Birth, cell fate and behavior of progenitors at the origin of the cardiac mitral valve

Congenital heart malformations often include mitral valve defects which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal (AVC) undergoes endothelial to mesenchymal transition (EndMT) to give rise to mitral valvular cells. However, the identity, fate decisions of these progenitors as well as the distribution of their derivatives in valve leaflets remain unknown. Here, we use scRNA-seq of genetically labeled mouse AVC endocardial cells and of micro-dissected embryonic and postnatal mitral valves to characterize the developmental road. We uncovered the genetic, cell signaling and metabolic processes underlying specification of the progenitors and how they contribute to subtypes of endothelial and interstitial embryonic and postnatal valvular cells. Using clonal genetic tracing with multicolor reporter, we describe specific modes of growth of endocardial cell-derived clones which build up in a proper manner functional valve leaflets. Our data reveal how both genetic and metabolic specification mechanisms specifically drive the fate of a subset of endocardial cells toward valve progenitors and 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先天性心脏畸形常伴发二尖瓣病变，此类病变的发病机制迄今尚未得到充分阐释。胚胎发育过程中，房室管（atrioventricular canal, AVC）内的特定受限心内膜细胞群会经历内皮-间充质转化（endothelial to mesenchymal transition, EndMT），进而分化为二尖瓣瓣叶细胞。然而，此类祖细胞的身份、命运决定及其子代细胞在瓣叶中的分布情况仍未明确。 本研究通过对基因标记的小鼠房室管心内膜细胞，以及经显微解剖获取的胚胎期与出生后二尖瓣瓣叶样本开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），解析其发育路径。本研究阐明了祖细胞特化所依赖的遗传、细胞信号转导及代谢调控通路，以及此类祖细胞如何分化为内皮型与间质型的胚胎期及出生后瓣叶细胞亚型。本研究借助多色报告基因开展克隆示踪，揭示了心内膜细胞来源克隆的特定增殖模式，此类克隆可有序组装为具备生理功能的二尖瓣瓣叶。 本研究数据揭示了遗传与代谢特化机制如何特异性地促使部分心内膜细胞定向分化为二尖瓣祖细胞，以及此类祖细胞通过独特的克隆贡献方式参与瓣膜形成。本研究通过显微解剖获取小鼠房室管与胚胎期二尖瓣瓣叶，从Tie2Cre×Rosa26-tdTomato双转基因雌性小鼠的胚胎中分离目标细胞。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）分离目标细胞，并利用10X Chromium平台完成后续实验。