GSE135246
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, HPSCs are generated from hemogenic endothelial cells that undergo an endothelial-to-hematopoietic transition (EHT). Growth factors and epigenetic changes can promote EHT, 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 a new 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. miR-223 targets alg2 (alpha 1,3/ 1,6 mannosyltransferase) in the AGM endothelium to restrict hemogenic and HSPC specification. This enzyme is involved in the attachment of the N-glycans,  a common co-translational modification9-12 that influences several pathophysiological processes, but has not yet been implicated in EHT. Using an N-glycosensor, we demonstrate that abundant protein N-glycan attachment occurs in vascular cells during EHT, and this process is required for HSPC production. High-throughput glycome analysis upon loss of miR-223 revealed that terminal alpha 1,3/6 mannose modifications are increased at the expense of alpha 2,3/6 sialic acid sugars. 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 regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants to restrict the hematopoietic fate. Overall design: Single cell RNA sequencing for zebrafish Tg(kdrl:hras-mCherry)s896 endothelial cells at 27 hpf for wildtype and miR-223(ya303) backgrounds