Turning it down a Notch

In this issue of Blood, Souilhol et al pinpoint the requirement for Notch signaling to precisely defined stages in hematopoietic stem cell (HSC) emergence and implicate both Notch1 and Notch2 in this process.¹ 

Definitive HSCs have been used in transplantation therapies for blood-related disorders for decades. The inability to expand them in vitro, or generate bona fide HSCs from pluripotent stem cells, has sparked a great interest in understanding how HSCs are generated de novo during embryonic development.^2,3  A mechanistic understanding of this holds promise for the development of robust in vitro culture methods to generate HSCs for research and ultimately for therapeutic purposes such as autologous gene correction for single gene hematologic disorders. During embryonic development, HSCs are generated autonomously in the aorta-gonad-mesonephros region (AGM), where they emerge in clusters of hematopoietic cells that bud from the hemogenic endothelium in the ventral wall of the dorsal aorta.⁴  The emergence of HSCs is controlled by several signaling pathways, including Notch. Notch signaling is required for early hematopoietic specification of the HSC lineage. Later on, its activity is downregulated in the aortic hematopoietic clusters,⁵  but the precise cellular stage at which it is active remained to be established (see Butko et al⁶  for a recent review).

Medvinsky and coworkers⁷  recently identified the direct cellular precursors to the definitive HSC, the pre-HSC type I and II. These cells express the hematopoietic marker CD41 and lack in vitro endothelial potential, indicating that they have already diverged from the hemogenic endothelium. The group showed that on ex vivo culture, pre-HSC type I mature via pre-HSC type II into definitive, transplantable HSCs. Building on from this, Souilhol et al set out to address where in the HSC lineage Notch signaling is active and required, using several complementary in vitro and in vivo approaches. Notch activity was assessed through ex vivo culture and transplantation of Hes1-green fluorescent protein–positive and –negative pre-HSC type I and II (Hes1 is a downstream target of Notch signaling). The Notch pathway was active in all pre-HSCs type I, but activity decreased on maturation of the cells into definitive HSCs (see also the model in figure 6 of Souilhol et al). To assess at which stage Notch is required, AGM explants were cultured in the presence of a chemical Notch inhibitor. This blocked HSC maturation from pre-HSC type I, but much less so from pre-HSC type II, revealing a critical requirement for Notch signaling in pre-HSC type I, but not pre-HSC type II. These results were in line with in vitro conditional deletion of the Notch transcriptional partner RBP-jκ, which showed that Notch signaling is no longer required toward the end of AGM HSC maturation. In a set of converse experiments, AGM pre-HSCs were exposed to constitutive Notch signaling in culture. Pre-HSC type I exposed to these conditions no longer could develop into functional HSCs. Pre-HSCs type II, in contrast, were not sensitive to constitutive Notch signaling. Together these experiments demonstrate that Notch signaling is indispensable in pre-HSC type I, but then needs to be downregulated to allow the cells to mature to pre-HSC type II.

Surprisingly, the authors found that not only the Notch1 receptor but also Notch2 is expressed by the HSC lineage. Interestingly, Notch1 and Notch2 show inverse expression patterns. Notch1 is expressed by all pre-HSCs type I and II, with the highest expression seen in type I, whereas Notch2 is not expressed on pre-HSC type I, but is on part of the pre-HSC type II. Addition of blocking antibodies to AGM explants confirmed a role for both Notch receptors in HSC development. The authors suggest that the weaker signaling strength reported for the Notch2 receptor may play a role in the downregulation of Notch signaling activity seen on maturation.

How does this compare with hematopoietic differentiation of human pluripotent cells? Also in that system, Notch activity is required for the endothelial-to-hematopoietic transition of hemogenic precursors,^8,9  specifically in the early stages of the process.⁸  It will be interesting to see whether fine regulation of Notch signaling in the progenitors that emerge in these cultures affects the cell types generated and how these compare with the pre-HSCs type I and II of the embryo.