The crucial role of individual Notch receptors and the mechanism by which they maintain intestinal crypt progenitor cells were assessed by using a series of inducible gut-specific Notch mutant mice. We found that Notch1 and Notch2 receptors function redundantly in the gut, as only simultaneous loss of both receptors results in complete conversion of proliferating crypt progenitors into post-mitotic goblet cells. This conversion correlates with the loss of Hes1 expression and derepression of the cyclin-dependent kinase (CDK) inhibitors p27 Kip1 and p57 Kip2 . We also found that the promoter of both CDK inhibitor genes is occupied by the Notch effector Hes1 in wild-type crypt progenitor cells. Thus, our results indicate that Notch-mediated Hes1 expression contributes to the maintenance of the proliferative crypt compartment of the small intestine by transcriptionally repressing two CDK inhibitors.

The Notch pathway is an evolutionary conserved, intercellular signaling pathway that plays an important role in cell fate specification and the embryonic development of many organs, including the liver. In humans, mutations in the Notch receptor ligand Jagged1 gene result in defective intrahepatic bile duct (IHBD) development in Alagille syndrome. Developmental abnormalities of IHBD in mice doubly heterozygous for Jagged1 and Notch2 mutations propose that interactions of Jagged1 and its receptor Notch2 are crucial for normal IHBD development. Because different cell types in the liver are involved in IHBD development and morphogenesis, the cell-specific role of Notch signaling is not entirely understood. We investigated the effect of combined or single targeted disruption of Notch1 and Notch2 specifically in hepatoblasts and hepatoblast-derived lineage cells on liver development using AlbCre transgenic mice. Hepatocyte differentiation and homeostasis were not impaired in mice after combined deletion of Notch1 and Notch2 (N1N2 F/F AlbCre). However, we detected irregular ductal plate structures in N1N2 Later, parts of the ductal plate reduplicate and dilate to form tubular structures that are subsequently incorporated in the portal mesenchyme. The remaining nontubular single-layered cells of the ductal plate are eliminated via apoptosis while the tubular structures further undergo a branching process to form the biliary tree. This process of ductal plate remodeling starts at the portal vein at apAbbreviations: AGS, Alagille syndrome; IFN-␣, interferon-␣; IHBD, intrahepatic bile duct; P, postnatal day; WT, wild-type; X-gal, X-galactosidase. From the

Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2–DL1–mediated signaling does not allow further T cell maturation beyond the CD25+ stage due to a lack of T cell receptor β expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1–DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch–Delta interactions in which N1–DL4 exhibits the greatest capacity to induce and support T cell development.

Pancreatic cancer is one of the most fatal malignancies lacking effective therapies. Notch signaling is a key regulator of cell fate specification and pancreatic cancer development; however, the role of individual Notch receptors and downstream signaling is largely unknown. Here, we show that Notch2 is predominantly expressed in ductal cells and pancreatic intraepithelial neoplasia (PanIN) lesions. Using genetically engineered mice, we demonstrate the effect of conditional Notch receptor ablation in Kras G12D-driven pancreatic carcinogenesis. Deficiency of Notch2 but not Notch1 stops PanIN progression, prolongs survival, and leads to a phenotypical switch toward anaplastic pancreatic cancer with epithelial-mesenchymal transition. By expression profiling, we identified increased Myc signaling regulated by Notch2 during tumor development, placing Notch2 as a central regulator of PanIN progression and malignant transformation. Our study supports the concept of distinctive roles of individual Notch receptors in cancer development.genetically engineered mice | K-Ras | Myc | Notch | pancreatic cancer P ancreatic ductal adenocarcinoma (PDAC) remains a devastating disease despite tremendous therapeutical efforts. PDAC derives from several preneoplastic lesions, including pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm, and mucinous cystic neoplasm (MCN), of which PanINs are the most common precursors (1). PanINs typically progress through defined histological and molecular stages, with the most advanced PanIN3 lesion being defined as carcinoma in situ (2). Because of early metastatic spread, PanIN3 represents the latest curable precursor lesion. Thus, defining the regulators of PanIN initiation and progression is of utmost importance.

Epstein‐Barr virus nuclear antigen 2 (EBNA‐2) plays a crucial role in B cell immortalization by Epstein‐Barr virus (EBV), most probably by its ability to transactivate several cellular and viral genes. Recently, we showed that EBNA‐2 interacts with the TP1 promoter of EBV through a cellular protein. In this report we provide evidence that this protein is recombination signal binding protein (RBP)‐J kappa, highly conserved in evolution, and originally isolated by its ability to bind to the J kappa‐type V(D)J recombination signal sequence. To identify the cellular protein interacting with the TP1 promoter, we performed electrophoretic mobility shift assays using binding sequences of known transcription factors, that carry partial homology to the crucial sequences of the EBNA‐2 responsive element (EBNA‐2RE), as competitor. Competition assays revealed the RBP‐J kappa recognition site as a very efficient competitor of cellular TP1 promoter binding protein. In parallel, we purified the protein to homogeneity from Raji cells by two ion‐exchange columns and affinity purification using the EBNA‐2RE coupled to magnetic beads. Affinity purified fractions separated on SDS‐PAGE revealed a single predominant band after silver staining which was recognized by anti‐RBP‐J kappa monoclonal antibody. These purified fractions exhibited binding specificity for EBNA‐2RE and EBNA‐2. In vitro‐translated murine RBP‐2 cDNA reacted with EBNA‐2RE and EBNA‐2 in the same fashion as the affinity purified protein. The interaction between RBP‐J kappa and EBNA‐2 is a prerequisite for EBNA‐2‐mediated transactivation of the TP1 promoter.