Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45(-) and CD45(+) pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications.

No abstract

Graphical Abstract Highlights d Single-cell transcriptome profiling constructs lncRNA landscape of HSC development d Computational and functional screening identifies 6 lncRNAs affecting hematopoiesis d Loss of H19 lncRNA results in failed HSC generation from endothelium in AGM region d H19 deficiency leads to promoter hypermethylation of Runx1 and Spi1 in pre-HSCs conceived and supervised the study; J.Z. and L.Z. performed the pre-HSC-and HSC-related experiments with help from J.H. and F.Z.; J.X. performed the bioinformatics analysis with help from X. Wen, Y.M., and Z.L.; S.L. performed shRNA construct and H19 mechanism study with help from Y.M., F.W., X. Wang, and Y.S.; X.L. performed single-cell RNAseq with support from F.T.; Q.L. performed DNA methylation sequencing with support from F.T.; Y.N. performed the flow cytometry with help from P.Z. and C.L.; M.B. provided H19-DMR flDMR/flDMR mice and edited the manuscript; and Y.L., J.Z., J.X., and Y.M. wrote the manuscript with help from J.Y. and B.L.

Key Points• ATF4 positively regulates expansion of functional HSCs in mouse FL.• ATF4-Angptl3 axis in niche cells is pivotal for HSC maintenance in FL.The fetal liver (FL) serves as a predominant site for expansion of functional hematopoietic stem cells (HSCs) during mouse embryogenesis. However, the mechanisms for HSC development in FL remain poorly understood. In this study, we demonstrate that deletion of activating transcription factor 4 (ATF4) significantly impaired hematopoietic development and reduced HSC self-renewal in FL. In contrast, generation of the first HSC population in the aorta-gonad-mesonephros region was not affected. The migration activity of ATF4 2/2 HSCs was moderately reduced. Interestingly, the HSC-supporting ability of both endothelial and stromal cells in FL was significantly compromised in the absence of ATF4. Gene profiling using RNA-seq revealed downregulated expression of a panel of cytokines in ATF4 2/2 stromal cells, including angiopoietin-like protein 3 (Angptl3) and vascular endothelial growth factor A (VEGFA).Addition of Angptl3, but not VEGFA, partially rescued the repopulating defect of ATF4 2/2 HSCs in the culture. Furthermore, chromatin immunoprecipitation assay in conjunction with silencing RNA-mediated silencing and complementary DNA overexpression showed transcriptional control of Angptl3 by ATF4. To summarize, ATF4 plays a pivotal role in functional expansion and repopulating efficiency of HSCs in developing FL, and it acts through upregulating transcription of cytokines such as Angptl3 in the microenvironment. (Blood. 2015;126(21):2383-2391

Whereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2– CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.