Single cell profiling reveals three endothelial to hematopoietic transitions with divergent isoform expression landscapes [RNA-seq]

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 remain 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. Here we identify and localized three differentiation trajectories, each containing a distinct HE subset: erythro-myeloid progenitor-primed HE in the YS plexus, lympho-myeloid progenitor-primed HE in large YS arteries, and HSPC-primed HE in the AGM. Chromatin modifiers and spliceosome components were enriched in AGM HE. This correlated with a higher isoform complexity of the AGM HE transcriptome. Distinct HEAGM specific isoform expression patterns were observed for a broad range of genes, including stemness-associated factors like Runx1. Our data forms a unique resource for studying cell fate decisions in different HE populations. 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-). Based on analyses of GEO accession GSE274544 cells selected for nanopore sequencing came from HEAGM (clusters AGMc3 andAGMc4), HEYSA (cluster YSm1 and YSm2), HEYSP (clusters YSb2 and YSb3) early yolk sac hematopoietic progenitor cluster p1 and p2