Heart development is a continuous process involving significant remodeling during embryogenesis and neonatal stages. To date, several groups have used single-cell sequencing to characterize the heart transcriptomes but failed to capture the progression of heart development at most stages. This has left gaps in understanding the contribution of each cell type across cardiac development. Here, we report the transcriptional profile of the murine heart from early embryogenesis to late neonatal stages. Through further analysis of this dataset, we identify several transcriptional features. We identify gene expression modules enriched at early embryonic and neonatal stages; multiple cell types in the left and right atriums are transcriptionally distinct at neonatal stages; many congenital heart defect-associated genes have cell type-specific expression; stage-unique ligand-receptor interactions are mostly between epicardial cells and other cell types at neonatal stages; and mutants of epicardium-expressed genesWt1andTbx18have different heart defects. Assessment of this dataset serves as an invaluable source of information for studies of heart development.

Endothelial cells line the inner surface of vasculature and play an important role in normal physiology and disease progression. Although most tissue is known to have a heterogeneous population of endothelial cells, transcriptional differences in organ specific endothelial cells have not been systematically analyzed at the single cell level. The Tabula Muris project profiled mouse single cells from 20 organs. We found 10 of the organs profiled by this Consortium have endothelial cells. Unsupervised analysis of these endothelial cells revealed that they were mainly grouped by organs, and organ-specific cells were further partially correlated by germ layers. Unexpectedly, we found all lymphatic endothelial cells grouped together regardless of their resident organs. To further understand the cellular heterogeneity in organ-specific endothelial cells, we used the heart as an example. As a pump of the circulation system, the heart has multiple types of endothelial cells. Detailed analysis of these cells identified an endocardial endothelial cell population, a coronary vascular endothelial cell population, and an aorta-specific cell population. Through integrated analysis of the single cell data from another two studies analyzing the aorta, we identified conserved cell populations and molecular markers across the datasets. In summary, by reanalyzing the existing endothelial cell single-cell data, we identified organ-specific molecular signatures and heart-specific subpopulations and molecular markers. We expect these findings will pave the way for a deeper understanding of vascular biology and endothelial cell-related diseases.

Heart organoids have the potential to generate primary heart-like anatomical structures and hold great promise as in vitro models for cardiac disease. However, their properties have not yet been fully studied, which hinders their wide spread application. Here we report the development of differentiation systems for ventricular and atrial heart organoids, enabling the study of heart diseases with chamber defects. We show that our systems generate chamber-specific organoids comprising of the major cardiac cell types, and we use single cell RNA sequencing together with sample multiplexing to characterize the cells we generate. To that end, we developed a machine learning label transfer approach leveraging cell type, chamber, and laterality annotations available for primary human fetal heart cells. We then used this model to analyze organoid cells from an isogeneic line carrying an Ebstein’s anomaly associated genetic variant in NKX2-5, and we successfully recapitulated the disease’s atrialized ventricular defects. In summary, we have established a workflow integrating heart organoids and computational analysis to model heart development in normal and disease states.

Choline acetyltransferase (ChAT)-positive neurons in neural stem cell (NSC) niches can evoke adult neurogenesis (AN) and restore impaired brain function after injury, such as acute ischemic stroke (AIS). However, the relevant mechanism by which ChAT⁺ neurons develop in NSC niches is poorly understood. Our RNA-seq analysis revealed that dimethylarginine dimethylaminohydrolase 1 (DDAH1), a hydrolase for asymmetric N^G,N^G-dimethylarginine (ADMA), regulated genes responsible for the synthesis and transportation of acetylcholine (ACh) (Chat, Slc5a7 and Slc18a3) after stroke insult. The dual-luciferase reporter assay further suggested that DDAH1 controlled the activity of ChAT, possibly through hypoxia-inducible factor 1α (HIF-1α). KC7F2, an inhibitor of HIF-1α, abolished DDAH1-induced ChAT expression and suppressed neurogenesis. As expected, DDAH1 was clinically elevated in the blood of AIS patients and was positively correlated with AIS severity. By comparing the results among Ddah1 general knockout (KO) mice, transgenic (TG) mice and wild-type (WT) mice, we discovered that DDAH1 upregulated the proliferation and neural differentiation of NSCs in the subgranular zone (SGZ) under ischemic insult. As a result, DDAH1 may promote cognitive and motor function recovery against stroke impairment, while these neuroprotective effects are dramatically suppressed by NSC conditional knockout of Ddah1 in mice.