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PubMedDate not supplied

Lack of requirement for presenilin1 in Notch1 signaling.

Studies in invertebrates have indicated a functional requirement for presenilin (PS) genes in the Notch pathway [1-5]. One model of Notch signal transduction suggests that proteolysis releases an activated Notch fragment that migrates to the nucleus and regulates gene transcription in concert with CBF1/Su(H)/lag1 (CSL) proteins [6-9]. Recent studies suggest that PS genes control the proteolysis and nuclear access of the Notch intracellular domain [3,4,10,11], offering a basis for the functional interaction of PS and Notch genes [12]. Here, we report that Notch1 signaling elicited by the ligand Delta1 was quantitatively unchanged in PS1-deficient primary embryonic fibroblasts (PEFs). Notch1 signals were measured by both the activation of the hairy/enhancer of split (HES1) promoter and by the antagonism of MyoD-induced muscle creatine kinase (MCK) promoter activity. A membrane-tethered ligand-independent Notch1 construct also showed full efficacy in both assays, despite its presumed requirement for cleavage. Although signaling through Notch1 persisted in PS1-deficient cells, we found a marked reduction in the appearance of a complex of a cleaved, intracellular Notch fragment (NICD) and a CSL protein, as previously reported [6] [10]. These studies reveal that PS1 is not required for ligand-dependent Notch signaling, and that PS1 and PS2 may be redundant. Our data also suggest that the identified NICD fragment may not be necessary for Notch signal transduction [9].

Animals↗
PubMed2004

Mastermind critically regulates Notch-mediated lymphoid cell fate decisions.

During lymphoid development, Notch1 plays a critical role in the T-cell/B-cell lineage decision, while Notch2 is essential for marginal zone B-cell (MZB) development. Notch pathway activation induces translocation of intracellular Notch (ICN) to the nucleus, where it interacts with the transcription factor CSL (CBF1/RBP-Jk, Suppressor of Hairless, Lag-1). In vitro, ICN binds Mastermind-like proteins, which act as potent Notch coactivators. Three MAML family members (MAML1-3) have been identified in mammals, but their importance in vivo is unknown. To investigate the function of MAMLs in hematopoietic development, we introduced a dominant negative (DN) mutant of MAML1, capable of inhibiting Notch1-4, in murine hematopoietic stem cells. DNMAML1 resulted in early inhibition of T-cell development and the appearance of intrathymic B cells, phenotypes consistent with Notch1 inhibition. The T-cell differentiation block was as profound as that produced by enforced expression of the Notch modulator Deltex1. In DNMAML1-transduced spleen cells, a dramatic decrease in MZB cells was present, consistent with Notch2 inhibition. In contrast, Deltex1 did not decrease MZB cell numbers. These results suggest a critical role for MAMLs during Notch-mediated cell fate decisions in vivo and indicate that DNMAML1, but not Deltex1, can be used to interfere with the function of multiple Notch family members.

Animals↗
PubMed1997

Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing.

The veins in the Drosophila wing have a characteristic width, which is regulated by the activity of the Notch pathway. The expression of the Notch-ligand Delta is restricted to the developing veins, and coincides with places where Notch transcription is lower. We find that this asymmetrical distribution of ligand and receptor leads to activation of Notch on both sides of each vein within a territory of Delta-expressing cells, and to the establishment of boundary cells that separate the vein from adjacent interveins. In these cells, the expression of the Enhancer of split gene m beta is activated and the transcription of the vein-promoting gene veinlet is repressed, thus restricting vein differentiation. We propose that the establishment of vein thickness utilises a combination of mechanisms that include: (1) independent regulation of Notch and Delta expression in intervein and vein territories, (2) Notch activation by Delta in cells where Notch and Delta expression overlaps, (3) positive feedback on Notch transcription in cells where Notch has been activated and (4) repression of veinlet transcription by E(spl)m beta and maintenance of Delta expression by veinlet/torpedo activity.

Animals↗
PubMed2003

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis.

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.

Arteries↗
PubMed2005

Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs.

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.

3' Untranslated Regions↗
PubMed2005

Regulation of Notch signalling by non-visual beta-arrestin.

Signalling activity of the Notch receptor, which plays a fundamental role in metazoan cell fate determination, is controlled at multiple levels. We uncovered a Notch signal-controlling mechanism that depends on the ability of the non-visual beta-arrestin, Kurtz (Krz), to influence the degradation and, consequently, the function of the Notch receptor. We identified Krz as a binding partner of a known Notch-pathway modulator, Deltex (Dx), and demonstrated the existence of a trimeric Notch-Dx-Krz protein complex. This complex mediates the degradation of the Notch receptor through a ubiquitination-dependent pathway. Our results establish a novel mode of regulation of Notch signalling and define a new function for non-visual beta-arrestins.

Animals↗
PubMed1999

The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 class ubiquitin ligase.

During development, the Notch receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. We have previously defined Su(dx) genetically as a negative regulator of Notch signaling. Here we present the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. We have identified a seven-codon deletion in a Su(dx) mutant allele and we show that expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling.

Amino Acid Sequence↗
PubMed2002

Isolation and characterization of Xenopus Hey-1: a downstream mediator of Notch signaling.

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.

Animals↗
PubMed2002

Presenilin-dependent gamma-secretase activity modulates neurite outgrowth.

Demonstration that cleavage of both APP and Notch are dependent on the product of the early onset Alzheimer's disease gene, presenilin-1 (PS1), has raised the possibility that Notch function may be altered in AD. This finding also suggests that Notch may be affected by APPgamma-secretase inhibitors under development for the treatment of Alzheimer's disease, as these target PS1. Data that address these questions have been lacking, due to inability to specifically modulate PS1 activity in a system directly relevant to the adult human brain. Using novel highly specific inhibitors of PS1/gamma-secretase, we demonstrate that modulation of PS1 activity in human CNS neurons not only affects Abeta generation, but also has unanticipated effects on Notch and its activity. We demonstrate that intracellular trafficking of Notch in human CNS neurons is altered by inhibition of PS1 and is accompanied by dramatic changes in neurite morphology, consistent with inhibition of Notch activity. These data, together with immunohistochemical evidence of elevation of Notch pathway expression in AD brain, suggest that Notch dysregulation may contribute to the neuritic dystrophy characteristically seen in Alzheimer's disease brain. In addition, they raise the possibility that inhibition of gamma-secretase/PS1 may have clinically beneficial effects on the neuritic pathology of AD, in addition to its expected effect to reduce amyloid burden.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis.

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.

Animals↗
PubMed1997

Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane.

Previous models for signal transduction via the Notch pathway have depicted the full-length Notch receptor expressed at the cell surface. We present evidence demonstrating that the Notch receptor on the plasma membrane is cleaved. This cleavage is an evolutionarily conserved, general property of Notch and occurs in the trans-Golgi network as the receptor traffics toward the plasma membrane. Although full-length Notch is detectable in the cell, it does not reach the surface. Cleavage results in a C-terminal fragment, N(TM), that appears to be cleaved N-terminal to the transmembrane domain, and an N-terminal fragment, N(EC), that contains most of the extracellular region. We provide evidence that these fragments are tethered together on the plasma membrane by a link that is sensitive to reducing conditions, forming a heterodimeric receptor.

Blotting, Western↗
PubMed2003

Drosophila blood cells.

Drosophila blood cells or haemocytes belong to three lineages: plasmatocytes, crystal cells and lamellocytes. There is no equivalent of a lymphoid lineage in insects which have no adaptive immunity. Haematopoiesis is under the control of a number of transcription factors and signalling pathways (such as GATA factors, JAK/STAT or Notch pathways) most of which have homologues which participate in the control of mammalian haematopoiesis. Drosophila plasmatocytes are professional phagocytes reminiscent of the cells from the mammalian monocyte/macrophage lineage. Several receptors responsible for recognition of microorganisms or apoptotic corpses have been identified, which include a Scavenger Receptor, a CD36 homologue and a peptidoglycan recognition protein. Crystal cells contain the enzymes necessary for humoral melanization that accompanies a number of immune reactions. The production of melanin generates, as by-products, cytotoxic free radicals that are believed to participate in the killing of pathogens. Finally, lamellocytes represent a cell type that specifically differentiates after parasitism of Drosophila larvae and forms a capsule around the invader. Encapsulation together with melanization eventually kill the parasite within the capsule.

Animals↗
PubMed2002

Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex.

Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL enhancer-binding proteins. Here we show that chromatin-dependent transactivation by the recombinant Notch ICD-CBF1 enhancer complex in vitro requires an additional coactivator, Mastermind (MAM). MAM provides two activation domains necessary for Notch signaling in mammalian cells and in Xenopus embryos. We show that the central MAM activation domain (TAD1) recruits CBP/p300 to promote nucleosome acetylation at Notch enhancers and activate transcription in vitro. We also find that MAM expression induces phosphorylation and relocalization of endogenous CBP/p300 proteins to nuclear foci in vivo. Moreover, we show that coexpression with MAM and CBF1 strongly enhances phosphorylation and proteolytic turnover of the Notch ICD in vivo. Enhanced phosphorylation of the ICD and p300 requires a glutamine-rich region of MAM (TAD2) that is essential for Notch transcription in vivo. Thus MAM may function as a timer to couple transcription activation with disassembly of the Notch enhancer complex on chromatin.

Animals↗
PubMed2006

Coordination of symmetric cyclic gene expression during somitogenesis by Suppressor of Hairless involves regulation of retinoic acid catabolism.

Vertebrate embryos faithfully produce bilaterally symmetric somites that give rise to repetitive body structures such as vertebrae and skeletal muscle. Body segmentation is regulated by a cyclic gene expression system, containing the Delta-Notch pathway and targets, which generates bilaterally symmetric oscillations across the Pre-Somitic Mesoderm (PSM). The position of the forming somite boundary is controlled by interaction of this oscillator with a determination front comprised of opposing gradients of FGF and retinoic acid (RA) signalling. Disruption of RA production leads to asymmetries in cyclic gene expression, but the link between RA and the oscillator is unknown. In somitogenesis, Notch signalling activates target genes through the transcription factor Suppressor of Hairless (Su(H)). Here, we report that two Su(H) genes coordinate bilaterally symmetric positioning of somite boundaries in the zebrafish embryo. Combined Su(H) gene knockdown caused defects in visceral left/right asymmetry, neurogenic lateral inhibition, and symmetrical failure of the segmentation oscillator. However, by selectively down-regulating Su(H)2 or Su(H)1 function using specific antisense morpholinos, we observed asymmetric defects in anterior or posterior somite boundaries, respectively. These morphological abnormalities were reflected by underlying asymmetric cyclic gene expression waves in the presomitic mesoderm, indicating a key role for Su(H) in coordinating the left-right symmetry of this process. Strikingly, expression of the RA-degrading enzyme cyp26a1 in the tailbud was controlled by Su(H) activity, and morpholino knockdown of cyp26a1 alone caused asymmetric cyclic dlc expression, suggesting that excess RA in the tailbud may contribute to the cyclic asymmetries. Indeed, exogenous RA was sufficient to generate asymmetric expression of all cyclic genes. Our observations indicate that one element of the Notch signalling pathway, Su(H), is required for control of RA metabolism in the tailbud and that this regulation is involved in bilateral symmetry of cyclic gene expression and somitogenesis.

Amino Acid Sequence↗
PubMed2006

Role of Sox2 in the development of the mouse neocortex.

The mammalian neocortex is established from neural stem and progenitor cells that utilize specific transcriptional and environmental factors to create functional neurons and astrocytes. Here, we examined the mechanism of Sox2 action during neocortical neurogenesis and gliogenesis. We established a robust Sox2 expression in neural stem and progenitor cells within the ventricular zone, which persisted until the cells exited the cell cycle. Overexpression of constitutively active Sox2 in neural progenitors resulted in upregulation of Notch1, recombination signal-sequence binding protein-J (RBP-J) and hairy enhancer of split 5 (Hes5) transcripts and the Sox2 high mobility group (HMG) domain seemed sufficient to confer these effects. While Sox2 overexpression permitted the differentiation of progenitors into astroglia, it inhibited neurogenesis, unless the Notch pathway was blocked. Moreover, neuronal precursors engaged a serine protease(s) to eliminate the overexpressed Sox2 protein and relieve the repression of neurogenesis. Glial precursors and differentiated astrocytes, on the other hand, maintained Sox2 expression until they reached a quiescent state. Sox2 expression was re-activated by signals that triggered astrocytic proliferation (i.e., injury, mitogenic and gliogenic factors). Taken together, Sox2 appears to act upstream of the Notch signaling pathway to maintain the cell proliferative potential and to ensure the generation of sufficient cell numbers and phenotypes in the developing neocortex.

Animals↗
PubMed2005

[Notch signaling in the regulation of hematopoiesis].

Notch signaling defines an evolutionarily ancient cell interaction mechanism. The signals transmitted through the Notch receptor, in combination with other cellular factors influence differentiation, proliferation and apoptotic events at all stages of development. Recent advances have elucidated both the biochemical mechanism regulating receptor activation and the molecular participants forming the intracellular signaling cascade. Authors present description of the main signaling components involved in the Notch pathway and how it can affect the growth and function of lymphocytes. Notch signaling is critical during lymphocyte development, and dysregulation of the pathway can give rise to leukemia. It is conceivable that appropriate manipulation of Notch signaling may become a useful tool in addressing a variety of human dysplastic condition and tissue regeneration.

Animals↗
PubMed2002

Regulation of notch signaling by o-linked fucose.

Notch and its ligands are modified by a protein O-fucosyltransferase (OFUT1) that attaches fucose to a Serine or Threonine within EGF domains. By using RNAi to decrease Ofut1 expression in Drosophila, we demonstrate that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for Ofut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. The transcription of Ofut1 is developmentally regulated, and surprisingly, overexpression of Ofut1 inhibits Notch signaling. Together, these results indicate that OFUT1 is a core component of the Notch pathway, which is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development.

Animals↗
PubMed2006

Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis.

Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.

Animals↗