AVCu, proliferating atrioventricular cushion mesenchymal cells; SMC, smooth muscle cells; FB, fibroblasts; C20, cluster number 20

AVCu, proliferating atrioventricular cushion mesenchymal cells; SMC, smooth muscle cells; FB, fibroblasts; C20, cluster number 20. Transcriptional characteristics of cardiomyocytes, endothelial cells, and valve development CMs, endothelial cells, smooth muscle cells, mesenchymal cells, macrophages, and epicardial cells had cluster sizes that ranged from 63 to 4,716 cells (Fig. progenitors transition to a mature cell type is unknown. Xiao et al. demonstrated that Hippo kinases Lats1/2 promote MAC13772 epicardial-fibroblast transition which is essential for maintaining proper extracellular milieu and coronary vessel development. INTRODUCTION The epicardium, cells covering the outer layer of the heart, originates from the extra-cardiac proepicardium. The proepicardium is compartmentalized into populations that give rise to cardiac endothelium and mesenchymal cells: fibroblasts and smooth muscle (Katz et al., 2012; Acharya et al., 2012). At mouse embryonic day (E)9.5, proepicardial cells attach to myocardium, spread as a continuous epithelial sheet, and form a single cell layer covering the entire myocardium. The epicardium expresses a number of important genes including signaling molecules such as Retinaldehyde dehydrogenase 2 (function in epicardial progenitor cell diversification. A high-throughput single cell (sc) RNA-sequence (seq) platform, Drop-seq, was adopted to characterize E13.5 and E14.5 cardiac cellular composition and heterogeneity in deficient and control hearts (Macosko et al., 2015). Our data revealed that Lats1/2 activity is required for EPDC progression from a transient subepicardial mesenchyme to fully differentiated cardiac fibroblasts and provide insight into mechanisms coordinating fibroblast development with coronary vascular remodeling in heart development. RESULTS Epicardial deletion of results in defective MAC13772 coronary vessel development We deleted in E11.5 epicardium using the allele (Zhou et al., 2008). MAC13772 conditional knock out (CKO) embryos failed to survive past E15.5 (Fig. S1A). CKO E14.5 hearts appeared normal (Fig. S1B,C), but E15.5 mutant hearts were smaller, with less compacted myocardium (Fig. 1A, Fig. S1B). CKO embryos also displayed skin hemorrhages, as well as, herniated livers and intestines (Fig. S1DCF). Open in a separate window Figure 1 Lats1/2 deficiency results in defective heart development. See also Figure S1 and Figure S2(A) E15.5 histology showed reduced compacted myocardium in CKO had decreased vessel coverage (asterisks) and blood islands (arrows) on ventral and lateral heart. (C) Pecam-1 IF. (D) Quantitation of vasculature in Fig. 1C. (E) Podoplanin labels epicardium and hearts had increased nuclear Yap in epicardium (white arrowheads) and subepicardium (yellow arrowheads). (F) Quantification of Yap subcellular localization. (G) CKO had decreased p-Yap in epicardium (white arrows) and subepicardium (yellow arrows). (HCI) hearts with reduced were normal at E15.5. Scale bar: A left panels 400m; right panels 80m; B 500m; C upper panels 200m, bottom panels 100m, E 25m, G 50m, H 200 m. Data: means SD. *CKO hearts revealed reduced vessel coverage and density with blood island-like structures (Fig. 1B). Pecam-1 immunofluorescence (IF) staining with confocal microscopy and automated quantification MAC13772 revealed dorsal vasculature had decreased branching and reduced vessel coverage with fewer junctions and increased lacunarity (Fig. 1C,D). As controls, we injected tamoxifen to and Cre negative littermates. Coronary MAC13772 vessel development in controls was normal (Fig. S2A,B). We examined Yap sub-cellular localization and Yap phosphorylation (p-Yap) as a readout of Lats kinase activity. Yap localization in CKO hearts, detected by total Yap and Podoplanin IF, revealed increased nuclear Yap in both epicardium and subepicardium (Fig. 1E,F). IF revealed decreased p-Yap in CKO epicardium and subepicardium but no change in CMs since we inactivated in the epicardial lineage (Fig. 1G). Podoplanin, restricted to the epicardium in control embryos, was also expressed in CKO subepicardium suggesting that EMT occurred prior to repression TFR2 of the epicardial program (Fig. 1G). Recent work indicated that epicardial deletion of and led to defective EMT (Singh et al., 2016). hybridization with EMT markers revealed that was elevated in CKO hearts, while was unchanged (Fig. S2C,D). Tgf-signaling that promotes epicardial EMT (Sridurongrit et al., 2008) was elevated in CKO epicardium as determined.