Complex regulation of HSC emergence by the Notch signaling pathway

Abstract

Hematopoietic stem cells are formed during embryonic development, and serve as the foundation of the definitive blood program for life. Notch signaling has been well established as an essential direct contributor to HSC specification. However, several recent studies have indicated that the contribution of Notch signaling is complex. HSC specification requires multiple Notch signaling inputs, some received directly by hematopoietic precursors, and others that occur indirectly within neighboring somites. Of note, proinflammatory signals provided by primitive myeloid cells are needed for HSC specification via upregulation of the Notch pathway in hemogenic endothelium. In addition to multiple requirements for Notch activation, recent studies indicate that Notch signaling must subsequently be repressed to permit HSC emergence. Finally, Notch must then be reactivated to maintain HSC fate. In this review, we discuss the growing understanding of the dynamic contributions of Notch signaling to the establishment of hematopoiesis during development.

Keywords: Hematopoiesis; Hematopoietic stem cell; Hemogenic endothelium; Notch signaling.

Figure 1. Overview of Notch Signaling

A. Notch receptors are activated by binding of the Notch extracellular domain (NECD) to Notch ligands of the Delta/Serrate/Lag-2 families on adjacent signal-sending cell. Ubiquitination of Notch ligands by the Mindbomb and Neuralized E3 ubiquitin ligases promotes endocytosis, facilitating cleavage by ADAM family metalloproteases through exposure of the S2 proteolytic cleavage site. S2 cleavage results in the separation of ligand-bound NECD, and the remaining transmembrane receptor. Subsequently, the ligand-NECD complex is taken up by the signal-sending cell. The remaining membrane-bound Notch receptor is cleaved at the S3 and S4 proteolytic sites by Gamma-Secretase, releasing the Notch intracellular domain (NICD) from the membrane tether and allowing NICD to translocate to the nucleus. In the absence of Notch receptor activation, Notch transcriptional targets are bound by RBPjK/CSL and transcriptional corepressors (Co-R), and held in a transcriptionally repressed state. Nuclear NICD binds RBPjK, displacing the corepressor complex and allowing for the recruitment of transcriptional partner Mastermind (MAM) and additional coactivators (Co-A), allowing for transcriptional activation of Notch target genes. B. In some cases RBPjK occupies Notch transcriptional target sites in a more dynamic manner. In this case, RBPjK does not occupy the Notch target site in the absence of Notch activation, and is instead recruited upon activation alongside NICD and transcriptional co-activators.

Figure 2. Multiple contributions of Notch signaling surrounding HSC specification in zebrafish embryos

A. Establishment of Arterial Identity. Hedgehog secreted by the notochord stimulates the production of Vegfa from the somites, initiating the arterial program including Notch ligand and receptor expression. B. Somite-Intrinsic Notch Signaling. Non-canonical Wnt ligand Wnt16 controls the pro-hematopoietic somitic expression of Notch ligands deltaC and deltaD. Fgf signaling downstream of Wnt16 is required for somitic deltaC, but not deltaD expression. Activation of the somitic Notch3 receptor, possibly via the DeltaC/D ligands, promotes HSPC specification, possibly by regulation of the organization or function of the sclerotome compartment. C. Somite-to-PLM Notch signaling. During medial convergence of the posterior lateral plate mesoderm, direct cell contact allows for Notch signaling between DeltaC and DeltaD ligands on the ventral face of the somite and Notch receptors on migrating arterial cells. D. Vasculogenesis. Direct arterial Notch target EphrinB2, together with venous EphB4, promotes arteriovenous segregation and the vasculogenesis. Formation of intact vasculature is required for the establishment of circulation and for circulation-dependent hemogenic endothelial maintenance. F. Proinflammatory signaling. Production of TNFα by primitive neutrophils signals through Tnfr2 to promote arterial expression of jag1a. Jag1a is required for HSPC formation, possibly through endothelialintrinsic signaling with the Notch1a receptor.

Figure 3. Notch activity in budding hematopoietic cells

A. Notch signaling is inactivated in the budding hematopoietic clusters of higher vertebrates. Green coloring indicates cells in a “Notch-on” active Notch signaling state. Grey coloring indicates cells in a “Notch-off” inactive Notch signaling state. B. Confocal microscopy of HSPC budding in transgenic zebrafish. Kdrl:Cre; bactin:DsRed labels vascular and vascular-derived cells, and Tp1:GFP labels cells responsive to Notch activity. White arrows indicate cells undergoing EHT. Notch signaling is active throughout the dorsal aorta, and budding HSPCs have high levels of Notch reporter GFP at 48hpf. C. Due to the rapid development of the zebrafish embryo, it remains unclear whether Tp1:GFP+ budding HSPCs are truly Notch-active, or whether these cells have inactivated Notch signaling while the GFP protein persists.