Expression profiling by high throughput sequencing
Summary
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+).
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