Divergent expression of Neurl3 from hemogenic endothelial cells to hematopoietic stem progenitor cells during development.

Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.

Adult-repopulating lymphoid potential of yolk sac blood vessels is not confined to arterial endothelial cells.

During embryogenesis, hematopoietic stem progenitor cells (HSPCs) are believed to be derived from hemogenic endothelial cells (HECs). Moreover, arterial feature is proposed to be a prerequisite for HECs to generate HSPCs with lymphoid potential. Although the molecular basis of hematopoietic stem cell-competent HECs has been delicately elucidated within the embryo proper, the functional and molecular characteristics of HECs in the extraembryonic yolk sac (YS) remain largely unresolved. In this study, we initially identified six molecularly different endothelial populations in the midgestational YS through integrated analysis of several single-cell RNA sequencing (scRNA-seq) datasets and validated the arterial vasculature distribution of Gja5+ ECs using a Gja5-EGFP reporter mouse model. Further, we explored the hemogenic potential of different EC populations based on their Gja5-EGFP and CD44 expression levels. The hemogenic potential was ubiquitously detected in spatiotemporally different vascular beds on embryonic days (E)8.5-E9.5 and gradually concentrated in CD44-positive ECs from E10.0. Unexpectedly, B-lymphoid potential was detected in the YS ECs as early as E8.5 regardless of their arterial features. Furthermore, the capacity for generating hematopoietic progenitors with in vivo lymphoid potential was found in nonarterial as well as arterial YS ECs on E10.0-E10.5. Importantly, the distinct identities of E10.0-E10.5 HECs between YS and intraembryonic caudal region were revealed by further scRNA-seq analysis. Cumulatively, these findings extend our knowledge regarding the hemogenic potential of ECs from anatomically and molecularly different vascular beds, providing a theoretical basis for better understanding the sources of HSPCs during mammalian development.