Single cell profiling of hemogenic endothelium and its surrounding niche in the mouse dorsal aorta. Single cell profiling of hemogenic endothelium and its surrounding niche in the mouse dorsal aorta

The first hematopoietic stem cells originate from hemogenic endothelium (HE), that trans-differentiate into the lumen to form hematopoietic clusters. The molecular mechanisms driving this transition are only poorly understood. Here, we performed single cell RNA-seq profiling of HE cells utilising a RUNX1 and GFI1 reporter mouse line.This allowed for a detailed characterisation of HE cells during endothelial-to-hematopoietic transition. Additionally we profiled the cells surrounding the dorsal aorta. These dataset present a comprehensive profile at single cell resolution of cells invovled in the endothelial-to-hematopoietic transition and the surrounding dorsal aorta niche. Overall design: Single cell RNA-seq of endothelium, hemogenic endothelium, cells undergoing endothelial-to-hematopoietic transition, and intra aortic hematopoietic clusters of E10.5 and E11.5. Surrounding mesenchymal cells of E10.5 were also profiled. AGM of E10.5 and E11.5 embryos were dissected and scRNA-seq (Smart-seq2) was performed on phenotypically defined FACS populations. Utilizing the Gfi1/Gfi1b transgenic reporter mouse line, the following FACS population were sorted from E10.5 and E11.5 embryos; endothelial cells (FACS-ENDO, TER119-CD41-CD45-KIT-CDH5+GFI1-GFI1b-), hemogenic endothelium (FACS-HE, TER119-CD41-CD45-KIT-CDH5+GFI1+GFI1b-), cells undergoing endothelial-to-hematopoietic transition (FACS-EHT, TER119-CD41+CD45-KIT+CDH5+GFI1+GFI1b-), and intra-aortic hematopoietic clusters (FACS-IAHC, TER119-CD41+CD45-KIT+CDH5+GFI1+GFI1b+). Additionally, cells were isolated from E10.5 embryos utilising the Runx1b transgenic reporter to include hemogenic endothelium (FACS-HE, TER119-CD41-CD45-KIT-CDH5+RUNX1b+) and Runx1b+ mesenchymal cells surrounding the dorsal aorta (FACS-MES, TER119-CD41-CD45-KIT-CDH5-RUNX1b+). Finally, scRNA-seq was also performed on cells sorted based on ACE (CD143), encompassing endothelial cells (FACS_ENDO, TER119-CD41-CD45-KIT-CDH5+ACE-GFI1-), ACE positive cells (FACS-ACE_SinglePos, TER119-CD41-CD45-KIT-CDH5+ACE+GFI1-) and hemogenic endothelium (FACS-HE_DoublePos, TER119-CD41-CD45-KIT-CDH5+ACE+GFI1+).