Single-cell RNA-sequencing of angioblasts and endothelial cells from E9.5-E11.5 mouse embryos

Background: The formation of the lymphatic system has been a subject of controversy for over a century. Although the accepted view is that lymphatic endothelial cells originate exclusively from veins, there have been reports of non-venous derived lymphatics in multiple tissues. Methods: We performed single-cell RNA sequencing analyses on ~20000 individual cells isolated from Pax3Cre/+;Rosa26tdTomato mouse embryos at E9.5, E10.5 and E11.5, the temporal window in which lymphatic endothelium initially emerges. Results: These analyses revealed the existence of populations of lymphangioblasts which arise from paraxial mesoderm and directly incorporate into the lymph sacs. Our findings support the notion that lymphatic endothelial cells also have non-venous origins and explain the discrepancies between previous studies. Endothelial progenitors and endothelial cells were isolated from Pax3Cre/+;Rosa26tdTomato embryos at ~E9.5, ~E10.5 and ~E11.5, to define transcriptional differences between paraxial mesoderm-derived and and non-paraxial mesoderm-derived endothelium. Single cell gene expression was profiled using the 10x Genomics Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit v3.1.