Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling

2000 ◽  
Vol 113 (23) ◽  
pp. 4313-4318 ◽  
Author(s):  
B. Varnum-Finney ◽  
L. Wu ◽  
M. Yu ◽  
C. Brashem-Stein ◽  
S. Staats ◽  
...  
Keyword(s):  
Cell Fate ◽  
Extracellular Domain ◽  
Notch Ligand ◽  
Cell Fate Decisions ◽  
Plastic Surface ◽  
Ligand Stabilization ◽  
Potential Activity ◽  
Inhibition Of Differentiation ◽  
Notch Ligands ◽  
Notch Activation

Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates. However, the mechanisms involved in ligand induced Notch activation are unknown. Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell. These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1(ext-myc)) or to the Fc portion of human IgG-1 (Delta-1(ext-IgG)), respectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized). Furthermore, both non-immobilized Delta-1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta. These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function. These results imply that ligand stabilization may be essential for Notch activation.

scholarly journals Density of the Notch ligand Delta1 determines generation of B and T cell precursors from hematopoietic stem cells

2005 ◽  
Vol 201 (9) ◽  
pp. 1361-1366 ◽  
Author(s):  
Mari H. Dallas ◽  
Barbara Varnum-Finney ◽  
Colleen Delaney ◽  
Keizo Kato ◽  
Irwin D. Bernstein
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Fate ◽  
Precursor Cells ◽  
Hematopoietic Stem ◽  
Notch Ligand ◽  
Murine Bone Marrow ◽  
Cell Fate Decisions ◽  
Multiple Cell ◽  
B Cell Precursors

Notch signaling regulates multiple cell fate decisions by hematopoietic precursors. To address whether different amounts of Notch ligand influence lineage choices, we cultured murine bone marrow lin−Sca-1+c-kit+ cells with increasing densities of immobilized Delta1ext-IgG consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1. We found that relatively lower densities of Delta1ext-IgG enhanced the generation of Sca-1+c-kit+ cells, Thy1+CD25+ early T cell precursors, and B220+CD43−/lo cells that, when cocultured with OP9 stroma cells, differentiated into CD19+ early B cell precursors. Higher densities of Delta1ext-IgG also enhanced the generation of Sca-1+c-kit+ precursor cells and promoted the development of Thy1+CD25+ cells, but inhibited the development of B220+CD43−/lo cells. Analyses of further isolated precursor populations suggested that the enhanced generation of T and B cell precursors resulted from the effects on multipotent rather than lymphoid-committed precursors. The results demonstrate the density-dependent effects of Delta1 on fate decisions of hematopoietic precursors at multiple maturational stages and substantiate the previously unrecognized ability of Delta1 to enhance the development of both early B and T precursor cells.

scholarly journals Jagged2 acts as a Delta-like Notch ligand during early hematopoietic cell fate decisions

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4449-4459 ◽  
Author(s):  
Inge Van de Walle ◽  
Greet De Smet ◽  
Martina Gärtner ◽  
Magda De Smedt ◽  
Els Waegemans ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Promoter Activation ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Lineage Differentiation ◽  
Hematopoietic Lineage ◽  
Myeloid Development ◽  
Dose Dependent ◽  
Notch Ligands

Abstract Notch signaling critically mediates various hematopoietic lineage decisions and is induced in mammals by Notch ligands that are classified into 2 families, Delta-like (Delta-like-1, -3 and -4) and Jagged (Jagged1 and Jagged2), based on structural homology with both Drosophila ligands Delta and Serrate, respectively. Because the functional differences between mammalian Notch ligands were still unclear, we have investigated their influence on early human hematopoiesis and show that Jagged2 affects hematopoietic lineage decisions very similarly as Delta-like-1 and -4, but very different from Jagged1. OP9 coculture experiments revealed that Jagged2, like Delta-like ligands, induces T-lineage differentiation and inhibits B-cell and myeloid development. However, dose-dependent Notch activation studies, gene expression analysis, and promoter activation assays indicated that Jagged2 is a weaker Notch1-activator compared with the Delta-like ligands, revealing a Notch1 specific signal strength hierarchy for mammalian Notch ligands. Strikingly, Lunatic-Fringe– mediated glycosylation of Notch1 potentiated Notch signaling through Delta-like ligands and also Jagged2, in contrast to Jagged1. Thus, our results reveal a unique role for Jagged1 in preventing the induction of T-lineage differentiation in hematopoietic stem cells and show an unexpected functional similarity between Jagged2 and the Delta-like ligands.

The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1402-1407 ◽  
Author(s):  
Kohshi Ohishi ◽  
Barbara Varnum-Finney ◽  
Rita E. Serda ◽  
Claudio Anasetti ◽  
Irwin D. Bernstein
Keyword(s):  
Dendritic Cells ◽  
Notch Signaling ◽  
Cell Fate ◽  
Interleukin 4 ◽  
Cellular Interactions ◽  
Blood Monocytes ◽  
Notch Ligand ◽  
Cell Fate Decisions ◽  
Gm Csf ◽  
Tnf Α

Notch-mediated cellular interactions are known to regulate cell fate decisions in various developmental systems. A previous report indicated that monocytes express relatively high amounts of Notch-1 and Notch-2 and that the immobilized extracellular domain of the Notch ligand, Delta-1 (Deltaext-myc), induces apoptosis in peripheral blood monocytes cultured with macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage CSF (GM-CSF). The present study determined the effect of Notch signaling on monocyte differentiation into macrophages and dendritic cells. Results showed that immobilized Deltaext-myc inhibited differentiation of monocytes into mature macrophages (CD1a+/−CD14+/− CD64+) with GM-CSF. However, Deltaext-myc permitted differentiation into immature dendritic cells (CD1a+CD14−CD64−) with GM-CSF and interleukin 4 (IL-4), and further differentiation into mature dendritic cells (CD1a+CD83+) with GM-CSF, IL-4, and tumor necrosis factor-α (TNF-α). Notch signaling affected the differentiation of CD1a−CD14+macrophage/dendritic cell precursors derived in vitro from CD34+ cells. With GM-CSF and TNF-α, exposure to Deltaext-myc increased the proportion of precursors that differentiated into CD1a+CD14− dendritic cells (51% in the presence of Deltaext-myc versus 10% in control cultures), whereas a decreased proportion differentiated into CD1a−CD14+ macrophages (6% versus 65%). These data indicate a role for Notch signaling in regulating cell fate decisions by bipotent macrophage/dendritic precursors.

Monocytes express high amounts of Notch and undergo cytokine specific apoptosis following interaction with the Notch ligand, Delta-1

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2847-2854 ◽  
Author(s):  
Kohshi Ohishi ◽  
Barbara Varnum-Finney ◽  
David Flowers ◽  
Claudio Anasetti ◽  
David Myerson ◽  
...  
Keyword(s):  
Cell Fate ◽  
Colony Stimulating Factor ◽  
Notch 1 ◽  
Notch Ligand ◽  
Cell Fate Decisions ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Induced Apoptosis ◽  
Stimulating Factor

Notch signaling has been shown to play a key role in cell fate decisions in numerous developmental systems. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we reported the expression of human Notch-1 in CD34+ progenitors. In this study, we evaluated the expression of human Notch-1 and Notch-2 protein by hematopoietic cells. In immunofluoresence study, we detected low amounts of Notch-1 and Notch-2 protein in both CD34+ and CD34+Lin− cells, high amounts in CD14+ monocytes as well as B and T cells, but no expression in CD15+ granulocytes. We further found that an immobilized truncated form of the Notch ligand, Delta-1, induced apoptosis in monocytes in the presence of macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage colony-stimulating factor (GM-CSF). The widespread expressions of Notch proteins suggest multiple functions for this receptor during hematopoiesis. These studies further indicate a novel role for Notch in regulating monocyte survival.

scholarly journals Modifications of the notch function by Abruptex mutations in Drosophila melanogaster.

Genetics ◽  
1994 ◽  
Vol 136 (1) ◽  
pp. 183-194 ◽  
Author(s):  
J F de Celis ◽  
A Garcia-Bellido
Keyword(s):  
Transmembrane Protein ◽  
Extracellular Domain ◽  
Cell Fates ◽  
Notch Protein ◽  
Protein Functions ◽  
Notch Gene ◽  
Epidermal Growth ◽  
Notch Locus ◽  
Notch Ligands ◽  
Notch Activation

Abstract The function of the Notch gene is required in cell interactions defining alternative cell fates in several developmental processes. The Notch gene encodes a transmembrane protein with 36 epidermal growth factor (EGF)-like repeats in its extracellular domain. This protein functions as a receptor that interacts with other transmembrane proteins, such as Serrate and Delta, which also have EGF repeats in their extracellular domain. The Abruptex mutations of the Notch locus are associated with amino acid substitutions in the EGF repeats 24-29 of the Notch protein. We have studied, in genetic combinations, the modifications of Notch function caused by Abruptex mutations. These mutations lead to phenotypes which are opposite to those caused by Notch deletions. The Abruptex phenotypes are modified by the presence of mutations in other loci, in particular in the genes Serrate and Delta as well as Hairless, and groucho. The results suggest that all Abruptex mutations cause stronger than normal Notch activation by the Delta protein. Some Abruptex alleles also display an insufficiency of N function. Abruptex alleles which produce stronger enhancement of Notch activation also display stronger Notch insufficiency. This insufficiency could be due to reduced ability of Abruptex proteins to interact with Notch ligands and/or to form functional Notch dimers.

scholarly journals Enhanced T-cell reconstitution by hematopoietic progenitors expanded ex vivo using the Notch ligand Delta1

Blood ◽  
2007 ◽  
Vol 109 (8) ◽  
pp. 3579-3587 ◽  
Author(s):  
Mari H. Dallas ◽  
Barbara Varnum-Finney ◽  
Paul J. Martin ◽  
Irwin D. Bernstein
Keyword(s):  
T Cell ◽  
Cell Fate ◽  
Hematopoietic Cell Transplantation ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
Hematopoietic Progenitors ◽  
Notch Ligand ◽  
Cell Fate Decisions ◽  
T Cell Reconstitution

Abstract A physiologic role for Notch signaling in hematopoiesis has been clearly defined in lymphoid differentiation, with evidence suggesting a critical role in T-cell versus B-cell fate decisions. Previously, we demonstrated that activation of endogenous Notch receptors by culture of murine lin−Sca-1+c-kit+ (LSK) hematopoietic progenitors with exogenously presented Notch ligand, Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1, promoted early T-cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show that culture of LSK precursors with Delta1ext-IgG increases the number of progenitors that are able to rapidly repopulate the thymus and accelerate early T-cell reconstitution with a diversified T-cell receptor repertoire. Most of the early T-cell reconstitution originated from cells that expressed lymphoid-associated antigens: B220, Thy1, CD25, and/or IL7Rα, whereas the most efficient thymic repopulation on a per cell basis originated from the smaller number of cultured cells that did not express lymphoid-associated antigens. These findings demonstrate the potential of Delta1ext-IgG-cultured cells for accelerating early immune reconstitution after hematopoietic cell transplantation.

scholarly journals Positive autoregulation of lag-1 in response to LIN-12 activation in cell fate decisions during C. elegans reproductive system development

Development ◽  
2020 ◽  
Vol 147 (18) ◽  
pp. dev193482
Author(s):  
Katherine Leisan Luo ◽  
Ryan S. Underwood ◽  
Iva Greenwald
Keyword(s):  
Cell Fate ◽  
Reproductive System ◽  
Target Gene ◽  
System Development ◽  
Animal Development ◽  
Cell Fate Decisions ◽  
C Elegans ◽  
Spatiotemporal Regulation ◽  
Target Gene Transcription ◽  
Notch Activation

ABSTRACTDuring animal development, ligand binding releases the intracellular domain of LIN-12/Notch by proteolytic cleavage to translocate to the nucleus, where it associates with the DNA-binding protein LAG-1/CSL to activate target gene transcription. We investigated the spatiotemporal regulation of LAG-1/CSL expression in Caenorhabditis elegans and observed that an increase in endogenous LAG-1 levels correlates with LIN-12/Notch activation in different cell contexts during reproductive system development. We show that this increase is via transcriptional upregulation by creating a synthetic endogenous operon, and identified an enhancer region that contains multiple LAG-1 binding sites (LBSs) embedded in a more extensively conserved high occupancy target (HOT) region. We show that these LBSs are necessary for upregulation in response to LIN-12/Notch activity, indicating that lag-1 engages in direct positive autoregulation. Deletion of the HOT region from endogenous lag-1 reduced LAG-1 levels and abrogated positive autoregulation, but did not cause hallmark cell fate transformations associated with loss of lin-12/Notch or lag-1 activity. Instead, later somatic reproductive system defects suggest that proper transcriptional regulation of lag-1 confers robustness to somatic reproductive system development.

scholarly journals Random birth order and bursty Notch ligand expression drive the stochastic AC/VU cell fate decision inC. elegans

2019 ◽  
Author(s):  
Simone Kienle ◽  
Nicola Gritti ◽  
Jason R. Kroll ◽  
Ana Kriselj ◽  
Yvonne Goos ◽  
...  
Keyword(s):  
Birth Order ◽  
Cell Fate ◽  
Single Molecule ◽  
Gonad Development ◽  
Time Lapse ◽  
Notch Signaling Pathway ◽  
Noise Sources ◽  
Notch Ligand ◽  
Cell Fate Decisions ◽  
Molecular Noise

AbstractCells in developing organisms must robustly assume the correct fate in order to fulfill their specific function. At the same time, cells are strongly affected by molecular fluctuations, i.e. ‘noise’, leading to inherent variability in individual cells. During development, some cells are thought to exploit such molecular noise to drive stochastic cell fate decisions, with cells randomly picking one cell fate out of several possible ones. Yet, how molecular noise drives such decisions is an open question. We address this question using a novel quantitative approach to study one of the best-understood stochastic cell fate decisions: the AC/VU decision inC. elegansgonad development. Here, two initially equivalent cells, Z1.ppp and Z4.aaa, interact, so that one cell becomes the anchor cell (AC) and the other a ventral uterine precursor cell (VU). It is thought that the symmetry is broken when small molecular fluctuations are amplified into cell fate by positive feedback loops in the Notch signaling pathway. To identify the noise sources that drive the AC/VU decision, we used a novel time-lapse microscopy approach to follow expression dynamics in live animals and single molecule FISH to quantify gene expression with single mRNA resolution. We found not only that random Z1.ppp/Z4.aaa birth order biased the decision outcome, with the first-born cell typically assuming VU fate, but that the strength of this bias and the speed of the decision decreased as the two cells were born closer together in time. Moreover, we find that the Notch ligandlag-2/Deltaexhibited strongly stochastic expression already in the two mother cells, Z1.pp/Z4.aa. Combining experiments with mathematical models, we showed that the resulting asymmetry inlag-2/Deltalevels inherited by the daughter cells, Z1.ppp/Z4.aaa, stochastic symmetry breaking when both cells are born at similar times. Together, our results suggest that two independent noise sources, birth order and stochasticlag-2/Deltaexpression, are exploited to amplify noise into cell fate in a manner that ensures a robust decision.

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