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

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 cilia exhibit a blood-flow dependent accumulation of KLF4 in these regions, independent of upstream left-right abnormalities, resulting in impaired cushion cellularization. snRNA-seq revealed that cilia KO endocardium fails to progress to late-EndoMT, retains endothelial markers and has reduced EndoMT/mesenchymal genes that KLF4 antagonizes. Together, these data identify a mechanosensory role for endocardial primary cilia in cushion development through regional regulation of KLF4. Whole hearts of e9.5 mouse embryos (Ift20+/+ (wildtype), Ift20+/-, Ift20-/-) were microdissected and flashfrozen. Five flashfrozen hearts of each genotype were pooled and prepared for single nuclei RNA-sequencing. Ift20+/+ had 3 replicates of this process; Ift20+/- and Ift20-/- had 2.血流对于心脏瓣膜形成至关重要，而细胞机械感受器是将血流信号转化为发育过程中转录调控的核心元件。本研究明确了原纤毛（primary cilia）在体内对心垫形成——心脏瓣膜发育的第一阶段——的空间调控作用，其通过调控Kruppel样因子4（Kruppel-like Factor 4, Klf4），区域性控制内皮细胞向间充质转化（Endothelial to Mesenchymal Transition, EndoMT）。研究发现，心脏内高剪切应力区域在心垫发育进程中会降低心内膜的纤毛发生，该现象与KLF4的下调及EndoMT的进展密切相关。组成型缺失纤毛的小鼠胚胎在上述区域出现了依赖于血流的KLF4积累，且该过程不受上游左右轴发育异常的影响，最终导致心垫细胞化过程受损。单细胞核RNA测序（single nuclei RNA-sequencing, snRNA-seq）结果显示，纤毛敲除（KO）的心内膜细胞无法进展至晚期EndoMT阶段，仍保留内皮细胞标志物，且KLF4所拮抗的EndoMT相关及间充质基因的表达水平显著降低。综上，上述数据明确了心内膜原纤毛通过区域性调控KLF4，在心脏心垫发育中发挥机械感知功能。本研究对胚胎发育第9.5天（E9.5）的小鼠完整心脏（Ift20+/+、Ift20+/-、Ift20-/-，其中Ift20+/+为野生型（wildtype））进行显微解剖并快速冷冻；将每种基因型的5个快速冷冻心脏混合，制备用于单细胞核RNA测序的样本。其中Ift20+/+组设置3个生物学重复，Ift20+/-与Ift20-/-组各设置2个生物学重复。