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Coordination of Endothelial Cell Positioning and Fate Specification By ARTICLE https://doi.org/10.1038/s41467-021-24414-z OPEN Coordination of endothelial cell positioning and fate specification by the epicardium Pearl Quijada 1,8, Michael A. Trembley1, Adwiteeya Misra1,2, Jacquelyn A. Myers 3,4, Cameron D. Baker 3,4, Marta Pérez-Hernández 5, Jason R. Myers3,4, Ronald A. Dirkx Jr.1, ✉ Ethan David Cohen6, Mario Delmar5, John M. Ashton 3,4 & Eric M. Small 1,2,7 The organization of an integrated coronary vasculature requires the specification of immature 1234567890():,; 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. 1 Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. 2 Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA. 3 Genomics Research Center, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. 4 Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. 5 Leon H. Charney Division of Cardiology, Department of Medicine, New York University School of Medicine, New York, NY, USA. 6 Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. 7 Department of Pharmacology and Physiology, University of Rochester, Rochester, NY, USA. 8Present address: Department of Integrative Biology and Physiology, Molecular Biology Institute, Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA Cardiovascular Theme, David Geffen School of Medicine, University of California, Los ✉ Angeles, CA, USA. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:4155 | https://doi.org/10.1038/s41467-021-24414-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24414-z oronary endothelial cells (ECs) organize into an intricate enrichment (Aldh1a2, Tbx18, Tcf21, Wt1) and did not express Cvascular network that provides the heart with oxygen and high levels of cardiomyocyte genes (Tnnt2, Myh7)(Supplementary nutrients and removes metabolic waste. During embryonic Fig. 1e). Increased expression of the mesenchymal cell marker development, localized specification of immature ECs into arterial Pdgfra was observed in a number of GFP+ cells at E16.5, consistent and venous fates occurs within the compact myocardium and sub- with the acquisition of a motile phenotype and differentiation into epicardium, respectively1.ECfatespecification and maturation are interstitial cell types (Supplementary Fig. 1f–h). accomplished through cell-intrinsic transcriptional programs that Single-cell RNA-sequencing (scRNA-seq) was performed on facilitate appropriate interconnections of the blood-supplying and EPDCs captured using the 10× Genomics platform (Fig. 1d). We blood-draining vascular beds2. Analyses of intersomitic and retinal excluded cell doublets based upon unique molecular identifier vessel development identified a population of endothelial tip cells counts, and mitochondrial and ribosomal gene expression that are particularly responsive to secreted angiogenic factors patterns were analyzed and filtered to obtain 3405 (E12.5) and such as vascular endothelial growth factor (Vegf), highlighting 2436 (E16.5) single EPDCs (Supplementary Fig. 2a, b). To define the functional heterogeneity of ECs and revealing mechanisms the cellular heterogeneity within the epicardium, we performed governing vascular pathfinding, remodeling, and maturation3. an integration of E12.5 and E16.5 data sets using canonical Clinical studies aimed at improving perfusion of the ischemic heart correlation analysis (CCA) followed by uniform manifold and skeletal muscle have attempted to recapitulate these develop- approximation and projection (UMAP) using Seurat to rule out mental angiogenic programs4. However, the mechanisms through batch effects (Supplementary Fig. 3a–d), and present a merged which coronary ECs coordinate positional information and fate analysis of E12.5 and E16.5 cells (Fig. 1e).Thisanalysisrevealed specification remain elusive, and current therapeutic strategies have 8 distinct populations (C1-C8) with a considerable contribution failed to generate a robust, functional vascular network. of proliferation state and developmental age to cellular The epicardium consists of a single layer of mesothelial cells on phenotype (Fig. 1e, f and Supplementary Fig. 4a, b). To facilitate the surface of the heart that harbors a population of multipotent the identification of epicardial cell phenotypes, E12.5 and E16.5 progenitors. Following epithelial-to-mesenchymal transition data were merged and a CCA was performed with previously (EMT), epicardium-derived cells (EPDCs) migrate into the compact published scRNA-seq data obtained from the postnatal day 1 myocardium and differentiate into cardiac fibroblast and vascular mouse heart17 (Supplementary Fig. 4c, d). Within these mural cell lineages5–7. Construction of the coronary plexus requires populations, five broad identities emerged, consistent with the integration of epicardium-derived mural cells with arterial and marker genes identified by hierarchical clustering and gene venous ECs derived from the sinus venosus and endocardium5,8,9. ontology (GO) analysis (Fig. 1g–k and Supplementary 5a, b; Genetic or mechanical disruption of the epicardium has also Supplementary Dataset 1): (1) early developmental stage revealed important paracrine contributions to cardiomyocyte progenitor (C1, C3); (2) early EMT (C5); (3) late developmental growth10 and coronary plexus formation11,12. Our previous study stage mesothelial (C2, C4); (4) late developmental stage EMT/ found that epicardial EMT is required for coronary blood vessel mesenchymal (C6, C7); and (5) a rare population of approxi- maturation and integrity, at least partially via contributing vascular mately 40 cells (C8, 1.64% of total) that display an enrichment pericytes to the growing plexus7. in IGF pathway genes and express high levels of Cav1 previously In this study, we performed single-cell RNA-sequencing of implicated in zebrafish heart regeneration13. EPDCs and coronary ECs at critical developmental stages to EPDCs in C1 and C3 are characterized by robust expression of gain insight into the mechanisms responsible for patterning of epicardial gene markers Tbx18 and Upk3b18,19, and GO terms the developing coronary vasculature via distinct epicardial cell associated with proliferation and epithelial cell differentiation, populations13–15. We found that epicardial EMT is not only defining populations of self-replicating E12.5 mesothelial cells responsible for the differentiation of EPDCs into vascular mural (Fig. 1g, h). In contrast, C5 is enriched in E12.5 EPDCs that lineages7, but also restricts the expression of chemotactic signals exhibit a predisposition towards EMT prior to the acquisition of a to discrete populations of mural cells that provide detailed posi- motile gene program, based on the expression of early markers of tional information, reminiscent of the guidepost neuron16. the mesenchymal and smooth muscle phenotype, including Genetic disruption of epicardial EMT in mice leads to profound Pdgfra, Itgb5, Sox9, and Tagln220,21 (Fig. 1g, i). C2 and C4 alterations in EC developmental trajectory, which includes the contain an over-abundance of E16.5 EPDCs that express genes accumulation of an immature EC population within the sub- related to maintenance of mesothelial characteristics (Maf, Btg2), epicardium. Importantly, EC maturation and migration are both likely representing cells that remain on the cardiac surface22,23 directly controlled by angiogenic chemokines, providing a para- (Fig. 1g, j). Genes encoding extracellular matrix proteins such as digm that coordinates EC localization and arteriovenous (AV) Postn, Dpt,andCol3a1 are robustly expressed in E16.5 EPDCs specification. Harnessing the principles that define the spatial in C6 and C7, consistent with the acquisition of a mesenchymal architecture of the developing coronary vasculature may provide phenotype20 (Fig. 1g, k). Of note, these data also revealed unique strategies to stimulate angiogenesis and improve perfusion of vascular programs based upon the emergence of angiogenesis ischemic
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