Integration of single-cell transcriptomes and biological function reveals distinct behavioral patterns in bone marrow endothelium.
During blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreERT2;R26FUCCI2aR reportermice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-β signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-β1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate.
Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue's homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs' ECs to decode mechanistic information and explore therapeutics.