Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

Highlights d Artery endothelial cells (ECs) of neonatal hearts have a unique response to injury d Injury stimulates artery cell migration and reassembly into collateral arteries d CXCL12-CXCR4 signaling guides artery reassembly, facilitating heart regeneration d Adult artery ECs can be induced to undergo artery reassembly with exogenous CXCL12

Background: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. While these functional differences are presumably imprinted in the transcriptome, the pathways and networks which sustain EC heterogeneity have not been fully delineated. Methods: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA-sequencing (scRNA-seq) data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We employed a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from scRNA-seq data. Results: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either scRNA-seq or bulk RNA-seq, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (e.g. liver, brain) whereas others (i.e. adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. In addition, we found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. Finally, we identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. Conclusions: In summary, we use scRNA-seq data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.

Highlights d GSK-3b inhibition-mediated hiPSC-cardiomyocyte proliferation is cell density dependent d GSK-3b inhibition with reduced cell-cell contact massively expands hiPSC-cardiomyocytes d LEF/TCF activity inhibits hiPSC-cardiomyocyte maturation without promoting cell cycling d Long-term expansion does not alter cardiomyocyte contractile function

During development, the formation of a mature, well-functioning heart requires transformation of the ventricular wall from a loose trabecular network into a dense compact myocardium at mid-gestation. Failure to compact is associated in humans with congenital diseases such as left ventricular non-compaction (LVNC). The mechanisms regulating myocardial compaction are however still poorly understood. Here, we show that deletion of the Ino80 chromatin remodeler in vascular endothelial cells prevents ventricular compaction in the developing mouse heart. This correlates with defective coronary vascularization, and specific deletion of Ino80 in the two major coronary progenitor tissues-sinus venosus and endocardium-causes intermediate phenotypes. In vitro, endothelial cells promote myocardial expansion independently of blood flow in an Ino80-dependent manner. Ino80 deletion increases the expression of E2F-activated genes and endothelial cell S-phase occupancy. Thus, Ino80 is essential for coronary angiogenesis and allows coronary vessels to support proper compaction of the heart wall.