Abstract
The Notch signaling pathway is involved in a broad spectrum of cell fate decisions during development, and in the hematopoietic system, it is known to favor T cell- vs B cell lineage commitment. However, its role in myeloid lineage development is less well understood. We have shown, using heterotypic co-cultures of murine primary hematopoietic stem cells (Lin-Sca-1+ckit+ HSCs) and OP9 stromal cells expressing the Notch ligand Delta1 (OP9-DL1), that Notch signaling derived from cell non-autonomous cues acts as a positive regulator of megakaryocyte fate from LSK cells. Bone marrow transplantation experiments with a constitutively active Notch mutant resulted in enhanced megakaryopoiesis in vivo, with increased MEP numbers and megakaryocyte colony formation. In contrast, expression of dnMAML using a conditional ROSA26 knock-in mouse model significantly impaired megakaryopoiesis in vivo, with a marked decrease in megakaryocyte progenitors. In order to understand the cellular differentiation pathways controlled by Notch, we first examined the ability of various purified progenitor populations to differentiate toward megakaryocytes upon Notch stimulation in vitro. We observed that CMP and MEP, but not GMP, can engage megakaryopoiesis upon Notch stimulation. Our results were consistent with expression analysis of Notch signaling genes in these purified progenitors and were supported by the observation that transgenic Notch reporter mice display higher levels of reporter (i.e. GFP) expression in HSC and MEP, vs. CMP and GMP in vivo. Furthermore, purified progenitors with high GFP expression gave rise to increased numbers of megakarocyte-containing colonies when plated in vitro compared to GFP-negative progenitors. In addition, further purification of the HSC population into long-term (LT), short-term (ST), and lymphoid-primed myeloid progenitors (LMPP) before plating on OP9-DL1 stroma showed that LMPP have a reduced ability to give rise to megakaryocytes compared to the other two populations. These data support the hypothesis that there is an early commitment to erythro/megakaryocytic fate from HSC prior to lymphoid commitment. To gain insight into the molecular mechanism underlying Notch-induced megakaryopoiesis, we performed global gene expression analysis that demonstrated the engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 treated with gamma-secretase inhibitor. Of interest, Runx1 was among the most upregulated genes in HSC co-cultured on OP9-DL1 stroma. To assess whether Notch signaling engages megakaryocytic fate through induction of Runx1, we plated HSC from Runx1 −/− mice on OP9-DL1 stroma. Compared to WT cells, Runx1 −/− HSC had a severely reduced ability to develop into CD41+ cells. In contrast, overexpression of Runx1 in WT HSC was sufficient to induce megakaryocyte fate on OP9 stroma without Notch stimulation. Together, our results indicate that Notch pathway activation induced by stromal cells is an important regulator of cell fate decisions in early progenitors. We show that Notch signaling is upstream of Runx1 during Notch-induced megakaryocyte differentiation and that Runx1 is an essential target of Notch signaling. We believe that these results provide important insight into the pathways controlling megakaryocyte differentiation, and may have important therapeutic potential for megakaryocyte lineage-related disorders.
Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2.
