Endocardial primary cilia and blood flow regulate EndoMT during endocardial cushion development [Single Nuc-RNAseq_NCX]

Blood flow is critical for heart valve formation, and cellular mechanosensors are essential to translate flow into transcriptional regulation of development. Here, we identify a role for primary cilia in vivo in the spatial regulation of cushion formation, the first stage of valve development, by regionally controlling endothelial to mesenchymal transition (EndoMT) via modulation of Kruppel-like Factor 4 (Klf4). We find that high shear stress intracardiac regions decrease endocardial ciliation over cushion development, correlating with KLF4 downregulation and EndoMT progression. Mouse embryos constitutively lacking heart contractility do not upregulate KLF4 or undergo EndoMT. snRNA-seq revealed that Ncx1-/- embryos failed to transition to early-EndoMT and have reduced mesenchymal markers. Whole hearts of e9.5 mouse embryos (Ncx1+/+ (wildtype), Ncx1+/-, Ncx1-/-) were microdissected and flashfrozen. Five flashfrozen hearts of each genotype were pooled and prepared for single nuclei RNA-sequencing, 1 technical replicate per genotype.