Study On The Labeling Of Neurl3 In The Development Of Mouse Intra-and Extra-embryonic Hematopoietic System Using Transcriptomics And Functional Analysis

The generation of hematopoietic stem cells（HSCs）is one of the most important events in the development of hematopoiesis,and plays an irreplaceable role in the maintenance of the whole blood system and the survival of life.In recent years,the origin of HSCs has been well understood.In the aorta-gonad-mesonephros region（AGM）during embryonic development,there exists a group of specialized endothelial cells,namely hemogenic endothelial cells（HECs）,which undergo an endothelial-to-hematopoietic transition（EHT）process to acquire a hematopoietic fate,gradually losing their endothelial morphology and transforming into mature HSCs with self-renewal and multilineage differentiation potential.In a recently study,we have been able to achieve efficient capture and precise resolution of the HECs in the AGM by combination of a series of cell surface markers,PK44（CD41^-CD43^-CD45^-CD31⁺CD201⁺Kit⁺CD44⁺）.In addition,the HSC-primed HECs could be highly enriched by the constructed Neurl3^EGFP reporter mouse model,but it is unclear whether Neurl3-EGFP can similarly label and enrich for other hematopoietic populations generated by HECs,including pre-HSCs and HSCs.Prior to the emergence of HSC,the yolk sac,as an important site for blood production,gave rise to a variety of hematopoietic progenitors,including erythroid-myeloid progenitors and lymphoid progenitors.These HSCs-independent hematopoietic progenitors,as the major hematopoietic cell population before birth,make a major contribution to the normal development and survival of the embryo.These hematopoietic progenitors are also derived from the flat Runx1⁺Kit^lo endothelial cells in yolk sac However,compared to the intra-embryonic HECs,the enrichment marker genes,molecular characteristics and the spatial location of HECs in extra-embryonic yolk sac have not been fully resolved.And the controversial question of whether the HECs with potential of lymphogenesis in yolk sac are more abundant in arterial endothelial cells or not,also limits the depth of our understanding of the hematopoiesis in yolk sac.In this study,by conjoint analysis of single-cell RNA sequencing data,flow cytometry analysis and functional assay,we revealed that Neurl3-EGFP not only marks the HSC-primed HECs in the intra-embryonic,but also labes pre-HSCs and efficiently enriches HSCs of fetal liver and adult bone marrow.Unexpectedly,the Neurl3 knockout did not affect the generation and development of HSCs.We also integrally analyzed multiple single-cell transcriptome sequencing datasets from different groups and combined them with different in vitro incubation function experiments and found that Neurl3-EGFP was also a single-gene enrichment marker for yolk sac-derived HECs,and can enrich all the blood-generating potential of yolk sac endothelial cells including myeloid,lymphoid and hematopoietic progenitor cells.Furthermore,we also found that although the HECs of the yolk sac exhibited weaker arterial features compared with the yolk sac arterial endothelial cells or the HECs from AGM by transcriptomic analysis,however,it can be naturally divided into two subpopulations with different arterial and venous characteristics.And there were more GO terms related to B and T cell development enriched in the subpopulation with higher arterial characteristics.To functionally validate the results of transcriptome analysis,we also introduced an Unc5b^{td Tomato} reporter mouse model capable of labeling arterial endothelium.Through functional experiments in vitro,we confirmed that the HECs of yolk sac are heterogeneous,and the subpopulation with arterial features is more able to enrich the lymphoid hematopoiesis potential.Notably,the hematopoietic potential of endothelial cell populations in intra-embryonic aorta and extra-embryonic yolk sac were predominatly enriched in Neurl3-EFFP⁺,whereas the lymphoid potential of the yolk sac lost the expression of Neurl3-EGFP during the hematopoietic progenitor stage,namely the lymphoid potential was exclusively detected in the Neurl3-EGFP negative hematopoietic progenitors in yolk sac.Furthermore,combining flow cytometric analysis of the physiological expression of Neurl3-EGFP in the B cells from fetal liver,adult bone marrow and in virto incubation products,as well as the bioinformatics analysis of the expression level of Neurl3 in fetal liver,adult bone marrow and adult peripheral blood,we deciphered the dynamic expression patterns of Neurl3 during B cell differentiation and maturation.In summary,based on the highly efficient enrichment of the HECs in the AGM region,we further enriched the HECs of yolk sac by Neurl3-EGFP and analyzed the heterogeneity of its lymphoid bias.The difference of B cells development from embryonic and adult stages also provides clues for us to further explore the lymphoid hematopoiesis.These findings also deepen our understanding of hematopoietic development,particularly the yolk sac hematopoiesis,and provide an alternative model mouse for us to more in-depth study the hematopoietic developmental events.