Notch Signaling Requires GATA-2 to Inhibit Myelopoiesis from Embryonic Stem Cells and Primary Hemopoietic Progenitors

Abstract

The bone marrow and thymus, although both hemopoietic environments, induce very distinct differentiation outcomes. The former supports hemopoietic stem cell self-renewal and multiple hemopoietic lineages, while the latter supports T lymphopoiesis almost exclusively. This distinction suggests that the thymic environment acts to restrict the hemopoietic fates available to thymic immigrants. In this study, we demonstrate that the addition of the Notch ligand Delta-like-1 (Dll-1) to an in vitro system that otherwise supports myelopoiesis, greatly reduces the myelopoietic potential of stem cells or uncommitted progenitors. In contrast, committed myeloid progenitors mature regardless of the presence of Dll-1. The block in myelopoiesis is the direct result of Notch signaling within the hemopoietic progenitor, and Dll-1-induced signals cause a rapid increase in the expression of the zinc finger transcription factor GATA-2. Importantly, in the absence of GATA-2, Dll-1-induced signals fail to inhibit commitment to the myeloid fate. Taken together, our results support a role for GATA-2 in allowing Dll-1 to restrict non-T cell lineage differentiation outcomes.

Direct Comparison of Dll1- and Dll4-Mediated Notch Activation Levels Shows Differential Lymphomyeloid Lineage Commitment Outcomes

“…In contrast, high levels of either Dll1 or Dll4 effectively inhibited the generation of CD11b + myeloid-lineage cells, similar to that observed in Fig. 1 and as previously reported (15). However, when OP9 cells expressed Dll at medium levels, we noted a clear increase in the generation of myeloid cells as compared with either low or high levels (Fig.…”

“…Viable HPCs were subsequently sorted by flow cytometry as CD117 + Sca-1 + cells. (15). Cells were harvested on day 6 for analysis or further passaged onto fresh plates containing OP9 cells and coculture media for analysis on days 9 and 12.…”

“…We used this strategy to generate OP9-DL1 cells, which could sustain all stages of T cell development from HPCs, while inhibiting B cell and myeloid lineage differentiation (12)(13)(14)(15). Both Dll1 and Dll4 were shown to be expressed in TECs (16,17), and this initially suggested that there could be redundancy between Dll1 and Dll4 function.…”

Rbm15 Modulates Notch-Induced Transcriptional Activation and Affects Myeloid Differentiation

“…After 5 min on ice, the lysates were sedimented by centrifugation, and the supernatant was used as the cytoplasmic extract. The pelleted nuclei were washed, and nuclear proteins were extracted with 2 packed cell volumes of nuclear extract buffer (20 mM HEPES [pH 8.0], 420 mM NaCl, 1.5 mM MgCl 2 , 0.5 mM DTT, 0.2 mM EDTA, and 25% glycerol) at 4°C for 45 min. Soluble material was pelleted, and the supernatant was dialyzed at 4°C for 1 h against Shapiro's buffer D (20 mM HEPES [pH 7.9], 20% glycerol, 100 mM KCl, 2 mM DTT, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.7 mg/liter pepstatin A, and 0.5 mg/liter leupeptin).…”

Haematopoietic and immune defects associated with GATA2 mutation

“…Insufficient GATA2 appears to allow HSCs to enter cell cycle and differentiate, thereby depleting self-renewal capacity (Ezoe et al, 2002;de Pater et al, 2013), while over-expression impairs haematopoiesis by blocking differentiation (Heyworth et al, 1999;Persons et al, 1999;Tipping et al, 2009). Fine-tuning of the balance between self-renewal and differentiation of HSC appears to be a critical function of GATA2, possibly through its role in mediating contact-dependent quiescence signals from the BM niche (de Pooter et al, 2006;Guiu et al, 2013). Direct effects on apoptosis are also thought to be mediated by an interaction of GATA2 with BCL2L1 (BCL-XL) (Rodrigues et al, 2005).…”