Embryonic endothelial evolution towards first hematopoietic stem cells revealed by single-cell transcriptomic and functional analyses

Abstract

Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational embryos. Due to the rare and transient nature, the HSC-competent HECs have never been stringently identified and accurately captured, let alone their genuine vascular precursors. Here, we first used high-precision single-cell transcriptomics to unbiasedly examine the relevant EC populations at continuous developmental stages with intervals of 0.5 days from embryonic day (E) 9.5 to E11.0. As a consequence, we transcriptomically identified two molecularly different arterial EC populations and putative HSCprimed HECs, whose number peaked at E10.0 and sharply decreased thereafter, in the dorsal aorta of the aorta-gonadmesonephros (AGM) region. Combining computational prediction and in vivo functional validation, we precisely captured HSCcompetent HECs by the newly constructed Neurl3-EGFP reporter mouse model, and realized the enrichment further by a combination of surface markers (Procr + Kit + CD44 + , PK44). Surprisingly, the endothelial-hematopoietic dual potential was rarely but reliably witnessed in the cultures of single HECs. Noteworthy, primitive vascular ECs from E8.0 experienced two-step fate choices to become HSC-primed HECs, namely an initial arterial fate choice followed by a hemogenic fate conversion. This finding resolves several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro.

Location, Location, Location: How Vascular Specialization Influences Hematopoietic Fates During Development

“…Similar to the first lymphoid progenitors in the yolk sac, which seem to arise from arterial vessels, recent studies have demonstrated that HE with multilineage hematopoietic and HSC potential first acquire the markers and the transcriptional signatures of the arterial endothelial program (Hadland et al, 2017;Uenishi et al, 2018;Slukvin and Uenishi, 2019;Hou et al, 2020). Arterial fate determination is a hallmark function of the Notch pathway, which is underscored by multiple loss-of-function studies for various Notch pathway members demonstrating defects in arterial specification and varying degrees of vascular malformation (Xue et al, 1999;Krebs et al, 2000Krebs et al, , 2004Duarte et al, 2004;North et al, 2009;Sorensen et al, 2009).…”

“…Intriguingly, Sox17 also works upstream of Notch to promote its activity, suggesting that Sox17 and Notch may participate in a feed-forward loop which drives the acquisition and the maintenance of arterial identity (Corada et al, 2013;Chiang et al, 2017). Although the relationship between HE and non-hemogenic arterial ECs has been debated (Ditadi et al, 2015;Gama-Norton et al, 2015), recent evidence using clonal analysis of lineage potential in vitro combined with single-cell RNA sequencing suggests that HSCcompetent HE and non-hemogenic arterial ECs diverge from a common arterial-like EC precursor shortly after E8 (Hou et al, 2020). Moreover, another recent study used single-cell RNA sequencing and ATAC-seq (assay for transposase-accessible chromatin sequencing) to identify a "pre-HE" state in a subset of arterial ECs with the potential to give rise to HSCs and LMPs in murine embryonic caudal arteries (dorsal aorta, umbilical artery, and vitelline artery) (Zhu et al, 2020).…”

Hematopoietic stem cells from pluripotent stem cells: Clinical potential, challenges, and future perspectives

“…Here, although hemodynamic forces seem critical for in vivo HSC formation, the exact molecular mechanism is not known and adaptation of biomechanical forces to ex vivo differentiation protocols for successful de novo HSC generation is lacking. A recent study demonstrated primitive vascular endothelial cells (at E8) follow a two‐step fate choice to become HSC‐primed HE cells; specification into arterial phenotype followed by a subsequent hemogenic conversion 58 . This transition from pre‐HE to HE stage was further demonstrated through trajectory analyses and genetic perturbation experiments 59 .…”

Biomechanical Regulation of Hematopoietic Stem Cells in the Developing Embryo

“…Further complicating our understanding of EHT and HSC development is the observation that extravascular hematopoiesis can occur during embryogenesis [45]. Single-cell analysis, however, has made it possible to better investigate lineage hierarchies of HE cells during EHT [23,41,[46][47][48]. Using this technology, researchers have demonstrated differences in the transcriptional landscape in the population of cells undergoing EHT, which is illustrated in part by changes in expression of the hematopoietic Wiskott-Aldrich Syndrome (WAS) gene [23].…”