Single-Cell Transcriptional Heterogeneity Landscapes of Third Heart Field Progenitor Cells

The transcriptional heterogeneity of third heart field cardiac progenitor cells (THF-CPCs) and their mechanisms of transcriptional regulation are currently unknown. This study aimed to determine the single-cell transcriptional landscapes and to reveal the transcriptional heterogeneity of THF-CPCs from embryonic and postnatal mouse hearts.Subsequently, scRNA-seq and analysis were performed with the Seurat and Monocle 3 packages in R software with standard procedures. Tbx18 and Nkx2.5 mRNA expression was determined with in situ hybridization. Immunofluorescence and β-galactosidase staining were performed to analyze reporter gene expression in mouse lineage tracing studies. Single-cell transcriptomic analysis revealed that the embryonic mouse heart contains a pool of multipotent CPCs, including first heart field, second heart field and third heart field (THF) CPC lineages. Furthermore, the key marker genes Tbx18, Wt1 and Tcf21 showed overlap in THF-CPCs, and this independent progenitor population showed unique transcriptional and spatiotemporal heterogeneity in the developing heart. According to transcriptional heterogeneity, THF-CPCs can be divided into typical, atypical and fusion cell sub-clusters. According to the spatiotemporal heterogeneity, at least, there are two types, “young”THF-CPCs, and “aged”THF-CPCs; furtherly, extracellular matrix (ECM)signal are sustained expressed in“aged”THF-CPCs, this maybe the key reasons of its loss of the pluripotent differentiation potential after birth. According to gene regulatory networks,THF-CPCs include the following cell types at least: cardiomyocyte and non-cardiomyocyte epicardial epithelial cells, which also includes components of cardiac progenitor cells.Interestingly, the transcriptional signals of epicardial epithelial cells and cardiomyocytes were clearly distinct; for example, the Tcf21 and Twist1 regulons were enriched in epicardial epithelial cells. The tbx18 regulon lacked gene regulatory networks involved in the regulation of myocardial structure, function, differentiation and proliferation. Acta1 and Tnni1 were enriched in the Hif1a regulon, which is involved in the regulation of myocardial cell structure and function. Snai1 and Tcf21 were enriched in the Twist1 regulon, which is involved in the transcriptional regulation of fibroblasts (Tgfbi and Cdh11) and smooth muscle cells (Myocd).It revealed that cellular diversity and heterogeneity of THF-CPCs was regulated by various regulons; the epithelial–mesenchymal transition (EMT) related regulons may play an important role in regulating the differentiation of fibroblasts and smooth muscle cells of THF-CPCs.Tbx18, Wt1 and TCF21-expressing cells should be defined as THF-CPCs. These cells also exist in cardiomyocytes during early embryonic development. Spatial and temporal heterogeneity may be an important reason explaining the controversy regarding THF-CPCs.