Distinct Transcriptional Signatures Distinguish the Emergence of Multipotent Progenitors and Hematopoietic Stem Cells from Endothelial Precursors in the Murine Embryo

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and transcriptional properties. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We previously found that the co-expression of two markers, CD61 and EPCR, identifies a subset of VE-Cadherin⁺ endothelial cells from the mouse P-Sp/AGM region (para-aortic splanchnopleura/aorta-gonad-mesonephros, where the first HSCs are generated from HE between E9 and E11 in development) enriched phenotypically for arterial endothelial surface markers (e.g. Dll4, CD44) and functionally for hemogenic precursors with HSC potential. However, this population remains heterogeneous, containing clonal hemogenic precursors with the potential for HSC as well as multilineage progenitor-restricted fates. Here, we report that expression of arterial marker CXCR4 further enriched for functional HSC potential in hemogenic precursors in the P-Sp/AGM between E9 and E10, when the first clonal HSC precursors are detected at rare frequency. In contrast, we detected more abundant clonal HE with multilineage hematopoietic potential (producing lymphoid, erythroid, and myeloid progeny in vitro but lacking HSC potential) at the same stage, which are distinguished by comparatively lower CXCR4 expression. To investigate transcriptional differences between HE populations differentially expressing CXCR4, we performed single-cell RNA sequencing of E9 P-Sp-derived VE-Cadherin⁺CD61⁺EPCR⁺ cells. Using an unbiased gene module analysis based on graph autocorrelation in the Monocle 3 platform to identify genes that co-vary over pseudotime, we found that Cxcr4 is uniquely expressed in a subset of cells simultaneously enriched for arterial-specific genes (including Dll4, Efnb2, Hey2, Sox17, Cd44) and genes with established roles in HSC maintenance and self-renewal (including Mecom, Cdkn1c, H19, Txnip, Kmt2a). Conversely, expression of these genes is decreased in cells undergoing the endothelial to hematopoietic transition at this stage based on pseudotemporal ordering, concomitant with increasing expression of hematopoietic-specifying transcription factors Runx1 and Gfi1, and other genes associated with definitive hematopoiesis (egs. Myb, Kit, Hlf, Gata2, Mpl, Lyl1). We also examined the aggregate expression of established HSC-specific signature genes from published data sets across pseudotime, and found that they exhibit similar expression dynamics to that of Cxcr4 and Dll4, reaching peak expression prior to the initiation of Runx1 and Gfi1 expression. Altogether, our studies support a model in which the initial populations of multipotent progenitors and HSCs emerge independently from HE in the P-Sp/AGM. Furthermore, our findings suggest that HE with HSC competence is uniquely defined by co-expression of arterial endothelial genes and genes encoding HSC self-renewal factors, providing insight into the earliest transcriptional programs that must be coordinated to drive HSC fate from endothelial precursors. Future studies will focus on identifying the signal pathways whose integration promotes expression of these HSC-defining transcriptional programs in endothelial cells, with the goal of advancing methods for HSC generation in vitro.