The Notch genes play a key role in cellular differentiation. The significance of Notch1 during thymocyte development is well characterized, but the function of Notch2 is poorly understood. Here we demonstrate that Notch2 but no other Notch family member is preferentially expressed in mature B cells and that conditionally targeted deletion of Notch2 results in the defect of marginal zone B (MZB) cells and their presumed precursors, CD1d(hi) fraction of type 2 transitional B cells. Among Notch target genes, the expression level of Deltex1 is prominent in MZB cells and strictly dependent on that of Notch2, suggesting that Deltex1 may play a role in MZB cell differentiation.

Hematopoietic stem cells (HSCs) are thought to arise in the aorta-gonad-mesonephros (AGM) region of embryo proper, although HSC activity can be detected in yolk sac (YS) and paraaortic splanchnopleura (P-Sp) when transplanted in newborn mice. We examined the role of Notch signaling in embryonic hematopoiesis. The activity of colony-forming cells in the YS from Notch1(-/-) embryos was comparable to that of wild-type embryos. However, in vitro and in vivo definitive hematopoietic activities from YS and P-Sp were severely impaired in Notch1(-/-) embryos. The population representing hemogenic endothelial cells, however, did not decrease. In contrast, Notch2(-/-) embryos showed no hematopoietic deficiency. These data indicate that Notch1, but not Notch2, is essential for generating hematopoietic stem cells from endothelial cells.

Mouse long-term hematopoietic reconstituting cells exist in the c-Kit ؉ Sca-1 ؉ Lin ؊ (KSL) cell population; among them, CD34 low/؊ cells represent the most highly purified population of hematopoietic stem cells in the adult bone marrow. Here, we demonstrate that retrovirus-mediated transduction of CD34 low/؊ c-Kit ؉ Sca-1 ؉ Lin ؊ (34 ؊ KSL) cells with the HES-1 gene, which encodes a basic helix-loophelix transcription factor functioning downstream of the Notch receptor, and is a key molecule for the growth phase of neural stem cells in the embryo, preserves the long-term reconstituting activity of these cells in vitro. We also show that cells derived from the HES-1-transduced 34 ؊ KSL population produce progenies characterized by negative Hoechst dye staining, which defines the side population, and by CD34 low/؊ profile in the bone marrow KSL population in each recipient mouse at ratios 3.5-and 7.8-fold those produced by nontransduced 34 ؊ KSL-derived competitor cells. We conclude that HES-1 preserves the long-term reconstituting hematopoietic activity of 34 ؊ KSL stem cells ex vivo. Up-regulation of HES-1 protein in the 34 ؊ KSL population before unnecessary cell division, that is, without retrovirus transduction, may represent a potent approach to absolute expansion of hematopoietic stem cells. IntroductionHematopoietic stem cells (HSCs) are generated during ontogeny and supply all mature hematopoietic lineages throughout life with their self-renewal and multilineage differentiation capacity. 1 Efforts have been made to expand HSCs ex vivo without loss of their original potency. Long-term reconstitution capacity of mouse and human HSCs is maintained for up to 2 to 3 weeks by coculture with certain stromal cells. [2][3][4] For expansion of HSCs without stromal cells, various combinations of cytokines that are active for immature hematopoietic progenitors have been surveyed. [5][6][7][8][9] Of interest are approaches using Notch signaling, since it has been shown to inhibit differentiation of diverse types of cells in vertebrates. [10][11][12][13][14] Notch signals are mediated by interactions between Notch receptors and their membrane-anchored ligands expressed in adjacent cells. 15 In the hematopoietic compartment, Notch receptors and ligands are expressed in hematopoietic progenitors and certain stromal cells, respectively. [16][17][18][19] It was recently reported that the Notch ligand Jagged-1 maintained the severe combined immunodeficiency (scid)-repopulating activity of human cord blood-derived CD34 ϩ CD38 Ϫ cells in vitro significantly longer than the control. 20 Further evidence implying the potential usefulness of Notch signaling in HSC expansion comes from the establishment of a line of cytokinedependent cells which differentiate into myeloid and lymphoid lineages in vivo when transplanted into syngeneic mice, by retroviral transduction of stem cell-enriched bone marrow cells with an activated form of Notch-1. 21 In these previous investigations, however, it was not certain whether HSC expansion was...