Single cell profiling reveals three endothelial to hematopoietic transitions with divergent isoform expression landscapes

Hemogenic endothelium (HE) is recognized as the origin of all definitive blood cells including hematopoietic stem cells (HSC). However, the mechanisms governing the hematopoietic progenitor versus HSC fate choice within the HE remains unknown. To explore this, we combined differentiation assays with full-length single-cell transcriptome data for extra-embryonic yolk sac (YS) and intra-embryonic aorta–gonad–mesonephros (AGM) region HE populations. We identified and localized three differentiation trajectories, each containing a distinct HE subset and potential: erythro-myeloid progenitor-primed HE in the YS plexus, lympho-myeloid progenitor-primed HE in large YS arteries, and HSC-primed HE in the AGM. Chromatin modifiers and spliceosome components were enriched AGM HE. Nanopore long-read sequencing identified AGM specific, functionally unique, splice variants of stemness-associated factors Runx1, Mecom, and Dnmt3b. Our data indicate that isoform expression changes are associated with HSC generation and provide a unique resource for studying cell fate decisions in HE. Overall design: Long Read single cell RNA-seq of cells undergoing hemogenic to endothelial transition in AGM at E9.5 and E10.5 and Yolk Sac (YS) at E9, E9.5, E10.5. To study the HE populations present at different anatomical locations and time points during the mouse development, we used transgenic reporter mouse lines GFI1-GFP and RUNX1B-RFP and performed Oxford Nanopore Long Read Sequencing (single cell RNA seq) on FACS isolated E9.5 and E10.5 AGM HE cells (RUNX1+, KIT-,CDH5+, GFI1+/-, CD41-, CD45-), and E9, E9.5, E10.5 YS HE cells (RUNX1+, KIT+, GFI1+/-, CD31+, CD41-, CD45-). Total 27 cells were used. We selected 9 cells from the middle of AGM_3 and AGM_4 cluster for AGM HE (HE-HSC), 9 cells from the middle of YS_m1 and YS_m2 as YSm HE (HE-LMP) and 9 cells from the middle of YS_b2 and YS_b3 as YSb HE (HE-EMP).