = 0.0017, OFT e9.5 versus AVC e9.5 P = 0.0322, OFT e10 versus AVC e10 P = 0.0189, AVC e8.5 versus e9.0 P = 0.018, AVC e9.0 versus e9.5 P = 0.0618, AVC e9.5 versus e10 P = 0.0003). e, scRNA-seq27 gene set enrichment for EndoMT progression (554 cells). f, UMAP plot of scRNA-seq27 endocardial cell/vEC/EndoMT clusters (554 cells) with pseudotime lineage from Slingshot (top) and Slingshot pseudotime for EndoMT progression (bottom) of endocardial cell/vEC/EndoMT clusters (554 cells). g, Total Klf4 mRNA expression over EndoMT pseudostages from scRNA-seq27 (554 cells). Statistics: NS, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. The statistical test used for d was a two-sided t test with Welch’s correction. The statistical test used for g was a two-tailed Student’s t test. Data are represented as mean ± s.e.m. Unless otherwise stated, n is the number of embryos. EC, endocardium; Mes, mesenchyme.
