Definitive hematopoietic stem and progenitor cells (HSPCs) arise from the transdifferentiation of hemogenic endothelial cells (hemECs). The mechanisms of this endothelial-to-hematopoietic transition (EHT) are poorly understood. We show that microRNA-223 (miR-223)–mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid-myeloid lineages by suppressing the mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in protein N-glycosylation. ECs that lack miR-223 showed a decrease of high mannose versus sialylated sugars on N-glycoproteins such as the metalloprotease Adam10. EC-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied miR-223 mutant defects. Thus, the N-glycome is an intrinsic regulator of EHT, serving as a key determinant of the hematopoietic fate.

No 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

Endothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing.Knockdown (KD) in ECs of single Gα proteins had little effect but combined depletion of Gαi and Gαq/11 blocked all known PECAM1-dependent responses. Re-expression of Gαi2 and Gαq but not Gαi1 and Gαi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gαi2 and Gq/11 (Q306 in Gαi1/3), determines participation in flow signaling. We developed pull-down assays for measuring Gα activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gα's, which identified latrophilins (ADGRLs) as specific upstream interactors for Gαi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gαi2 and Gαq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of Gα proteins.Latrophilins are thus central mediators of junctional shear stress mechanotransduction via Gα protein-dependent and -independent mechanisms.