Notch: of mice and men?

A recent study has demonstrated that Notch is a positive regulator of murine megakaryopoiesis.¹  In this issue of Blood, Poirault-Chassac et al report the opposite: Notch inhibits the terminal differentiation of human megakaryocytes.²  Does this discrepancy arise from species-specific activities of Notch or a difference in experimental design?

Notch signaling plays critical roles in cell fate determination, proliferation, and survival. In the hematopoietic system, Notch is perhaps best known as a positive regulator of normal T-cell development and, when mutated, as a driver of T-cell leukemia.³  A recent study by Mercher and colleagues revealed that Notch is a positive regulator of megakaryocyte development.¹  In their study, murine LSK hematopoietic progenitor cells plated on OP9 stroma expressing the Delta-like1 (DL1) ligand of Notch gave rise to far more CD41⁺ megakaryocytes than progenitors cultured on OP9-GFP cells. Furthermore, megakaryocytes derived from OP9-DL1 cultures stained intensely with acetylcholinesterase and showed increased expression of CD42, elevated ploidy and an increased capacity to form megakaryocyte colonies (CFU-MK) relative to cells derived from OP9-GFP stroma. These effects were diminished when canonical Notch signaling was inhibited with either a gamma secretase inhibitor or by expression of dominant-negative MAML1, whereas activation of Notch signaling by expression of ICN4 led to increased CFU-MK colonies and increased absolute numbers of megakaryocytes.

In contrast to these observations in mice, a previous study reported that Notch1 activation inhibits megakaryocytic differentiation of human K562 cells and also Lin⁻Sca-1⁺ murine progenitors cultured on 3T3 cells expressing the Notch ligand Jagged1.⁴  Poirault-Chassac and colleagues confirm the human versus mouse discrepancy by providing new insights into Notch-regulated human megakaryopoiesis. They discovered that culturing human CD34⁺ cells with either an immobilized chimeric form of the Notch ligand Delta-like4 (Dll4Fc) or on OP9-DL4 and OP9-DL1 stroma led to significantly reduced production of mature CD41⁺CD42⁺ megakaryocytes and diminished proplatelet formation.²  These effects were reversed by culture with a gamma secretase inhibitor or by expression of dominant-negative MAML1. Of note, the negative effect of Notch required exposure to DL4 in the first 5 days of culture. This is surprising because activation of Notch signaling interfered with terminal differentiation, but did not alter production of CFU-MK or megakaryocyte erythroid precursors (MEPs) from CD34⁺ cells. Importantly, Poirault-Chassac and colleagues ruled out several obvious experimental differences and also verified that exposure of murine LSK cells to DL1 or DL4 enhanced production of CD41⁺ cells in culture.

What explains this species-selective effect of Notch on megakaryocytes? One explanation may lie in the starting material for the 2 studies: although they are both considered to be populations enriched for stem cells, human CD34⁺ and murine LSK cells are not equivalent. A more precise study might compare the effect of Notch activation on megakaryocyte differentiation from highly purified mouse versus human MEPs. Alternatively, the source of the progenitor cells might also be crucial determinant: Poirault-Chassac et al assayed the effect of Notch modulation on human megakaryocytes derived from cord blood or mobilized adult CD34⁺ cells, whereas Mercher et al examined differentiation of murine bone marrow progenitors.

It is possible that progenitors from different compartments retain a memory of their origin and respond to Notch differently. It is also formally possible that Notch activates overlapping, but distinct, downstream effectors in human and mouse myeloid progenitors. Additional experiments should address these issues. Finally, although there is a difference in the response of human and mouse progenitors to Notch, its dysregulation appears to have the same effect in both species: increased Notch activation has been observed in both humans with and a mouse model of t(1;22) acute megakaryoblastic leukemia.⁵  Thus, even though the precise function of Notch in normal megakaryopoiesis is uncertain, it is clear that Notch participates in regulation of hematopoietic cell differentiation beyond the lymphoid compartment.