Abstract
The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.
Highlights
The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart
To define the cellular heterogeneity within the epicardium, we performed an integration of E12.5 and E16.5 data sets using canonical correlation analysis (CCA) followed by uniform manifold approximation and projection (UMAP) using Seurat to rule out batch effects (Supplementary Fig. 3a–d), and present a merged analysis of E12.5 and E16.5 cells (Fig. 1e)
Five broad identities emerged, consistent with marker genes identified by hierarchical clustering and gene ontology (GO) analysis (Fig. 1g–k and Supplementary 5a, b; Supplementary Dataset 1): (1) early developmental stage progenitor (C1, C3); (2) early epithelial-tomesenchymal transition (EMT) (C5); (3) late developmental stage mesothelial (C2, C4); (4) late developmental stage EMT/ mesenchymal (C6, C7); and (5) a rare population of approximately 40 cells (C8, 1.64% of total) that display an enrichment in IGF pathway genes and express high levels of Cav[1] previously implicated in zebrafish heart regeneration[13]
Summary
The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. We performed single-cell RNA-sequencing of EPDCs and coronary ECs at critical developmental stages to gain insight into the mechanisms responsible for patterning of the developing coronary vasculature via distinct epicardial cell populations[13,14,15]. Harnessing the principles that define the spatial architecture of the developing coronary vasculature may provide strategies to stimulate angiogenesis and improve perfusion of ischemic heart tissue, a limiting aspect of regenerative medicine approaches
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