Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation

Abstract

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1 + cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

Endothelial-to-hematopoietic transition is induced by Notch glycosylation and upregulation of Mycn

“…Our findings differ from earlier reports in zebrafish embryos 14,15 and human pluripotent stem cells 21 as well as initial findings in mice 8,16 . Analysis of endothelial Notch genetic mosaics allowed us to find that increased Notch activity in DAECs inhibits EHT, a finding in line with more recent studies using distinct experimental approaches 34,60,61 . We show that single ECs with mildly decreased or elevated Notch signaling can both form the first embryonic arteries, but their subsequent contribution to Kit+ hematopoietic clusters is influenced by their Notch signaling status.…”

Multifaceted Actions of GFI1 and GFI1B in Hematopoietic Stem Cell Self-Renewal and Lineage Commitment

“…Although the role of master regulator of this conversion of HE cells pertains to RUNX1, its first action is to directly drive the expression of the GFI1/GFI1B factors, and it has been shown that forced expression of both can compensate for the loss of RUNX1 in this process (Lancrin et al, 2012). Indeed, the expression of RUNX1 is followed almost immediately by an increase in GFI1 in a subset of arterial endothelial cells of the AGM that defines them as hemogenic, despite the fact that they retain a fully endothelial phenotype (Thambyrajah et al, 2016;Porcheri et al, 2020). A subset of these GFI1positive HE cells are then recruited in a Notch/DL4-dependent manner to the nascent intra-aortic hematopoietic cluster (IAHC) where they then start to express KIT (c-Kit).…”

“…A subset of these GFI1positive HE cells are then recruited in a Notch/DL4-dependent manner to the nascent intra-aortic hematopoietic cluster (IAHC) where they then start to express KIT (c-Kit). This marks the beginning of the EHT process during which cells silence their endothelial program and acquire a hematopoietic gene signature (Porcheri et al, 2020).…”

Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation

“…Mouse G2V.Gpr56 and Gpr97 knockout ESCs were generated with CRISPR/Cas9 (Ran, Hsu et al, 2013). Guide(g)RNAs (Table EV2)…”

“…HECs undergo a highly-conserved 'endothelial-tohematopoietic transition' (EHT) process (Ciau-Uitz & Patient, 2019, Dzierzak & Bigas, 2018, Dzierzak & Speck, 2008, Jaffredo, Nottingham et al, 2005. Transcriptomics and loss-of-function studies have identified candidate regulators/signaling pathways in transitioning cells (Gao, Chen et al, 2020, Li, Gao et al, 2017, Lichtinger, Ingram et al, 2012, Moignard, Woodhouse et al, 2015, Porcheri, Golan et al, 2020, Swiers, Baumann et al, 2013, Zhou, Li et al, 2016.…”