Notch Signaling in Hematopoietic Precursor Cells Maintains the Expression of Genes Required for Stem Cell Self-Renewal and Promotes the Expression of Genes Associated with T cell Differentiation.

Notch Signaling in Hematopoietic Precursor Cells Maintains the Expression of Genes Required for Stem Cell Self-renewal and Promotes the Expression of Genes Associated with T cell Differentiation.

Keizo Kato, Barbara Varnum-Finney, Irwin D. Bernstein

Clinical Research Division, Fred Hutchinson Cancer Research Center,

Seattle, WA, USA

We have previously shown that Notch signaling promotes the self-renewal of hematopoietic precursors, including short-term repopulating cells, and induces early T-cell differentiation. Here we evaluate gene expression in murine Lin-Sca-1+c-kit+ Hoechst side population (SKSP) bone marrow cells during culture with the immobilized Notch ligand, Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human-IgG1 for 28 days with SCF, IL-6, IL-11 and Flt-3-ligand. We analyzed hematopoietic stem cell (HSC) associated genes, including polycomb genes, Bmi-1 and Rae28, required for HSC self-renewal, and Rex-1 required for embryonic stem cell self-renewal, together with early T-lymphoid differentiation associated genes such as GATA-3, pre-Ta and CD3e, by semi-quantitative or real-time RT-PCR. After culture for 7 or 14 days with Delta1ext-IgG, the expression of Bmi-1, Rae28 and Rex-1 was greater in cultures containing Delta1ext-IgG compared to those without. Bmi-1, Rae28 and Rex-1 were likely not direct targets of Notch signaling since the expression in SKSP cells was equivalent after 3 hours culture in the presence or absence of Notch ligand, whereas rapid up-regulation of the direct Notch target Hes1 was observed 3 hours after incubation with Delta1ext-IgG. Expression of GATA-3, pre-Ta and CD3e was induced by Notch signaling since their expression was seen by 7-14 days with but not without Delta1ext-IgG. Furthermore, the expression of these genes was dependent on Notch signaling since removal of cells from Notch ligand after culture for 28 days led to a rapid reduction of Hes1 expression within 3 hours, and a slower reduction in genes associated with self-renewal observed after 2 days. Our results suggest that Notch signaling regulates the self-renewal of hematopoietic precursors by maintaining the expression of genes known to be required for stem cell self-renewal, while also promoting the expression of T-cell differentiation-associated genes.