The N-Glycome regulates the endothelial-to-hematopoietic transition

Abstract

20Hematopoietic stem and progenitor cells (HSPCs) are required to establish and maintain the 21 adult blood system in vertebrates. During development, hemogenic endothelial cells undergo an 22 endothelial-to-hematopoietic transition (EHT) to generate HSPCs 1-4 . Growth factors and 23 epigenetic changes can promote EHT 1,3,5 , but these mechanisms do not explain its tight 24 spatiotemporal regulation during development. Here, we show that microRNA (miR) miR-223-25 mediated regulation of N-glycan biosynthesis intrinsically restrains EHT, representing the first 26 pathway that prevents excessive HSPC production. We find that miR-223 is uniquely expressed 27 in hemogenic endothelial cells undergoing EHT and in nascent HSPCs. Loss of miR-223 28 promotes the expansion of these cells in the zebrafish and mouse aorta-gonad-mesonephros 29 (AGM), where EHT occurs 6-8 . miR-223 targets alg2 (α1,3/ α1,6 mannosyltransferase) and 30 st3gal2 (α2,3 sialyltransferase) for repression in the AGM endothelium. These two enzymes are 31 involved in the biosynthesis of N-glycans, a common co-translational modification 9,10 that 32 influences several pathophysiological processes 11,12 , but has not yet been implicated in EHT. 33 Using an N-glycosensor, we demonstrate that vascular N-glycosylation increases during EHT, 34 and this process is disrupted upon loss of miR-223. Specifically, high-throughput glycome 35 analysis revealed terminal α1,3 mannose and α2,3 sialic acid modifications of membrane 36 proteins are altered upon loss of miR-223. Importantly, pharmacological manipulation targeting 37 these N-glycan types in wild-type embryos phenocopies the loss of miR-223 and enhances EHT 38 as well as HSPC production. Thus, the N-glycome plays a previously unappreciated role as an 39 intrinsic negative regulator of EHT, with specific mannose and sialic acid modifications serving 40as key endothelial determinants of the hematopoietic fate. 41 43Zebrafish endothelial-to-hematopoietic transition (EHT) has emerged as an instrumental 44 model to understand the formation and regulation of definitive hematopoietic stem and 45 progenitor cells (HSPCs) with long-term engraftment capability. As in their mammalian 46 counterparts, EHT occurs in endothelial cells that co-express vascular and hematopoietic genes 47

Definitive hematopoietic stem cells minimally contribute to embryonic hematopoiesis

“…In contrast, C6 cells had high expression of key hemogenic and HSC marker genes, including gata2b, meis1b, and myb, as well as expression of lineage-restricted genes indicative of multipotent priming, such as gata1a (erythroid) (Gutié rrez et al, 2020;Galloway et al, 2005), ikzf1 (lymphoid) (Huang et al, 2019;Willett et al, 2001), and co-ro1a and csf1rb (myeloid) (Li et al, 2012;Carstanjen et al, 2005;Song et al, 2004); we consequently designated cluster C6 as hemogenic endothelial cells generating HSCs (HE/HSCs). The C4 cells expressed erythroid and lymphoid markers with low expression of myeloid markers, suggesting this population is analogous to the recently described lympho-erythroid-primed progenitor (LEP) in zebrafish (Kasper et al, 2020). Cells with LMP expression profile occurred in two clusters: (1) an LMP population with higher macrophage marker expression (csf1ra, irf8, mfap4, and mpeg1.1) (Rojo et al, 2019;Li et al, 2011;Zakrzewska et al, 2010) (LMP-M, C8) and (2) an LMP population with higher granulocyte marker expression (mpx and lyz) (Hall et al, 2007;Renshaw et al, 2006) (LMP-G; C9).…”

Definitive Hematopoietic Stem Cells Minimally Contribute to Embryonic Hematopoiesis

“…In contrast, C6 cells had high expression of key hemogenic and HSC marker genes including gata2b, meis1b, and myb, as well as expression of lineage-restricted genes indicative of multipotent priming such as gata1a (erythroid) (Gutiérrez, et al, 2020;Galloway, et al, 2005), ikzf1 (lymphoid) Willett, et al, 2001), and coro1a and csf1rb (myeloid) (Li, et al, 2012;Carstanjen, et al, 2005;Song, et al, 2004); we consequently designated cluster C6 as hemogenic endothelial cells generating hematopoietic stem cells (HE/HSC). The C4 cells expressed erythroid and lymphoid markers with low expression of myeloid markers, suggesting this population is analogous to the recently described lympho-erythroid-primed progenitor (LEP) in zebrafish (Kasper, et al, 2020). Cells with lympho-myeloid progenitor (LMP) expression profile occurred in two clusters: an LMP population with higher macrophage marker expression (csf1ra, irf8, mfap4, mpeg1.1) (Rojo, et al, 2019;Li, et al, 2011;Zakrzewska, et al, 2010) (LMP-M, C8) and an LMP population with higher granulocyte marker expression (mpx, lyz) (Hall, et al, 2007;Renshaw, et al, 2006) (LMP-G, C9).…”

“…Most cells resided in clusters C1, C2, and C3 and predominantly expressed erythroid markers, which we broadly designated as erythroid progenitors (EryP). Together, our identified clusters encompass the range of HSPC-associated progenitor traits (Hou, et al, 2020;Kasper, et al, 2020;Zhu, et al, 2020;Xue, et al, 2019;Zhou, et al, 2016), here resolved into distinct progenitor clusters.…”

“…A main challenge of studying developmental HSC contribution originates from the difficulty of specifically distinguishing HSCs from embryonic hematopoietic progenitors, as their emergence is spatiotemporally overlapping and commonly used markers are expressed in both developing progenitor and HSC populations (Kasper, et al, 2020;Hadland and Yoshimoto, 2018;McGrath, et al, 2015;Chen, et al, 2011;Godin and Cumano, 2002). Through our scRNA-seq exploration of newly formed HSPCs, we document transcriptional heterogeneity that defines seven distinct HSPC populations including welldocumented subsets, such as Pre-HE, HE/HSC, and EryP, and new populations, such as LMP (He, et al, 2020a) and LEP (Kasper, et al, 2020) (Figure 1). This high-resolution transcriptional landscape of developmental HSPCs allowed us to infer differentiation trajectories and cell-type-specific gene enrichment networks relevant to early hematopoiesis.…”

Single-cell transcriptome of early hematopoiesis guides arterial endothelial-enhanced functional T cell generation from human PSCs

“…In addition to RUNX1, many other factors have been found to regulate EHT, including TFs (e.g., GATA2 and MEIS2) and cell signaling pathways [e.g., the transforming growth factor- (TGF-) and Notch pathways] (6)(7)(8)(9). More recently, the N-glycome and cell cycle regulation have been identified as drivers of EHT (10,11). Despite these discoveries, the mechanisms controlling EHT remain to be fully elucidated.…”