N-Glycome regulation limits the transdifferentiation of endothelial cells into hematopoietic stem cells

Hematopoietic stem and progenitor cells (HSPCs) are required to establish and maintain the adult blood system in vertebrates. During development, hemogenic endothelial cells undergo an endothelial-to-hematopoietic transition (EHT) to generate HSPCs 1–4 . Growth factors and epigenetic changes can promote EHT 1,3,5 , but these mechanisms do not explain its tight spatiotemporal regulation during development. Here, we show that microRNA (miR) miR-223-mediated regulation of N-glycan biosynthesis intrinsically restrains EHT, representing the first pathway that prevents excessive HSPC production. We find that miR-223 is uniquely expressed in hemogenic endothelial cells undergoing EHT and in nascent HSPCs. Loss of miR-223 promotes the expansion of these cells in the zebrafish and mouse aorta-gonad-mesonephros (AGM), where EHT occurs 6–8 . miR-223 targets alg2 (α1,3/ α1,6 mannosyltransferase) and st3gal2 (α2,3 sialyltransferase) for repression in the AGM endothelium. These two enzymes are involved in the biosynthesis of N-glycans, a common co-translational modification 9,10 that influences several pathophysiological processes 11,12 , but has not yet been implicated in EHT. Using an N-glycosensor, we demonstrate that vascular N-glycosylation increases during EHT, and this process is disrupted upon loss of miR-223. Specifically, high-throughput glycome analysis revealed terminal α1,3 mannose and α2,3 sialic acid modifications of membrane proteins are altered upon loss of miR-223. Importantly, pharmacological manipulation targeting these N-glycan types in wild-type embryos phenocopies the loss of miR-223 and enhances EHT as well as HSPC production. Thus, the N-glycome plays a previously unappreciated role as an intrinsic negative regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants of the hematopoietic fate.