Secreted forms of DELTA and SERRATE define antagonists of Notch signaling in Drosophila

Development ◽  
1997 ◽  
Vol 124 (17) ◽  
pp. 3439-3448 ◽  
Author(s):  
X. Sun ◽  
S. Artavanis-Tsakonas
Keyword(s):  
Notch Signaling ◽  
Molecular Level ◽  
Notch Pathway ◽  
Dominant Negative ◽  
Genetic Interactions ◽  
Wing Margin ◽  
Loss Of Function ◽  
Dominant Negative Mutations ◽  
Notch Ligands ◽  
Enhancer Of Split

We examined the function of secreted forms of the two known Drosophila Notch ligands, DELTA and SERRATE, by expressing them under various promoters in the Drosophila developing eye and wing. The phenotypes associated with the expression of secreted Delta (DlS) or secreted Serrate (SerS) forms mimic loss-of-function mutations in the Notch pathway. Both genetic interactions between DlS or SerS transgenics and duplications or loss-of-function mutations of Delta or Serrate indicate that DlS and SerS behave as dominant negative mutations. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split mdelta, a target of Notch signaling, is down-regulated by SERS. The antagonistic nature of the two mutant secreted ligand forms in the eye is consistent with their behavior in the wing, where they are capable of down-regulating wing margin specific genes opposite to the effects of the endogenous ligands. This analysis uncovers secreted molecular antagonists of Notch signaling and provides evidence of qualitative differences in the actions of the two ligands DLS and SERS.

The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2465-2474 ◽  
Author(s):  
X. Sun ◽  
S. Artavanis-Tsakonas
Keyword(s):  
Notch Pathway ◽  
Notch Signalling ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Wild Type ◽  
Mutant Forms ◽  
Intracellular Domains ◽  
Notch Ligands ◽  
Enhancer Of Split

We examined the function of the intracellular domains of the two known Drosophila Notch ligands, Delta and Serrate, by expressing wild-type and mutant forms in the developing Drosophila eye under the sevenless promoter. The expression of intracellularly truncated forms of either Delta (sev-DlTM) or Serrate (sev-SerTM) leads to extra photoreceptor phenotypes, similar to the eye phenotypes associated with loss-of-function mutations of either Notch or Delta. Consistent with the notion that the truncated ligands reduce. Notch signalling activity, the eye phenotypes of sev-DlTM and sev-SerTM are enhanced by loss-of-function mutations in the Notch pathway elements, Notch, Delta, mastermind, deltex and groucho, but are suppressed by a duplication of Delta or mutations in Hairless, a negative regulator of the pathway. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split m delta, a target of Notch signalling, is down-regulated by the truncated ligands highly expressed in neighbouring cells. We conclude that the truncated ligands act as antagonists of Notch signalling.

scholarly journals An EP overexpression screen for genetic modifiers of Notch pathway function in Drosophila melanogaster

2004 ◽  
Vol 83 (2) ◽  
pp. 71-82 ◽  
Author(s):  
LAUREN E. HALL ◽  
SHAUNA J. ALEXANDER ◽  
MICHAEL CHANG ◽  
NATHANIEL S. WOODLING ◽  
BARRY YEDVOBNICK
Keyword(s):  
Notch Signaling ◽  
Notch Pathway ◽  
Dominant Negative ◽  
Genetic Modifiers ◽  
Loss Of Function ◽  
Partial Loss ◽  
Dominant Negative Form ◽  
Nuclear Component ◽  
Pathway Function ◽  
Negative Form

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.

Serrate and Notch specify cell fates in the heart field by suppressing cardiomyogenesis

Development ◽  
2000 ◽  
Vol 127 (17) ◽  
pp. 3865-3876
Author(s):  
M.S. Rones ◽  
K.A. McLaughlin ◽  
M. Raffin ◽  
M. Mercola
Keyword(s):  
Gene Expression ◽  
Notch Signaling ◽  
Cell Fate ◽  
Notch Pathway ◽  
Cell Types ◽  
Myocardial Cell ◽  
Dominant Negative ◽  
Cell Fates ◽  
Cell Fate Decisions ◽  
Heart Field

Notch signaling mediates numerous developmental cell fate decisions in organisms ranging from flies to humans, resulting in the generation of multiple cell types from equipotential precursors. In this paper, we present evidence that activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof (Raffin, M., Leong, L. M., Rones, M. S., Sparrow, D., Mohun, T. and Mercola, M. (2000) Dev. Biol., 218, 326–340). The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Here we show that Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicated that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreased myocardial gene expression and increased expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling had the opposite effect. Activation or inhibition of Notch also regulated contribution of individual cells to the myocardium. Importantly, expression of Nkx2. 5 and Gata4 remained largely unaffected, indicating that Notch signaling functions downstream of heart field specification. We conclude that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates.

scholarly journals Caenorhabditis elegans SUR-5, a Novel but Conserved Protein, Negatively Regulates LET-60 Ras Activity during Vulval Induction

1998 ◽  
Vol 18 (8) ◽  
pp. 4556-4564 ◽  
Author(s):  
Trent Gu ◽  
Satoshi Orita ◽  
Min Han
Keyword(s):  
Caenorhabditis Elegans ◽  
Sequence Similarity ◽  
Signal Transduction Pathway ◽  
Specific Aspect ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Negative Function ◽  
Ras Activation ◽  
Dominant Negative Mutations ◽  
Novel Protein

ABSTRACT The let-60 ras gene acts in a signal transduction pathway to control vulval differentiation in Caenorhabditis elegans. By screening suppressors of a dominant negativelet-60 ras allele, we isolated three loss-of-function mutations in the sur-5 gene which appear to act as negative regulators of let-60 ras during vulval induction.sur-5 mutations do not cause an obvious mutant phenotype of their own, and they appear to specifically suppress only one of the two groups of let-60 ras dominant negative mutations, suggesting that the gene may be involved in a specific aspect of Ras activation. Consistent with its negative function, overexpressing sur-5 from an extragenic array partially suppresses the Multivulva phenotype of an activatedlet-60 ras mutation and causes synergistic phenotypes with a lin-45 raf mutation. We have clonedsur-5 and shown that it encodes a novel protein. We have also identified a potential mammalian SUR-5 homolog that is about 35% identical to the worm protein. SUR-5 also has some sequence similarity to acetyl coenzyme A synthetases and is predicted to contain ATP/GTP and AMP binding sites. Our results suggest thatsur-5 gene function may be conserved through evolution.

Notch Signaling Is Necessary and Sufficient for Runx1-Dependent Stem Cell Induction in the Embryonic Aorta-Gonad-Mesonephros (AGM) Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4161-4161
Author(s):  
Caroline Erter Burns ◽  
Leonard I. Zon
Keyword(s):  
Stem Cell ◽  
Notch Signaling ◽  
Cell Fate ◽  
Notch Pathway ◽  
Notch Receptor ◽  
Dorsal Aorta ◽  
Loss Of Function ◽  
Cardinal Vein ◽  
Hematopoietic Stem

Abstract Vertebrate hematopoiesis can be divided into two embryonic phases: a short primitive wave predominantly generating erythrocytes and a definitive (fetal/adult) wave producing long-term hematopoietic stem cells (HSCs). The definitive wave occurs in the embryonic aorta-gonad-mesonephros (AGM) region through the asymmetric induction of HSCs from the ventral, but not dorsal, aortic endothelial wall. Since Notch signaling is critical for orchestrating a variety of developmental cell fate choices from invertebrates to humans and has been implicated in affecting the differentiation of some hematopoietic lineages, we analyzed whether the Notch pathway regulates definitive HSC induction in vivo. The zebrafish mutant mindbomb harbors a mutation in an essential E3 ligase that ubiquitylates Delta, which in turn allows the Notch intercellular domain to be released and activate downstream target gene transcription. Thus, in the absence of Mindbomb function Notch signaling does not occur. We found that although mindbomb mutants show normal primitive hematopoiesis, definitive c-myb and runx1 HSC expression is lacking. Since embryos injected with synthetic morpholinos designed to inhibit proper splicing of runx1 RNA ( runx morphants) show the same hematopoietic phenotype as mindbomb mutants, we next addressed the epistatic relationship between notch and runx1 using classic gain-of-function and loss-of-function analyses. In runx1 morphants expression of a notch receptor, notch3, and a delta ligand, deltaC, in the developing dorsal aorta was normal. Moreover, injection of runx1 RNA rescued HSCs in the AGM of mindbomb mutants. Together, these results suggest that Runx1 functions downstream of Notch in promoting HSC fate. We next analyzed whether a constitutively activated form of Notch (NICD) is sufficient for HSC specification in the AGM using an inducible binary transgenic system. Zebrafish carrying the heat-shock promoter driving the activator gal4 were mated to animals carrying 6 gal4 -responsive tandem upstream activating sequences (UAS) driving NICD. At the 10 somite-stage the embryos were heat-shocked at 37°C for 1 hour to activate NICD throughout the double transgenic animals. Surprisingly, expression of both HSC markers, c-myb and runx1, were expanded from their normal restricted domain in the ventral endothelium to the entire circumference of the dorsal aorta. Most interestingly, the presence of ectopic c-myb and runx1 transcripts were observed in the developing post-cardinal vein, a vessel that normally does not produce HSCs. These data imply that activation of the Notch pathway generates increased numbers of HSCs in vivo. When runx1 RNA is injected into wild-type embryos a similar expansion of c-myb transcripts is seen throughout the entire dorsal aorta and post-cardinal vein, further indicating that Runx1 functions downstream of Notch in HSC induction. In summary, discovery of the molecular programs essential and sufficient for fetal/adult hematopoietic ontogeny will lead to a further understanding of the physiologic and pathologic processes regulating stem cell homeostasis and translate into more effective therapies for blood disorders.

scholarly journals The Notch Pathway Intermediate HES-1 Silences CD4 Gene Expression

1998 ◽  
Vol 18 (12) ◽  
pp. 7166-7175 ◽  
Author(s):  
Han K. Kim ◽  
Gerald Siu
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Notch Signaling ◽  
Signaling Pathway ◽  
Transcriptional Control ◽  
Notch Pathway ◽  
Notch Signaling Pathway ◽  
Thymic Development ◽  
Enhancer Function ◽  
Enhancer Of Split

ABSTRACT We have previously identified a transcriptional silencer that is critical for proper expression of the CD4 gene during T-cell development. Here we report that the Hairy/Enhancer of Split homologue HES-1, a transcription factor in the lin12/Notch signaling pathway, binds to an important functional site in the CD4 silencer. Overexpression of HES-1 leads to the silencer site-dependent repression of CD4 promoter and enhancer function as well as the downregulation of endogenous CD4 expression in CD4+ CD8−TH cells. Interestingly, overexpression of an activated form of Notch1 (NotchIC) leads to the repression of CD4 promoter and enhancer function both in the presence and absence of the silencer. NotchIC-mediated CD4 silencer function is not affected by the deletion of the HES-1-binding site, indicating that multiple factors binding to CD4 transcriptional control elements are responsive to signaling from this pathway, including other silencer-binding factors. Taken together, these data are consistent with the hypothesis that the lin12/Notch signaling pathway is important in thymic development and provide a molecular mechanism via the control of CD4 gene expression in which the lin12/Notch pathway affects T-cell developmental fate.

scholarly journals The Drosophila melanogaster Suppressor of deltex Gene, a Regulator of the Notch Receptor Signaling Pathway, Is an E3 Class Ubiquitin Ligase

Genetics ◽  
1999 ◽  
Vol 152 (2) ◽  
pp. 567-576 ◽  
Author(s):  
M Cornell ◽  
D A P Evans ◽  
R Mann ◽  
M Fostier ◽  
M Flasza ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Cell Fate ◽  
Ubiquitin Ligase ◽  
Notch Pathway ◽  
Notch Receptor ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Wing Vein ◽  
Cell Fate Decisions

Abstract 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.

A New Target in Multiple Myeloma: Inhibition of Notch Pathway Induces Apoptosis and Enhances Drug Sensitivity of Myeloma Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 841-841
Author(s):  
Yulia Nefedova ◽  
Krista Verali ◽  
Daniel M. Sullivan ◽  
William S. Dalton ◽  
Dmitry I. Gabrilovich
Keyword(s):  
Multiple Myeloma ◽  
Notch Signaling ◽  
Stromal Cells ◽  
Tumor Size ◽  
Critical Role ◽  
Notch Pathway ◽  
Control Group ◽  
Induced Apoptosis ◽  
Notch Ligands

Abstract Bone marrow (BM) microenvironment and particularly BM stromal cells play a critical role in de novo drug resistance of multiple myeloma (MM) cells. BM stromal cells express Notch ligands and activate Notch signaling in MM cells. We have previously demonstrated that Notch signaling is one of the major mechanisms of BM stroma mediated MM cell protection from chemotherapeutic drugs. Here we investigated whether the pharmacological inhibition of Notch signaling with γ-secretase inhibitor (GSI) could affect viability of MM cells and overcome BM stroma mediated resistance of MM cells to chemotherapy. GSI (6–8 μM) induced apoptosis of MM cells cultured in suspension or on monolayer of BM stroma via specific inhibition of Notch signaling. The effect of GSI was evaluated in four different MM cell lines and primary MM cells isolated from BM of four patients with MM. Treatment with GSI alone significantly reduced the viability of MM cells with IC50 almost 5-fold lower than that for peripheral blood mononuclear cells or BM cells from healthy donors. In addition, treatment of MM cells with GSI reversed BM stroma mediated protection of MM cells from drug-induced apoptosis. This effect of GSI was associated by dramatic up-regulation of pro-apoptotic protein NOXA and down-regulation of anti-apoptotic proteins bcl-xL, bcl-2, and Akt. Inhibition of NOXA with siRNA canceled the cytotoxic effect of GSI on MM cells indicating that NOXA could mediate the direct effect of GSI on MM cells. To test the effect of GSI on MM cells in vivo we used SCID/NOD mouse model. Mice were injected s.c. with human MM RPMI-8226 cells. These cells express both Notch ligands and Notch receptors so Notch signaling is activated by interaction between MM cells themselves. Tumor became visible in ~3 weeks after inoculation and grew as a solid tumor which allowed easily monitoring tumor size. Mice were treated with doxorubicin (1.5 mg/kg, i.p., once a 4 days, 3 times), GSI (5mg/kg/day i.p. for 14 days), combinations of doxorubicin and GSI, or vehicle control (PBS). Tumor size was constantly monitored during treatment and 3 weeks after the treatment. Treatment with doxorubicin and GSI alone resulted in moderate decreased in tumor burden as compared with control group. In contrast, combination of GSI and doxorubicin induced dramatic antitumor effect. Thus, our study, for the first time, demonstrates that inhibition of Notch signaling with GSI can be a new promising approach for therapeutic intervention in MM.

scholarly journals Dominant-negative mutations in human IL6ST underlie hyper-IgE syndrome

2020 ◽  
Vol 217 (6) ◽  
Author(s):  
Vivien Béziat ◽  
Simon J. Tavernier ◽  
Yin-Huai Chen ◽  
Cindy S. Ma ◽  
Marie Materna ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Mutant Proteins ◽  
Skeletal Abnormalities ◽  
Tyrosine Residues ◽  
Hyper Ige Syndrome ◽  
Dominant Negative Mutations ◽  
Allergic Manifestations ◽  
Stat3 Mutations

Autosomal dominant hyper-IgE syndrome (AD-HIES) is typically caused by dominant-negative (DN) STAT3 mutations. Patients suffer from cold staphylococcal lesions and mucocutaneous candidiasis, severe allergy, and skeletal abnormalities. We report 12 patients from 8 unrelated kindreds with AD-HIES due to DN IL6ST mutations. We identified seven different truncating mutations, one of which was recurrent. The mutant alleles encode GP130 receptors bearing the transmembrane domain but lacking both the recycling motif and all four STAT3-recruiting tyrosine residues. Upon overexpression, the mutant proteins accumulate at the cell surface and are loss of function and DN for cellular responses to IL-6, IL-11, LIF, and OSM. Moreover, the patients’ heterozygous leukocytes and fibroblasts respond poorly to IL-6 and IL-11. Consistently, patients with STAT3 and IL6ST mutations display infectious and allergic manifestations of IL-6R deficiency, and some of the skeletal abnormalities of IL-11R deficiency. DN STAT3 and IL6ST mutations thus appear to underlie clinical phenocopies through impairment of the IL-6 and IL-11 response pathways.

scholarly journals four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3533-3542
Author(s):  
Gerri R. Buckles ◽  
Cordelia Rauskolb ◽  
John Lee Villano ◽  
Flora N. Katz
Keyword(s):  
Notch Signaling ◽  
Molecular Basis ◽  
Feedback Loop ◽  
Regional Growth ◽  
Notch Pathway ◽  
Notch Signaling Pathway ◽  
Normal Pattern ◽  
Notch Ligands ◽  
Notch Activation

The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. We show that four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with our demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led us to further investigate the relationships between these pathways. Surprisingly, we find that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. We also show that four-jointed interacts with abelson, enabled and dachs, which leads us to suggest that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out.

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