Understanding the cell fate and behavior of progenitors at the origin of the mouse cardiac mitral valve

Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve. Cells were isolated from enbryos from Tie2crex Rosa26 tdTomato female mice after microdissection of the AVC and embryonic mitral valves. At least 60 embryos were used for each stage of development Cells were then FACS sorted using tomato as a reporter and used in 10X chromium

先天性心脏畸形包含二尖瓣缺损，其发病机制目前仍未得到充分阐释。在胚胎发育过程中，房室管（atrioventricular canal）内的特定心内膜细胞群会经历内皮细胞向间充质细胞转化（endothelial-to-mesenchymal transition）过程，进而分化为二尖瓣瓣膜细胞。然而，这些祖细胞的身份与命运决定，以及其衍生细胞在瓣膜小叶中的行为与分布，目前仍不明确。本研究通过对遗传标记的心内膜细胞以及显微切割获取的小鼠胚胎期与出生后二尖瓣瓣膜进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），以解析其发育轨迹。本研究明确了调控祖细胞特化的代谢过程，以及祖细胞对瓣膜细胞亚型的贡献。通过回顾性多色克隆分析，本研究阐明了心内膜细胞来源克隆的特定生长模式与行为特征，这些克隆以精准方式构建出功能性瓣膜小叶。本研究数据揭示了遗传与代谢机制如何特异性调控部分心内膜细胞的命运，使其通过独特的克隆贡献方式参与瓣膜形成。实验样本取自经显微切割获取的房室管（atrioventricular canal, AVC）与胚胎二尖瓣组织，分离自Tie2-Cre × Rosa26-tdTomato雌性小鼠的胚胎。每个发育阶段至少使用60枚胚胎，随后以tdTomato为报告基因通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）对细胞进行纯化，用于10× Chromium平台测序。