3027 – HSC-INDEPENDENT EMP CONTAIN ERYTHROID/MEGAKARYOCYTE AND INNATE LYMPHOID/MYELOID LINEAGE HETEROGENEITY PRIOR TO SEEDING THE FETAL LIVER

Hematopoietic ontogeny unfolds in two broad programs- a complex hematopoietic stem cell (HSC)-independent program followed by an HSC-dependent program. We have previously determined that HSC-independent, multi-lineage “erythro-myeloid” progenitors (EMP) emerge from hemogenic endothelium in the yolk sac of the murine embryo and engraft the fetal liver to generate the first definitive erythroid, granulocyte, monocyte, and NK cells prior to the establishment of HSC-derived hematopoiesis. Analysis of single cell transcriptomic data from E9.5 EMP and from hematopoietic cells in the E12.5 fetal liver provides a unique opportunity to tie predictions of progenitor identity to subsequent differentiation states. We analyzed 2,959 kit+CD41+CD16/32+ yolk sac EMP and 7,495 fetal liver hematopoietic cells pooled from 30 E9.5 and 3 E12.5 embryos, respectively, using the 10X Genomics and inDrops platforms. Dimensionality reduction coupled with graph-based clustering and analysis of differentially expressed marker genes allowed us to presumptively identify erythroid-, megakaryocyte-, macrophage-, and myeloid-biased clusters both in EMP and in the E12.5 fetal liver. Functional colony-forming assays confirm the existence of primary myeloid- and erythroid-biased EMP in E9.5 embryos. E9.5 EMP also contain a cluster of innate lymphoid-biased progenitors, consistent with the recently identified NK cell lineage potential of EMP. Integration of EMP and fetal liver datasets further supported the concept that EMP consist of a surprising, early complexity of lineage-biased progenitors, whose differentiation continue in the fetal liver. Interestingly, EMP also contained a cluster of hemogenic endothelial-biased progenitors that were not found in the fetal liver, and which serve as a source of diverging erythroid/megakaryocyte and innate lymphoid/myeloid pseudotime trajectories. Taken together, our data support the concept that EMP emerge from hemogenic endothelium and begin to differentiate into multiple lineage-biased progenitors before engrafting the fetal liver to complete their differentiation along erythroid/megakaryocytic and innate immune cell paths to meet the needs of the developing embryo prior to the establishment of HSC-derived blood cell production.