We examined the expression of two members of theNotch family, Notch-1 and Notch-2, and one Notch ligand, Jagged-1, in hematopoietic cells. Both Notch-1 and Notch-2 were detected in murine marrow precursors (Lin−Sca-1+c-kit+). The Notch ligand, Jagged-1, was not detected in whole marrow or in precursors. However, Jagged-1 was seen in cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma, and in stromal cell lines. These results indicate a potential role for Notch-Notch ligand interactions in hematopoiesis. To further test this possibility, the effect of Jagged-1 on murine marrow precursor cells was assessed by coculturing sorted precursor cells (Lin−Sca-1+c-kit+) with a 3T3 cell layer that expressed human Jagged-1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 (Jagged-1ext). We found that Jagged-1, presented both on the cell surface and on beads, promoted a twofold to threefold increase in the formation of primitive precursor cell populations. These results suggest a potential use for Notch ligands in expanding precursor cell populations in vitro.

Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates. However, the mechanisms involved in ligand induced Notch activation are unknown. Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell. These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1(ext-myc)) or to the Fc portion of human IgG-1 (Delta-1(ext-IgG)), respectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized). Furthermore, both non-immobilized Delta-1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta. These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function. These results imply that ligand stabilization may be essential for Notch activation.