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Fringe-dependent notch activation and tramtrack function are required for specification of the polar cells in Drosophila oogenesis.

During Drosophila oogenesis, each egg chamber is encapsulated through the coordinated signaling of multiple pathways, resulting in the formation of polar cells at the termini and a row of stalk cells in between each egg chamber. Notch signaling is required for specification of a precursor group containing both stalk and polar cells. Together, the Notch and JAK/STAT pathways specify the stalk cells as well as a group of prepolar cells, from within that group. The mechanism by which the polar cells differentiate from the prepolar group involves apoptosis, but the pathways which control that process are largely unknown. We now demonstrate that Notch signaling, activated by Delta and transduced by the transcription factor Tramtrack, is involved in the process of refining the prepolar cell group to two polar cells. The glycosyltransferase Fringe is expressed and required cell-autonomously in prepolar cells for this process. However, the transcription factor Mirror, which inhibits fringe expression in other tissues and stages of development, as well as Serrate, one of the two known ligands for Notch, are not required for maturation of prepolar cells. This finding suggests that Fringe is necessary for generating positional information in localizing a high-affinity interaction between Notch and its ligand Delta, even if a second ligand is not essential.

Animals↗

Notch synergizes with axin to regulate the activity of armadillo in Drosophila.

Cell fate decisions require the integration of various signalling inputs at the level of transcription and signal transduction. Wnt and Notch signalling are two important signalling systems that operate in concert in a variety of systems in vertebrates and invertebrates. There is evidence that the Notch receptor can modulate Wnt signalling and that its target is the activity and levels of Armadillo/beta-catenin. Here, we characterize this function of Notch in relation to Axin, a key element in the regulation of Wnt signalling that acts as a scaffold for the Shaggy/GSK3beta-dependent phosphorylation of Armadillo/beta-catenin. While Notch can regulate ectopic Wingless signalling caused by loss of function of Shaggy, it can only partially regulate the ectopic Wnt signalling induced by the loss of Axin function. The same interactions are observed in tissue culture cells where we observe a synergy in between Axin and Notch in the regulation of Armadillo/beta-catenin. Our results provide evidence for a function of Axin in the regulation of Armadillo that is different from its role as a scaffold for GSK3beta.

Adaptor Proteins, Signal Transducing↗

Gamma-secretase-like cleavages of Notch and beta APP are mutually exclusive in human cells.

Presenilins 1 and 2 are two transmembrane proteins that seem necessary for controlling the proteolytic cleavages of two substrates, betaAPP and Notch, giving rise to Abeta (amyloid beta-peptide) and NICD (Notch Intracellular Domain), respectively. It is a matter for discussion whether presenilins act directly as the cleaving enzyme (referred to as gamma-secretase) or indirectly as a regulator of the substrates/enzymes trafficking to the permissive cell compartment where gamma-secretase cleavage could occur. Here we examined whether betaAPP and Notch undergo mutually exclusive proteolytic events in HEK293 cells or whether they behave as substrates able to compete for a single protease. We show that the overexpression of mDeltaE-Notch-1 does not influence the endogenous recovery of secreted and intracellular Abeta nor those derived from betaAPP-overexpressing HEK293 cells. We establish, conversely, that increasing amounts of betaAPP do not modify the steady-state generation of NICD nor affect the kinetic of production. These data indicate that the proteolytic cleavages leading to the productions of Abeta and NICD are mutually exclusive events in HEK293 cells, and suggest that distinct proteolytic activities contribute to betaAPP and Notch processing.

Amyloid Precursor Protein Secretases↗

Notch signaling can inhibit Xath5 function in the neural plate and developing retina.

Neuronal differentiation is regulated by both positive and negative regulatory factors; however, precisely how these factors interact to regulate retinogenesis is still unclear. We have examined the ability of the Notch pathway to modulate the function of the basic helix-loop-helix factor Xath5. Overexpression of Xath5 by RNA injection into cleavage-stage blastomeres promotes ectopic neurogenesis at neural plate stages and ganglion cell differentiation in the developing retina. We found that these activities of Xath5 could be inhibited by coexpression of activated Notch. Notch inhibition of Xath5 function was reversed by coexpression with the zinc finger protein X-MyT1. The Notch effector enhancer-of-split related 1 (ESR1) also blocked Xath5 activity but efficient inhibition by ESR1 required the DNA binding basic domain and the conserved WRPW motif. In addition, ESR1 inhibited the ability of Xath5 to directly activate the expression of XBrn3d, a transcription factor involved in retinal ganglion cell development. Xath5 could upregulate expression of X-Delta-1, ESR1, and ESR3, suggesting that Xath5 participates in a regulatory loop with the Notch pathway.

Amino Acid Motifs↗

Relationship between Notch receptors and hyperoxia-induced lung injury in newborn rats.

To investigate role of Notch1 - 3 in hyperoxia-induced lung injury in newborn rat exposed to 85% O2, SD rat litters born on the 22th day were randomly divided into two groups: room air group and hyperoxia group. The animals were sacrificed 1, 4, 7, 10, 14 and 21 days after continued exposure to oxygen (n = 40, oxygen > 0.85) or room air (n = 40). 6 rats each group were used to assess lung histological changes by HE staining and expression of Notch in lungs by immunohistochemistry. Total RNA was extracted by Trizol reagent from frozen lung tissues. Notch mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that 7, 14 and 21 days after O2 exposure, hyperoxia group showed lung injury characterized by pulmonary edema, hemorrhage and lung development arrest. Positive staining for Notch1, Notch 2 in hyperoxia group was much lower than those in room air group at all time points (P < 0. 01, P < 0.05), but compared with the controls, the hyperoxia group showed higher expression of Notch3 (P > 0.05). Immunostained cells were typically airways epithelia, alveolar epithelial and inflammatory cells, and fibroblasts in hyperoxia group (P < 0.01). Notch mRNA levels showed similar change as protein level (P < 0.01). It is concluded that the prolonged exposure to 85% O2 resulted in abnormal expression of Notch receptors, which might contribute to the pathogenesis of hyperoxia-induced lung injury in newborn rats. The decreased inhibition of Notch1 might be one of the protective reaction and major mechanisms for proliferation/differentiation of type II alveolar epithelial cells. The up-regulation of Notch3 activity might result in the lung development arrest of the newborn rats.

Aerobiosis↗

Evidence for Notch-mediated lateral inhibition in organizing butterfly wing scales.

Here I present gene expression data that implicate a Notch-mediated lateral inhibition process in the spatial organization of butterfly wing scales. During early pupal development the receptor molecule Notch is expressed in a grid-like pattern in the wing epithelium, resulting in parallel rows of uniformly spaced cells with low Notch expression. Previous work has shown that these low-Notch cells express a homolog of the achaete-scute proneural transcription factors and develop into scales. All of these observations are consistent with the Drosophila model of Notch-mediated bristle determination and support the hypothesis that bristles and scales share an underlying patterning mechanism.

Animals↗

Immunohistological localization of Notch receptors and their ligands Delta and Jagged in synovial tissues of rheumatoid arthritis.

BACKGROUND: The interaction of Notch receptors with their transmembrane ligands Delta and Jagged plays an important role not only in the organization of a variety of tissues but also in several genetic disorders and cancer development. The functional involvement of the Notch signaling in rheumatoid arthritis (RA) has been reported previously, but the expression profile of Notch-related molecules, as well as their relation with clinicopathological parameters, remains unclear. METHODS: In this study, we analyzed the immunohistochemical staining pattern of four Notch receptors (Notch1-4) and their ligands (Delta1 and Jagged1) in 14 synovial tissues obtained from 14 RA patients. RESULTS: Notch2 and Notch4 were expressed in limited areas in a few samples or in small blood vessels, respectively. Notch1, Notch3, Delta1, and Jagged1 were overexpressed in the synovial lining and sublining cells on synovial hyperplastic lesions in all samples. Notch1 expression was also observed in T and B lymphocytes of lymphoid follicles independently. Notch1 and Notch3 expression overlapped with that of Jagged1, as determined by confocal microscopy. Activation of Notch1 signaling in the RA synovium was identified using a specific antibody to the cleaved form of Notch1. The expression of these molecules did not show any correlation with clinicopathological parameters. CONCLUSIONS: Our results suggest that Notch signaling is activated in RA synovium but does not necessarily reflect the pathological condition of RA.

Adolescent↗

Notch signaling in breast cancer and tumor angiogenesis: cross-talk and therapeutic potentials.

Notch signaling is an evolutionarily conserved pathway that regulates numerous physiological processes. Disruption of Notch has been implicated in multiple tumor types. Evidence from in vitro experiments, mouse models and human tumor samples indicates that Notch plays a predominantly oncogenic role in breast cancer and interacts with other pathways involved in tumorigenesis. In addition, Notch signaling is required for physiological angiogenesis and may promote tumor angiogenesis. A variety of strategies for blocking Notch signaling, in particular gamma-secretase inhibition, are discussed as potential therapies for breast cancer and tumor angiogenesis.

Amyloid Precursor Protein Secretases↗

Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor.

The Notch gene of Drosophila plays an important role in cell fate specification throughout development. To investigate the functions of specific structural domains of the Notch protein in vivo, a series of deletion mutants have been ectopically expressed under the hsp70 heat shock promoter. Two classes of dominant phenotypes are observed, one suggestive of Notch loss-of-function mutations and the other of Notch gain-of-function mutations. Dominant activated phenotypes result from overexpression of a protein lacking most extracellular sequences, while dominant negative phenotypes result from overexpression of a protein lacking most intracellular sequences. These results support the notion that Notch functions as a receptor whose extracellular domain mediates ligand binding, resulting in the transmission of developmental signals by the cytoplasmic domain. Finally, the phenotypes observed suggest that the cdc 10/ankyrin repeat region within the intracellular domain plays an essential role in the postulated signal transduction events.

Animals↗

Direct regulation of the Nrarp gene promoter by the Notch signaling pathway.

Nrarp encodes for an evolutionarily conserved small ankyrin repeat-containing protein that functions as a negative regulator of Notch signaling. Interestingly, increased Nrarp transcription was observed following induction of Notch signaling, suggesting the existence of a negative feedback loop. We show here that both mouse and human promoter regions of Nrarp share two conserved regions located approximately 2 and approximately 3 kb upstream of the transcription start site each containing a perfect putative binding site for the Notch-dependent transcription factor Su(H). A 4.4 kb genomic fragment of the mouse Nrarp locus containing those conserved regions and fused to a luciferase reporter gene showed basal promoter activity in 293T cells and this activity was strongly increased by the intracellular domain of Notch, NICD. NICD-dependent stimulation was attenuated by a dominant negative mutant of Su(H), Su(H)DBM, and was not observed in Su(H)-deficient cells (OT-11). Promoter bashing and gel shift assays revealed that the most distal putative Su(H) binding site located within the -3 kb conserved element plays a crucial role in this induction. Collectively, these results provide definitive support for direct regulation of the Nrarp gene by the Notch pathway.

Animals↗

Notch signaling from tumor cells: a new mechanism of angiogenesis.

Notch signaling is an evolutionarily conserved pathway and plays key roles in embryonic vascular development and angiogenesis. Multiple components of the Notch pathway are expressed in vasculature, and mice deficient for a variety of these components display embryonic lethality with vascular remodeling defects. Alteration of Notch signaling in various endothelial cells generates profound effects on angiogenesis in vitro. New evidence shows that Notch signaling from tumor cells is able to activate endothelial cells and trigger tumor angiogenesis in vitro and in a xenograft mouse tumor model. Selective interruption of Notch signaling within tumors may provide an antiangiogenic strategy.

Angiogenesis Inhibitors↗

Lasting longer without oxygen: The influence of hypoxia on Notch signaling.

Notch signaling has multiple functions during invertebrate and vertebrate development, where it plays important roles in cell fate determination, proliferation, survival, and border formation. The precise function of Notch signaling is context dependent, and one of the unsolved mysteries of Notch signaling is how a relatively stereotyped signal transduction pathway exerts such a wide variety of context-specific responses. Recent data from Gustaffson et al. provide important new information on this topic by showing that hypoxia enhances Notch signaling due to the association of Notch and HIF-1alpha. This interaction may have important consequences for tumor cell growth.

Animals↗

Regulation of Notch signalling by endocytosis and endosomal sorting.

Cell-cell signalling is an essential process in the formation of multicellular organisms. Notch is the receptor of an evolutionarily conserved signalling pathway regulating numerous developmental decisions. Indeed, its misregulation is linked to multiple developmental and physiological disorders. Notch and its ligands are distributed widely throughout development, yet Notch activity is highly controlled and restricted in time and space. Recent advances have highlighted that endocytosis followed by endosomal sorting of both the Notch receptor and its ligands is an essential mechanism by which Notch-mediated signalling is developmentally controlled.

Animals↗

Crystal structure of the CSL-Notch-Mastermind ternary complex bound to DNA.

Notch signaling mediates communication between cells and is essential for proper embryonic patterning and development. CSL is a DNA binding transcription factor that regulates transcription of Notch target genes by interacting with coregulators. Transcriptional activation requires the displacement of corepressors from CSL by the intracellular portion of the receptor Notch (NotchIC) and the recruitment of the coactivator protein Mastermind to the complex. Here we report the 3.1 A structure of the ternary complex formed by CSL, NotchIC, and Mastermind bound to DNA. As expected, the RAM domain of Notch interacts with the beta trefoil domain of CSL; however, the C-terminal domain of CSL has an unanticipated central role in the interface formed with the Notch ankyrin repeats and Mastermind. Ternary complex formation induces a substantial conformational change within CSL, suggesting a molecular mechanism for the conversion of CSL from a repressor to an activator.

Amino Acid Sequence↗

Bre1 is required for Notch signaling and histone modification.

Notch signaling controls numerous cell fate decisions during animal development. These typically involve a Notch-mediated switch in transcription of target genes, although the details of this molecular mechanism are poorly understood. Here, we identify dBre1 as a nuclear component required cell autonomously for the expression of Notch target genes in Drosophila development. dBre1 affects the levels of Su(H) in imaginal disc cells, and it stimulates the Su(H)-mediated transcription of a Notch-specific reporter in transfected Drosophila cells. Strikingly, dBre1 mutant clones show much reduced levels of methylated lysine 4 on histone 3 (H3K4m), a chromatin mark that has been implicated in transcriptional activation. Thus, dBre1 is the functional homolog of yeast Bre1p, an E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation. Our results indicate that histone modification is critical for the transcription of Notch target genes.

Animals↗

The Drosophila Notch inhibitor and tumor suppressor gene lethal (2) giant discs encodes a conserved regulator of endosomal trafficking.

Notch signaling is involved in many developmental and pathological processes, and its activity must be precisely controlled in order to prevent aberrant development and disease. We have previously shown that the tumor suppressor gene lethal (2) giant discs (lgd) is required to prevent ectopic activation of Notch in developmental processes in Drosophila. Here we show that lgd is required in all imaginal disc cells to suppress the activity of the Notch pathway. lgd encodes a member of a poorly characterized protein family present in all animals, which includes a member that is involved in an inheritable form of mental retardation in humans. Our analysis reveals that Lgd is required for endosomal trafficking of Notch and other proteins. In the absence of Lgd, Notch is activated in a ligand-independent manner in probably all imaginal disc cells in an endosomal compartment downstream of the block in hrs mutants.

Animals↗

Diverse effects of the Notch ligands Jagged1 and Delta1 on the growth and differentiation of primary acute myeloblastic leukemia cells.

OBJECTIVE: Notch signaling plays a role in regulating the self-renewal and differentiation of hematopoietic progenitors. Since acute myeloblastic leukemia (AML) originates from dysregulated hematopoietic progenitors, the Notch system may be involved in the abnormal growth. We previously reported that AML cells express Notch proteins. In this study, we examined the effects of recombinant human Notch ligand proteins, Jagged1 and Delta1, on the growth and differentiation of primary AML cells. MATERIALS AND METHODS: AML cells separated from blood from 12 patients were cultured in wells coated with Jagged1, Delta1, or control IgG. The short-term growth was evaluated using a colorimetric assay. The self-renewal capacity was evaluated by the clonogenic cells recovered, which were obtained via a colony assay involving cells cultured with the ligands or control IgG. Differentiation was evaluated by the morphology of the cultured cells and flow cytometric analysis. RESULTS: The ligand stimulation caused three types of response in the short-term growth of the primary AML cells, namely, promotion, suppression, or no significant effect. The self-renewal capacity was suppressed or not significantly affected by the ligands, even in cells showing short-term growth promotion. The ligand stimulation altered blast cells into macrophage-like cells from their morphology and increased the expression of differentiation markers such as CD13 or CD14 in some samples. CONCLUSIONS: The Notch ligands had diverse effects on the short-term growth of primary AML cells. The ligands did not promote the self-renewal capacity of any of the cells examined and instead tended to induce differentiation under the conditions used.

Adult↗

Mouse Nkd1, a Wnt antagonist, exhibits oscillatory gene expression in the PSM under the control of Notch signaling.

During vertebrate embryogenesis, the formation of reiterated structures along the body axis is dependent upon the generation of the somite by segmentation of the presomitic mesoderm (PSM). Notch signaling plays a crucial role in both the generation and regulation of the molecular clock that provides the spatial information for PSM cells to form somites. In a screen for novel genes involved in somitogenesis, we identified a gene encoding a Wnt antagonist, Nkd1, which is transcribed in an oscillatory manner, and may represent a new member of the molecular clock constituents. The transcription of nkd1 is extremely downregulated in the PSM of vestigial tail (vt/vt), a hypomorphic mutant of Wnt3a, whereas nkd1 oscillations have a similar phase to lunatic fringe (L-fng) transcription and they are arrested in Hes7 (a negative regulator of Notch signaling) deficient embryos. These results suggest that the transcription of nkd1 requires Wnt3a, and that its oscillation patterns depend upon the function of Hes7. Wnt signaling has been postulated to be upstream of Notch signaling but we demonstrate in this study that a Wnt-signal-related gene may also be regulated by Notch signaling. Collectively, our data suggest that the reciprocal interaction of Notch and Wnt signals, and of their respective negative feedback loops, function to organize the segmentation clock required for somitogenesis.

Adaptor Proteins, Signal Transducing↗