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.

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca 2؉ -dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However, the molecular mechanisms that regulate their differentiation, proliferation, and endothelial sheet formation remain to be elucidated. Here, we show that members of the transforming growth factor (TGF)-β superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5–days postcoitum embryos. TGF-β and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-β and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover, SB-431542 up-regulated the expression of claudin-5, an endothelial specific component of tight junctions. These results suggest that endogenous TGF-β/activin signals play important roles in regulating vascular growth and permeability.

T ransforming growth factor (TGF)-β is a multifunctional cytokine that regulates a wide range of cellular responses, including cell proliferation, differentiation, adhesion, migration, and apoptosis.(1,2) It is a potent inhibitor of various types of cells, including most epithelial cells, whereas it stimulates deposition of extracellular matrix proteins and induction of epithelialto-mesenchymal transition (EMT). TGF-β thus plays two distinct and opposing roles in cancer progression. In early stages of carcinogenesis, it acts as a tumor suppressor by preventing cell proliferation, although in advanced stages of cancer, tumor cells often become refractory to TGF-β-mediated growth inhibition. TGF-β is often overexpressed in tumor cells, and induces migration, invasion, and EMT of tumor cells and facilitates immunosuppression, deposition of extracellular matrix proteins and angiogenesis. TβR-II is the primary ligand-binding receptor at the cell surface and contains constitutively active kinase. Upon ligand binding, the TβR-II kinase transphosphorylates TβR-I, and TβR-I then transduces intracellular signals through various proteins, of which Smad proteins are the major signaling transducers for TGF-β.(6,7) Activated TβR-I phosphorylates receptor-regulated Smads (R-Smads), i.e. Smad2 and Smad3, which interact with common-mediator Smad (Smad4) and translocate to the nucleus where they regulate transcription of various target genes. Smad7 is an inhibitory Smad that inhibits TGF-β signaling through interaction with activated TβR-I and other mechanisms.TGF-β signaling can be regulated by various molecules, including antisense oligonucleotides to certain TGF-β isoforms, monoclonal antibodies to TGF-βs, soluble forms of TβR-II, and small-molecule compounds that act on TGF-β receptor kinases. (8) Of these, TβR-I kinase is one potential target for blockade of TGF-β signaling pathway. Recently, several small-molecule compounds that bind to and inhibit TβR-I kinase activity have been generated and shown to potently inhibit TGF-β activity in vitro and in vivo.(8) Small-molecule inhibitors of TβR-I kinase are highly specific for TβR-I, although they also inhibit kinase activities of closely related molecules, that is, type I receptors for activin and nodal (activin-receptor like kinase-4; ALK-4 and ALK-7). (9) An in vivo experimental bone metastasis model has been established using an intracardiac injection of cancer cells, including MDA-MB-231 cells. (10,11) In this model, growth of cancer cells arrested in bone is easily observed as the progression of osteolysis on radiographs. This model is thus frequently used to explore the molecular interactions between the cancer cells and bone microenvironment. Several studies using this in vivo model suggested that TGF-β and its transcriptional targets, including parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), are the essential mediators of bone metastasis. (12,13) More recent studies have revealed that Smad signaling is essential for the development of bone metas...