Endocardial primary cilia and blood flow regulate EndoMT during endocardial cushion development

Valvular heart disease affects 2.5% of the population and is frequently associated with congenital heart disease. Blood flow is critical for valve formation, but the cellular mechanosensors translating flow into the transcriptional regulation of valve development remain undiscovered. Here, we identify that primary cilia and blood flow in mouse embryos regulate early valve development in vivo by regionally controlling endothelial-to-mesenchymal transition (EndoMT) through the modulation of Krüppel-like factor 4 (Klf4) in the endocardial cushions. Endocardial ciliation decreases during cushion development in regions of high shear stress, correlating with KLF4 downregulation and EndoMT progression. Mouse embryos lacking cilia exhibit blood flow-dependent accumulation of KLF4 and impaired cushion cellularization. Single-nucleus RNA sequencing revealed that the cilia-knockout and contractility-knockout endocardium fails to progress through EndoMT pseudostages, retains endothelial markers, and has reduced EndoMT and mesenchymal genes that KLF4 antagonizes. These data indicate that endocardial primary cilia function as mechanosensors in cushion development through the regional regulation of KLF4.