P1332: all embryonic hematopoietic waves emerge from hemato-endothelial progenitors

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: The emergence of blood cells during mammalian embryonic development occurs in three independent waves of different origin. Importantly, the first blood cells emerge in the extra embryonic Yolk-sac (YS). The YS provides the embryo first with primitive erythrocytes (EryP, wave 1) and shortly after with erythro-myeloid progenitors (EMPs, wave 2). Both YS-derived hematopoietic waves are essential for embryonic development. Later on, hematopoietic stem cells (HSCs, wave 3), originate in the aorta-gonado-mesonephros region of the embryo proper. Both YS-derived EMPs and intra-embryonic blood progenitors including HSCs then colonize the fetal liver, where they expand and differentiate. Finally, before birth, hematopoietic progenitors seed the ultimate hematopoietic site, the bone marrow. After birth HSCs gradually take over for the hematopoiesis, however, they are dispensable during embryonic development. EryPs are believed to arise directly from mesoderm, while EMPs and HSCs are established to originate from hemogenic endothelium. The existence of primitive macrophages (MF) and megakaryocytes (Mk) derived from the first wave was described but remains controversial. The EMP wave is the major source of red blood cells during the second two trimesters of human pregnancy. Nearly all extremely low birth weight newborns develop anemia, therefore understanding the emergence of EMP-derived erythropoiesis is critical for future clinical applications in pre and early post-natal therapy. Aims: General goal of this project is to reveal the molecular mechanism of specification of yolk-sac derived hematopoietic lineages. Additionally, we aim to determine whether the first MFs and Mks emerge from EMPs or primitive hematopoietic wave and whether the EryPs emerge from an hemato-endothelial ancestor. Methods: We have performed single-cell RNA profiling of c-kit+ cells isolated from yolk sac of murine embryos at E7.5, E8.5 and E9.5 using the Illumina platform. The data from this experiment is validated using multi-parametric flow cytometry, qPCR analysis and via a novel transgenic mouse reporter system. Results: Using single-cell transcriptomic analysis of c-kit expressing YS progenitors, we show, that at E7.5, the progenitors of EryP (pEryP, wave 1) are very close to cells with hemato-endothelial expression profile. The developmental pathway trajectory analysis of E7.5-E9.5 YS c-kit+ hemato-endothelial cells suggests that EryPs emerge directly from E7.5 hemato-endothelial cells. Moreover, since the first macrophage progenitors (pre-MF) in our dataset were found at E9.5, way after the emergence of EMPs (E8.5), and in close proximity to the EMP clusters, we suggest, that first MFs derive from the EMP wave. Summary/Conclusion: In summary, we propose that all hematopoietic cells share a common hemato-endothelial ancestor and that the primitive wave generates solely cells with erythroid fate while the EMP wave produces erythroid as well as the first myeloid cells during embryonic development. Keywords: Fetal, Yolk sac, Hematopoiesis, RNA-seq