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Notch
Kinds of Notch Terms modified by Notch Selected AbstractsSequential activation of Notch family receptors during mouse spermatogenesisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2003Shintaro Mori The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells. [source] Zebrafish notch signalling pathway mutants exhibit trunk vessel patterning anomalies that are secondary to somite misregulationDEVELOPMENTAL DYNAMICS, Issue 10 2010Christina Therapontos Abstract The Notch signalling pathway mutants, after-eight (aei), beamter (bea), and deadly-seven (des) have previously been used to study somitogenesis and neurogenesis. Notch signalling has also been shown to have roles in vascular development. However, vascular development in each of these three Notch mutants has not been described, and so their potential usefulness for further understanding the role of Notch signalling in angiogenesis is unknown. Here we demonstrate each of the mutants also exhibit vascular defects in inter-somitic vessel (ISV) positioning and patterning. Ectopic filopodia were also observed on the ISVs of the mutants. Ectopic filopodia are not due to loss of dll4. Somite expression of known vascular guidance cues, efnb2, sema3a2, and plexinD1 are disrupted, suggesting that the ISV vascular phenotype is due to disruption of these cues. Developmental Dynamics 239:2761,2768, 2010. © 2010 Wiley-Liss, Inc. [source] Role for notch signaling in salivary acinar cell growth and differentiationDEVELOPMENTAL DYNAMICS, Issue 3 2009Howard Dang Abstract The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of ,-secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes-1 expression, and Hes-1 expression was inhibited by ,-secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes-1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724,731, 2009. © 2009 Wiley-Liss, Inc. [source] Relationship between delta-like and proneural bHLH genes during chick retinal developmentDEVELOPMENTAL DYNAMICS, Issue 6 2008Branden R. Nelson Abstract Notch signaling in the retina maintains a pool of progenitor cells throughout retinogenesis. However, two Notch-ligands from the Delta-like gene family, Dll1 and Dll4, are present in the developing retina. To understand their relationship, we characterized Dll1 and Dll4 expression with respect to proliferating progenitor cells and newborn neurons in the chick retina. Dll4 matched the pattern of neural differentiation. By contrast, Dll1 was primarily expressed in progenitor cells. We compared Dll1 and Dll4 kinetic profiles with that of the transiently up-regulated cascade of proneural basic helix,loop,helix (bHLH) genes after synchronized progenitor cell differentiation, which suggested a potential role for Ascl1 in the regulation of Delta-like genes. Gain-of-function assays demonstrate that Ascl1 does influence Delta-like gene expression and Notch signaling activity. These data suggest that multiple sources of Notch signaling from newborn neurons and progenitors themselves coordinate retinal histogenesis. Developmental Dynamics 237:1565,1580, 2008. © 2008 Wiley-Liss, Inc. [source] Isolation and characterization of Xenopus Hey-1: A downstream mediator of Notch signalingDEVELOPMENTAL DYNAMICS, Issue 4 2002M.S. Rones Abstract Regulation of Notch signaling likely occurs, at least in part, at the level of basic helix-loop-helix (bHLH) transcription factors that function downstream of Suppressor of Hairless (Su(H)) in the Notch pathway. To begin to characterize modulation of Notch signaling during organogenesis, we examined the bHLH transcription factor, XHey-1 (hairy related-1) in early Xenopus laevis embryos. XHey-1 is expressed in numerous tissues during early development including the somites, head, embryonic kidneys, and heart. Importantly, the expression of XHey-1 was significantly altered in response to perturbation of Notch signaling by means of inducible constructs that served to either activate or suppress Notch signaling through Su(H) in a temporally controlled manner. © 2002 Wiley-Liss, Inc. [source] Pivotal role of Notch signaling in regulation of erythroid maturation and proliferationEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2006Yoshimichi Tachikawa Abstract:, Notch signaling plays an important role in cell fate decisions in developmental systems. To clarify its role in committed hematopoietic progenitor cells, we investigated the effects of Notch signaling in erythroid colony forming cells (ECFCs) generated from peripheral blood. ECFCs express Notch receptors, Notch1 and Notch2, and Notch ligands Delta1, Delta4, and Jagged1. When we assayed the effects of Notch ligands on erythroid maturation by flow cytometry, we found that immobilized Delta1 and immobilized Delta4 in particular inhibited maturation, whereas Jagged1 had no effect. In addition, Delta4 inhibited proliferation without reducing cell viability. Increases in expression levels of the Notch target gene hairy enhancer of split (HES) -1 were evident by real-time PCR after stimulation with immobilized Delta4. The effect of soluble Delta4 on expression of HES-1 was less pronounced than that seen with the immobilized form, indicating that all surface-bound ligands are important for effective signal transduction. When ECFCs were cultured in the presence of soluble Delta4 at a low cell concentration, erythroid maturation was slightly inhibited, but at a high concentration, maturation was promoted via competition of soluble Delta4 with endogenous ligands. These results indicate a pivotal role of Notch signaling in regulating erythroid maturation and proliferation, and further suggest that cell,cell interactions modulate growth of erythroid progenitor cells via Notch system. [source] Notch signaling is activated by TLR stimulation and regulates macrophage functionsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Tanapat Palaga Dr. Abstract Notch signaling is a well-conserved pathway involved in cell fate decisions, proliferation and apoptosis. We report on the involvement of Notch signaling in regulating gene expression in activated macrophages. Toll-like receptors (TLR) agonists such as bacterial lipopeptide, polyI:C, lipopolysaccharide and unmethylated CpG DNA all induced up-regulation of Notch1 in primary and macrophage-like cell lines. Notch1 up-regulation was dependent on the MyD88 pathway when stimulated through TLR2, but not TLR4. Activated Notch1 and expression of the Notch target genes, Hes1 and Deltex, were detected in activated macrophages, suggesting that Notch signaling was activated upon stimulation. Inhibiting processing of Notch receptor by ,-secretase using a ,-secretase inhibitor (GSI), the expression of Notch1 was down-regulated to basal levels. This treatment significantly modulated expression of TNF-,, IL-6, and IL-10. In addition, the amount of nitric oxide produced was significantly lower and the expression of MHC class II was up-regulated in GSI-treated cells. Treatment with GSI or silencing Notch1 resulted in decreased translocation of NF-,Bp50 into nucleus upon stimulation. Taken together, stimulation of macrophages through the TLR signaling cascade triggered activation of Notch signaling, which in turn regulated gene expression patterns involved in pro-inflammatory responses, through activation of NF-,B. [source] Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially regulate activation of peripheral T helper cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2005Sascha Rutz Abstract The Notch pathway is involved in cell differentiation processes in various organs and at several developmental stages. The importance of Notch for early T lymphocyte development is well established. Recently, Notch has been implicated in directing naive T helper cell differentiation towards the Th1, Th2 or regulatory T cell lineages. However, the molecular events underlying these processes are poorly understood. We show that the Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially affect early T cell activation and proliferation following T cell receptor cross-linking. Delta-like1 and Jagged1 induce a dose-dependent inhibition of early activation markers CD69 and CD25, as well as inhibition of proliferation after anti-CD3 stimulation of purified CD4+ T cells. Similarly, the rapid activation of transcription factors NF-AT, AP-1 and NF-,B is suppressed. In contrast, triggering of Notch by Delta-like4 enhances T cell activation and proliferation. The observed effects are dependent on simultaneous cross-linking of TCR and Notch but independent of ,-secretase-mediated cleavage of Notch. These data suggest direct interference between Notch and early TCR signal transduction events, independent of the classical Notch pathway via release of the Notch intracellular domain. A Notch-mediated alteration of TCR signaling strength may contribute to the recently described modulation of naïve T cell differentiation by Notch ligands. [source] The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2005Gina Balciunaite Abstract We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCR, and surface TCR,/, and TCR,/, expressing T cells. Selection for cytoplasmic TCR,-positive cells is dependent on preT, expression. Both ,/, and ,/, TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7. [source] Inhibition of Notch signaling biases rat thymocyte development towards the NK cell lineageEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2004Jens van den Brandt Abstract Notch receptors are involved in directing the choice between alternative cell fates in developmental scenarios such as thymopoiesis. By pharmacological interference in rat fetal thymus organ culture we show that inhibition of Notch signaling arrests T,cell development at an early double-negative stage and is accompanied by a dramatic increase in the number of NK cells. These cells show an activated phenotype, lack recombination of the TCR, gene locus and express perforin. Similarly, in thymic lobes reconstituted with fetal liver cells, progenitors predominantly develop into NK cells both after pharmacological interference of Notch and after treatment with a recombinant rat Notch1/Fc chimera. Collectively, this identifies the lineage decision of NK/T precursor cells as an important site of Notch action in rat thymocytes. [source] A Comparison of Conventional Local Approach and the Short Crack Approach to Fatigue Crack Initiation at a Notch,ADVANCED ENGINEERING MATERIALS, Issue 9 2009Narayanaswami Ranganathan Methods to estimate fatigue crack initiation life at a notch tip are compared. The methods used determine the strain amplitudes at the notch tip using Neuber's or Glinka's approximation. In conventional approaches, equivalent-damage levels are determined, using appropriate strain-life relationships coupled with damage-summation models. In the short-crack approach, a crack-like defect is assumed to exist at the notch tip. It is shown that the short-crack concept can be successfully applied to predict crack-initiation behavior at a notch. Model predictions are compared with carefully designed experiments. It is shown that model predictions are very close to experimentally measured lives under an aircraft-wing loading spectrum. [source] Cryptic variation in butterfly eyespot development: the importance of sample size in gene expression studiesEVOLUTION AND DEVELOPMENT, Issue 1 2007Robert D. Reed SUMMARY Previous studies have shown that development can be robust to variation in parameters such as the timing or level of gene expression. This leads to the prediction that natural populations should be able to host developmental variation that has little phenotypic effect. Cryptic variation is of particular interest because it can result in selectable phenotypes when "released" by environmental or genetic factors. Currently, however, we have little idea of how variation is distributed between genes or over time in pattern formation processes. Here we survey expression of Notch (N), Spalt (Sal), and Engrailed (En) during butterfly eyespot determination to better understand how pattern formation may vary within a population. We observed substantial heterochronic variance in the progress of spatial expression patterns for all three proteins, suggesting some degree of developmental buffering in eyespot development. Peak variance for different proteins was found at both early and late stages of development, contrasting with previous models suggesting that the distribution of variance should be more temporally focused during pattern formation. We speculate that our observations are representative of a standing reservoir of cryptic variation that may contribute to phenotypic evolution under certain circumstances. Our results also provide a strong cautionary message that gene expression studies with limited sample sizes can be positively misleading in terms of inferring expression pattern time series, as well as for making cross-species phylogenetic comparisons. [source] Hes7: a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesodermGENES TO CELLS, Issue 2 2001Yasumasa Bessho Background: Whereas Notch signalling is essential for somitogenesis, mice deficient for the basic helix-loop-helix (bHLH) genes Hes1 and Hes5, downstream Notch effectors, display normal somite formation, indicating that there may be an as-yet unidentified Hes1 -related bHLH gene. Results: We identified a novel bHLH gene, designated Hes7, from mouse embryos. Hes7 has a conserved bHLH domain in the amino-terminal region and the WRPW domain at the carboxy-terminal end, like Hes1. The mouse Hes7 gene is located next to Aloxe3, which is mapped to a position 37.0 cM from the centromere on chromosome 11. In a transfection analysis, Hes7 represses transcription from the N box- and E box-containing promoters. In addition, Hes7 suppresses the E47-induced transcriptional activation. Promoter analysis indicated that Hes7 expression is controlled by Notch signalling. Strikingly, Hes7 is specifically expressed in the presomitic mesoderm in a dynamic manner. We also identified two related bHLH genes from human: one is closely related to mouse Hes7 and therefore designated hHes7 and the other designated hHes4. Conclusion: The structure, transcriptional activity and expression pattern in the presomitic mesoderm of Hes7 are very similar to those of Hes1, suggesting that Hes7, together with Hes1, may play a role in somite formation under the control of Notch signalling. [source] Notch signaling modulates the activation of microglial cellsGLIA, Issue 15 2007Luc Grandbarbe Abstract The Notch signaling pathway plays a crucial role in specifying cellular fate in metazoan development by regulating communication between adjacent cells. Correlative studies suggested an involvement of Notch in hematopoietic cell development. Here, we report that the Notch pathway is expressed and active in microglial cells. During inflammatory activation, the transcription of the Notch down-stream effector Hes1 is downregulated. When Notch1 transcription in microglia is inhibited, an upregulation of the expression of pro-inflammatory cytokines is observed. Notch stimulation in activated microglia, using a soluble form of its ligand Jagged1, induces a decrease in pro-inflammatory cytokines secretion and nitric oxide production as well as an increase in phagocytic activity. Notch-stimulation is accompanied by an increase in the rate of STAT3 phosphorylation and nuclear translocation. Our results show that the Notch pathway plays an important role in the control of inflammatory reactions in the CNS. © 2007 Wiley-Liss, Inc. [source] Liver stem cells and hepatocellular carcinoma,HEPATOLOGY, Issue 1 2009Lopa Mishra Although the existence of cancer stem cells (CSCs) was first proposed over 40 years ago, only in the past decade have these cells been identified in hematological malignancies, and more recently in solid tumors that include liver, breast, prostate, brain, and colon. Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood, including the precise cell(s) of origin, molecular genetics, and the mechanisms responsible for the highly aggressive clinical picture of hepatocellular carcinoma (HCC). Exploration of the difference between CSCs from normal stem cells is crucial not only for the understanding of tumor biology but also for the development of specific therapies that effectively target these cells in patients. These ideas have drawn attention to control of stem cell proliferation by the transforming growth factor beta (TGF-,), Notch, Wnt, and Hedgehog pathways. Recent evidence also suggests a key role for the TGF-, signaling pathway in both hepatocellular cancer suppression and endoderm formation, suggesting a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as HCC. (HEPATOLOGY 2009;49:318,329.) [source] Bile duct proliferation in Jag1/fringe heterozygous mice identifies candidate modifiers of the alagille syndrome hepatic phenotype,HEPATOLOGY, Issue 6 2008Matthew J. Ryan Alagille syndrome (AGS) is a heterogeneous developmental disorder associated with bile duct paucity and various organ anomalies. The syndrome is caused by mutations in JAG1, which encodes a ligand in the Notch signaling pathway, in the majority of cases and mutations in the NOTCH2 receptor gene in less than 1% of patients. Although a wide array of JAG1 mutations have been identified in the AGS population, these mutational variants have not accounted for the wide phenotypic variability observed in patients with this syndrome. The Fringe genes encode glycosyltransferases, which modify Notch and alter ligand-receptor affinity. In this study, we analyzed double heterozygous mouse models to examine the Fringe genes as potential modifiers of the Notch-mediated hepatic phenotype observed in AGS. We generated mice that were haploinsufficient for both Jag1 and one of three paralogous Fringe genes: Lunatic (Lfng), Radical (Rfng), and Manic (Mfng). Adult Jag1+/,Lfng+/, and Jag1+/,Rfng+/, mouse livers exhibited widespread bile duct proliferation beginning at 5 weeks of age and persisting up to 1 year. The Jag1+/,Mfng+/, livers showed a subtle, yet significant increase in bile duct numbers and bile duct to portal tract ratios. These abnormalities were not observed in the newborn period. Despite the portal tract expansion by bile ducts, fibrosis was not increased and epithelial to mesenchymal transition was not shown in the affected portal tracts. Conclusion: Mice heterozygous for mutations in Jag1 and the Fringe genes display striking bile duct proliferation, which is not apparent at birth. These findings suggest that the Fringe genes may regulate postnatal bile duct growth and remodeling, and serve as candidate modifiers of the hepatic phenotype in AGS. (HEPATOLOGY 2008;48:1989,1997.) [source] Bile duct proliferation in liver-specific Jag1 conditional knockout mice: Effects of gene dosage,HEPATOLOGY, Issue 2 2007Kathleen M. Loomes The Notch signaling pathway is involved in determination of cell fate and control of cell proliferation in multiple organ systems. Jag1 encodes a ligand in the Notch pathway and has been identified as the disease-causing gene for the developmental disorder Alagille syndrome. Evidence from the study of human disease and mouse models has implicated Jag1 as having an important role in the development of bile ducts. We have derived a conditional knockout allele (Jag1loxP) to study the role of Jag1 and Notch signaling in liver and bile duct development. We crossed Jag1loxP mice with a transgenic line carrying Cre recombinase under the control of the albumin promoter and ,-fetoprotein enhancer to ablate Jag1 in hepatoblasts. The liver-specific Jag1 conditional knockout mice showed normal bile duct development. To further decrease Notch pathway function, we crossed the Jag1 conditional knockout mice with mice carrying the hypomorphic Notch2 allele, and bile duct anatomy remained normal. When Jag1 conditional mice were crossed with mice carrying the Jag1 null allele, the adult progeny exhibited striking bile duct proliferation. Conclusion: These results indicate that Notch signaling in the liver is sensitive to Jag1 gene dosage and suggest a role for the Notch pathway in postnatal growth and morphogenesis of bile ducts. (HEPATOLOGY 2007.) [source] Nuclear and membrane expression of the angiogenesis regulator delta-like ligand 4 (DLL4) in normal and malignant human tissuesHISTOPATHOLOGY, Issue 5 2009Juan Carlos Martinez Aims:, Delta-like ligand 4 (DLL4) is one of five known Notch ligands in mammals and interacts predominantly with Notch 1. DLL4 is induced by vascular endothelial growth factor (VEGF) and acts downstream of VEGF as a ,brake' on VEGF-induced vessel growth, forming an autoregulatory negative feedback loop inactivating VEGF. This action was believed to occur only in vascular development, raising hopes that DLL4 could be a specific drug target for controlling vessel growth in tumours and other pathological conditions. Our aim was to pursue this by raising a monoclonal antibody to the internal domain of DLL4 and assess its distribution in normal and malignant tissues in comparison with antibodies against the external domain of DLL4. Methods and results:, The anti-DLL4 monoclonal antibody was raised using conventional mouse hybridoma techniques. The antibody has been fully characterized by Western blotting and transfectant immunostaining. It has also been comprehensively compared with other antibodies against both the internal and external domains of DLL4. The antigen is widely expressed on human tissues not only on endothelium but also on epithelium and stromal cells. Indeed, in our comprehensive survey only pulmonary alveoli failed to express DLL4. Of a wide range of malignancies, most also expressed DLL4 on tumour cells with a predominantly cytoplasmic pattern, although a number also displayed nuclear positivity. Conclusions:, Contrary to previous beliefs, DLL4 is widely distributed in tissues other than vessels including many malignancies. Furthermore, the molecule is internalized on binding its receptor and often transported to the nucleus. These findings raise many interesting possibilities for further study of DLL4 and its potential as a therapeutic target. [source] Microenvironmental influences on human B-cell developmentIMMUNOLOGICAL REVIEWS, Issue 1 2000F. E. Bertrand Summary: Mammalian B-cell development can be viewed as a developmental performance with several acts. The acts are represented by checkpoints centered around commitment to the B-lineage and functional Ig gene rearrangement , culminating in expression of the pre-B-cell receptor (pre-BCR) and the BCR. Progression of cells through these checkpoints is profoundly influenced by the fetal liver and adult bone marrow (BM) stromal cell microenvironments. Our laboratory has developed a model of human B-cell development that utilizes freshly isolated/non-transformed human BM stromal cells as an in vitro microenvironment. Human CD34+ hematopoietic stem cells plated in this human BM stromal cell microenvironment commit to the B lineage and progress through the pre-BCR and BCR checkpoints. This human BM stromal cell microenvironment also provides survival signals that prevent apoptosis in human B-lineage cells. Human B-lineage cells exhibit differential expression of Notch receptors and human BM stromal cells express the Notch ligand Jagged-1. These results suggest a potential role for Notch in regulating B-lineage commitment and/or progression through the pre-BCR and BCR checkpoints. [source] Regulation of signal transduction by glycosylationINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Robert S. Haltiwanger The incredible diversity found in cell-surface glycoconjugate structures led researchers over 30 years ago to propose that complexity in carbohydrates must play a role in cellular communication. Recent studies from a number of laboratories have confirmed this hypothesis, demonstrating that cell-surface glycoconjugates play significant roles in signal transduction events. One striking example is the effect of O -fucose modifications on the Notch-signalling pathway. Notch is a cell-surface receptor that is essential for proper development. The extracellular domain of Notch contains up to 36-tandem epidermal growth factor-like (EGF) repeats, many of which are predicted to be modified at putative consensus sequences with O -fucose and O -glucose saccharides. Genetic alterations (by knockout or RNAi methodologies) in the enzyme responsible for the addition of O -fucose to Notch, protein O -fucosyltransferase-1, result in severe, embryonic lethal phenotypes resembling Notch mutants. Thus, O -fucosylation appears to be essential for proper Notch function. Elongation of the O -fucose monosaccharide by the ,1,3- N -acetylglucosaminyltransferase, Fringe, modulates Notch function, either increasing or decreasing response from ligands depending on context. Although it is now clear that O -fucose modifications affect Notch signalling, the molecular mechanism by which this occurs is not known. As an initial step in understanding how O -fucose glycans affect Notch function, we are mapping O -fucose modifications to specific sites on Notch. Already, we have demonstrated that O -fucose modifies one of the EGF repeats involved in ligand binding, suggesting that the sugars may play a role in Notch,ligand interactions. Experiments to test the role of O -fucose modifications at specific sites are in progress. We have also found that Fringe modifies O -fucose on some EGF repeats but not others. Our initial analyses suggest that the basis of this specificity is encoded within the sequences of the individual EGF repeats. Site mapping has also confirmed the presence of O -glucose saccharides on Notch. The evolutionarily conserved, predicted O -glucose sites on Notch are as numerous as those for O -fucose, suggesting that the O -glucose modifications will play an equally important role in Notch biology. We have recently identified an enzymatic activity capable of catalyzing the addition of O -glucose to EGF repeats. Purification of the protein O -glucosyltransferase is underway. These and other results will be discussed. [source] The distribution of Notch receptors and their ligands during articular cartilage developmentJOURNAL OF ANATOMY, Issue 6 2003A. J. Hayes Abstract We examined the distribution of Notch family members and their ligands during the development of articular cartilage and the growth plate. Notch 1 was expressed by the chondrocytes of the developing articular surface but became increasingly restricted to the deeper layers after birth whilst expression of this family member was restricted to hypertrophic chondrocytes in the growth plate. Notch 2 and 4, Delta and Jagged 2 showed a broadly similar distribution, being present throughout the articular cartilage during development and becoming increasingly restricted to deeper layers with age. Hypertrophic chondrocytes within the growth plate also expressed Notch 2 and 4, Delta and Jagged 2 (which was also expressed in prehypertrophs). Notch 3 and Jagged 1 were absent from developing articular cartilage but were present in deeper layers at later time points (> 1 month) and both receptor and ligand were expressed in hypertrophic chondrocytes at all ages examined. These results highlight the complex Notch signalling interactions that result in the formation of the heterogeneous articular cartilage and allow for the co-ordinated ossification and elongation of the growth plate. Mechanisms by which these processes are controlled are discussed in light of recent advances in the understanding of Notch signalling pathways. [source] A role for the transcription factor HEY1 in glioblastomaJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1 2009Esther Hulleman Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk. [source] Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2001Toru Mizuguchi Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood-forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow-derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co-cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell,cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcirption factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3-dioxygenase, are improved in hepatocyte/BMSC co-cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro. © 2001 Wiley-Liss, Inc. [source] Relative roles of TGF-,1 and Wnt in the systemic regulation and aging of satellite cell responsesAGING CELL, Issue 6 2009Morgan E. Carlson Summary Muscle stem (satellite) cells are relatively resistant to cell-autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P-Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor-beta (TGF-,) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age-dependent myogenic activity of sera TGF-,1, and its potential cross-talk with systemic Wnt. We found that sera TGF-,1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF-,1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF-,1 were inhibitory and young sera suppressed myogenesis if TGF-,1 was activated. Our data suggest that platelet-derived sera TGF-,1 levels, or endocrine TGF-,1 levels, do not explain the age-dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF-, neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF-,1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF-, receptor kinase inhibitor, which attenuated TGF-, signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF-,1-dependent block on muscle regeneration, identify physiological modalities of age-imposed changes in TGF-,1, and introduce new therapeutic strategies for the broad restoration of aged organ repair. [source] Notch signaling promotes astrogliogenesis via direct CSL-mediated glial gene activationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2002Weihong Ge Abstract In the developing central nervous system (CNS), Notch signaling preserves progenitor pools and inhibits neurogenesis and oligodendroglial differentiation. It has recently been postulated that Notch instructively drives astrocyte differentiation. Whether the role of Notch signaling in promoting astroglial differentiation is permissive or instructive has been debated. We report here that the astrogliogenic role of Notch is in part mediated by direct binding of the Notch intracellular domain to the CSL DNA binding protein, forming a transcriptional activation complex onto the astrocyte marker gene, glial fibrillary acidic protein (GFAP). In addition, we found that, in CSL,/, neural stem cell cultures, astrocyte differentiation was delayed but continued at a normal rate once initiated, suggesting that CSL is involved in regulating the onset of astrogliogenesis. Importantly, although the classical CSL-dependent Notch signaling pathway is intact and able to activate the Notch canonical target promoter during the neurogenic phase, it is unable to activate the GFAP promoter during neurogenesis. Therefore, the effect of Notch signaling on target genes is influenced by cellular context in regulation of neurogenesis and gliogenesis. © 2002 Wiley-Liss, Inc. [source] Expression of morphogenic genes in mature ovarian and testicular tissues: Potential stem-cell niche markers and patterning factorsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2006Kristian R. von Schalburg Abstract Morphogens are developmental regulators that modulate different tissue patterning, proliferation, differentiation, or remodeling processes in embryonic and adult tissues. Morphogens may also evoke specific regulatory programs in stem cells. Some of the morphogens involved in these processes have been characterized, while others remain unidentified. A microarray containing 3,557 salmonid cDNAs was used to compare the transcriptomes of rainbow trout precocious ovary at three different stages during second year (June, August, and October) with a reference (June normal ovary) transcriptome. During this study, we detected morphogen transcript hybridizations to salmonid elements and the study was enlarged to investigate these activities in various developmental stages of both ovary and testis. Genes from diverse development regulator families such as Anterior gradient-2, BMP, Epimorphin, Flightless, Frizzled, Notch, Tiarin, Twisted gastrulation, and Wnt were demonstrated to be expressed in the adult trout gonads. In mice or rats, expression of mammalian bmp-4, epimorphin, flightless, twisted gastrulation, and GW112 transcripts were localized to cell types isolated from the developed ovary and testis. Comparisons of salmonid and mammalian morphogens at the amino acid residue level show high similarities, suggesting functional conservation. This report provides evidence for local regulation by various morphogens and their potential to control distinct programs of gene expression in the gametes and their accessory cells during gametogenesis. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] , -secretase inhibitors exerts antitumor activity via down-regulation of Notch and Nuclear factor kappa B in human tongue carcinoma cellsORAL DISEASES, Issue 6 2007J Yao Objective:, To investigate the effect of the , -secretase inhibitors (GSIs) on the growth of human tongue carcinoma cells and to provide the molecular mechanism for potential application of GSIs in the treatment of tongue carcinoma. Materials and methods:, Human tongue carcinoma Tca8113 cells were cultured with the GSI L-685 458. Cell growth was determined by the methylthiazole tetrazolium method. Cell cycle and apoptosis were analyzed by flow cytometry and/or confocal microscopy. RT-PCR and Western blot were employed to determine the intracellular expression levels. Nuclear factor kappa B (NF- ,B) activation was examined by electrophoretic mobility shift assay. Results:, L-685,458 dose-dependently inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G0,G1 cell cycle arrest and apoptosis. The mRNA and protein levels of Hairy/Enhancer of Split-1, a target of Notch activation, were decreased dose-dependently by L-685,458. Furthermore, L-685,458 down-regulated cyclin D1, B-cell lymphocytic-leukemia proto-oncogene 2 and c-Myc expressions, which are regulated by the transcription factor NF- ,B. Coincident with this observation, L-685,458 induced a dose-dependent reduction of constitutive NF- ,B activation in Tca8113 cells. Conclusions:, The GSI L-685,458 may have a therapeutic value for the treatment of human tongue carcinoma. Moreover, the effects of L-685,458 in tumor inhibition may act partially via the modulation of Notch and NF- ,B. [source] Structure and stability of the ankyrin domain of the Drosophila Notch receptorPROTEIN SCIENCE, Issue 11 2003Mark E. Zweifel RMSD, root mean square deviation; CD, circular dichroism spectroscopy Abstract The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 Å resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions. [source] Involvement of the notch pathway in the regulation of matrix metalloproteinase 13 and the dedifferentiation of articular chondrocytes in murine cartilageARTHRITIS & RHEUMATISM, Issue 2 2009Régis Blaise Objective To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. Methods Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch -expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription,polymerase chain reaction, immunoblotting, and immunocytochemistry. Results Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. Conclusion The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch. [source] Expression of Notch signalling-related genes in normal and differentiating rat dental pulp cellsAUSTRALIAN ENDODONTIC JOURNAL, Issue 2 2010Hantang Sun dds Abstract Notch signalling is of fundamental importance to various processes during embryonic development and in adults. The possible role of Hey1, an important Notch signalling component, in odontoblast differentiation was evaluated in this study. Primary cultured dental pulp cells, derived from upper incisors of 5-week-old Wistar rats, were placed in ,-modification of Eagle's minimal essential medium supplemented with 10% Fetal Bovine Serum (FBS), and ascorbic acid (AA) and ,-glycerophosphate (,-GP), with or without dexamethasone, and cultured on dishes coated with collagen type IA for 7 days. Conventional and real-time Polymerase Chain Reaction (PCR) was performed to determine the expression of Notch-related genes and dentin sialophosphoprotein as a marker of odontoblast differentiation. Dentin sialophosphoprotein and Hey1 expression was significantly increased and decreased in the presence of AA + ,-GP compared with controls, respectively. These findings suggest that Hey1 may be a negative regulator in odontoblast differentiation. [source] | ||||||||||||||||||||||||||||||||||||||||