scholarly journals Optogenetic inhibition of Delta reveals digital Notch signaling output during tissue differentiation

2019 ◽  
Author(s):  
Ranjith Viswanathan ◽  
Aleksandar Necakov ◽  
Mateusz Trylinski ◽  
Rohit Krishnan Harish ◽  
Daniel Krueger ◽  
...  
Keyword(s):  
Cellular Response ◽  
Genome Engineering ◽  
Notch Pathway ◽  
Tissue Level ◽  
Notch Signalling ◽  
Tissue Differentiation ◽  
Type Locus ◽  
Spatio Temporal ◽  
Notch Activation

AbstractSpatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue- and cell-scale regulatory modes. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.

scholarly journals Drosophila IMP regulates Kuzbanian to control the timing of Notch signalling in the follicle cells

2018 ◽  
Author(s):  
Weronika Fic ◽  
Celia Faria ◽  
Daniel St Johnston
Keyword(s):  
Rna Binding ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Follicle Cells ◽  
Over Expression ◽  
Apical Domain ◽  
Late Endosomes ◽  
Notch Cleavage ◽  
Mirna Pathway ◽  
Notch Activation

AbstractThe timing of Drosophila egg chamber development is controlled by a germline Delta signal that activates Notch in the follicle cells to induce them to cease proliferation and differentiate. Here we report that follicle cells lacking the RNA-binding protein IMP go through one extra division due to a delay in the Delta-dependent S2 cleavage of Notch. The timing of Notch activation has previously been shown to be controlled by cis-inhibition by Delta in the follicle cells, which is relieved when the miRNA pathway represses Delta expression. imp mutants are epistatic to Delta mutants and give an additive phenotype with belle and dicer mutants, indicating that IMP functions independently of both cis-inhibition and the miRNA pathway. We find that the imp phenotype is rescued by over-expression of Kuzbanian, the metalloprotease that mediates the Notch S2 cleavage. Furthermore, Kuzbanian is not enriched at the apical membrane in imp mutants, accumulating instead in late endosomes. Thus, IMP regulates Notch signalling by controlling the localisation of Kuzbanian to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.SummaryIMP regulates Notch signalling in follicle cells by controlling Kuzbanian localisation to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.

scholarly journals Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
N. Träber ◽  
K. Uhlmann ◽  
S. Girardo ◽  
G. Kesavan ◽  
K. Wagner ◽  
...  
Keyword(s):  
Computational Analysis ◽  
Tissue Level ◽  
Quantitative Investigation ◽  
Pulsatile Pressure ◽  
Spatio Temporal ◽  
Stress Sensing ◽  
Living Organisms ◽  
Insight Into

AbstractMechanical stress exerted and experienced by cells during tissue morphogenesis and organ formation plays an important role in embryonic development. While techniques to quantify mechanical stresses in vitro are available, few methods exist for studying stresses in living organisms. Here, we describe and characterize cell-like polyacrylamide (PAAm) bead sensors with well-defined elastic properties and size for in vivo quantification of cell-scale stresses. The beads were injected into developing zebrafish embryos and their deformations were computationally analyzed to delineate spatio-temporal local acting stresses. With this computational analysis-based cell-scale stress sensing (COMPAX) we are able to detect pulsatile pressure propagation in the developing neural rod potentially originating from polarized midline cell divisions and continuous tissue flow. COMPAX is expected to provide novel spatio-temporal insight into developmental processes at the local tissue level and to facilitate quantitative investigation and a better understanding of morphogenetic processes.

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

Development ◽  
2001 ◽  
Vol 128 (1) ◽  
pp. 107-116 ◽  
Author(s):  
E. Hirsinger ◽  
P. Malapert ◽  
J. Dubrulle ◽  
M.C. Delfini ◽  
D. Duprez ◽  
...  
Keyword(s):  
Myogenic Differentiation ◽  
Muscle Differentiation ◽  
Detailed Examination ◽  
Notch Signalling ◽  
Myogenic Cells ◽  
Myogenic Markers ◽  
Notch Activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.

scholarly journals Effect of Hes1 on AML Cells: A Potential Therapeutic Target

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5208-5208
Author(s):  
Chen Tian ◽  
Yongsheng Jia ◽  
Dongzhi Hu ◽  
Yizhuo Zhang
Keyword(s):  
Conflicts Of Interest ◽  
Growth Arrest ◽  
Notch Pathway ◽  
Growth And Survival ◽  
Downstream Effector ◽  
Cancer Types ◽  
Notch Signal Pathway ◽  
Notch Activation

Abstract Notch signal pathway is an important mediator of growth and survival in several cancer types, while Notch pathway genes function as oncogenes or tumor suppressors in different cancers. Although aberrant Notch activation contributes to leukemogenesis in T cells, the role of Notch pathway in acute myeloid leukemia (AML) remains unclear. To address this problem, we investigated the expression levels of its downstream effector Hes1 and p21 in primary AML cells and cell lines by realtime PCR and western, and found that both of them was weak in these cells. Induced activation of Hes1 consisently led to AML growth arrest and apoptosis, which was associated with enhanced p21 expression. Besides, overexpression of Hes1 inhibited growth of AML cells in vivo. In conclusion, we reported that Hes1 mediated growth arrest and apoptosis of human AML cells, and demonstrated a novel tumor suppressor role for Hes1 in AML. Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals NOTCH activity differentially affects alternative cell fate acquisition and maintenance

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Leonard Cheung ◽  
Paul Le Tissier ◽  
Sam GJ Goldsmith ◽  
Mathias Treier ◽  
Robin Lovell-Badge ◽  
...  
Keyword(s):  
Cell Fate ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Differential Sensitivity ◽  
Endocrine Gland ◽  
Direct Role ◽  
Cell Lineages ◽  
Pituitary Development ◽  
Multiple Processes ◽  
Notch Activation

The pituitary is an essential endocrine gland regulating multiple processes. Regeneration of endocrine cells is of therapeutic interest and recent studies are promising, but mechanisms of endocrine cell fate acquisition need to be better characterised. The NOTCH pathway is important during pituitary development. Here, we further characterise its role in the murine pituitary, revealing differential sensitivity within and between lineages. In progenitors, NOTCH activation blocks cell fate acquisition, with time-dependant modulation. In differentiating cells, response to activation is blunted in the POU1F1 lineage, with apparently normal cell fate specification, while POMC cells remain sensitive. Absence of apparent defects in Pou1f1-Cre; Rbpjfl/fl mice further suggests no direct role for NOTCH signalling in POU1F1 cell fate acquisition. In contrast, in the POMC lineage, NICD expression induces a regression towards a progenitor-like state, suggesting that the NOTCH pathway specifically blocks POMC cell differentiation. These results have implications for pituitary development, plasticity and regeneration. Activation of NOTCH signalling in different cell lineages of the embryonic murine pituitary uncovers an unexpected differential sensitivity, and this consequently reveals new aspects of endocrine lineages development and plasticity.

Involvement of Dll1/Notch Interaction In MM Drug Resistance, Clonogenic Growth and In Vivo Engraftment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2966-2966
Author(s):  
Dehui Xu ◽  
Jinsong Hu ◽  
Elke De Bruyne ◽  
Eline Menu ◽  
Ben Van Camp ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Stromal Cells ◽  
Target Genes ◽  
Notch Pathway ◽  
Bortezomib Treatment ◽  
Clonogenic Growth ◽  
Pathway Activation ◽  
Notch Activation

Abstract Abstract 2966 The great challenges in multiple myeloma (MM) treatment are to overcome drug resistance and to prevent relapse. The bone marrow (BM) microenvironment plays a critical role in MM cell growth and survival. The Notch pathway was found to be activated in MM. In this study we aimed at investigating the role of Dll1/Notch interaction in MM clonogenic growth and in vivo engraftment, as well as the role of Notch pathway in BM-induced drug resistance. Dll1 is a Notch ligand expressed in BM stromal cells. Cocultures were performed using murine 5T33MM cells with Dll1 ligand or MS5.Dll1 (Dll1-overexpressing) stromal cells. Notch downstream target genes (Hes1, Hes5, Hey1, Hey2, and HeyL) were investigated for Notch pathway activation, showing that 5T33MM cells expressed Notch target genes and that Hes5 and HeyL were up-regulated both on mRNA (Real-Time PCR) and protein level (western blot) after Dll1/Notch interaction. This up-regulation could be reverted by blocking Notch signaling with DAPT. Moreover, only adhesive cell-cell direct interaction was necessary for MM Notch activation and no soluble factors in the coculture system were involved. Colony-forming-cell (CFC) assays and in vivo engraftment studies were performed to investigate MM clonogenic ability and MM disease initiation. 5T33MM cells cocultured for several days on MS5.Dll1 cells were able to form more tumor colonies compared to 5T33MM cells cocultured on MS5 control cells. Furthermore, these cocultured 5T33MM cells were injected intravenously into naive mice to investigate disease development. Mice injected with Dll1-activated 5T33MM cells developed faster MM disease than those injected with control cells, and also had a higher paraprotein concentration (1.98 ± 0.44 g/dl, n=3) compared to control mice (0.95 ± 0.02 g/dl, n=3). 5T33MM cells pretreated with DAPT for 6 days, to inhibit constitutive Notch pathway activation, had a decreased ability to form colonies in the CFC assay compared to control cells. In addition, mice were injected with 5T33MM cells pretreated with DAPT or DMSO. After 59 days, the DMSO control group became sick with an average M paraprotein of (1.78±0.14g/dl, n=3), while in the DAPT group, all mice were healthy and no M paraprotein could be detected at that time. The DAPT treated group did develop MM disease, but significantly delayed (average of 161 days after injection) with a lower M paraprotein amount (0.35±0.13g/dl, n=3). All these results suggest that the Notch pathway is involved in MM in vitro clonogenic growth and in vivo engraftment. Further studies revealed that Notch pathway activation with Dll1 could induce drug resistance to Bortezomib treatment. Cell viability assay showed that 72.6% of 5T33MM cells stimulated with Dll1 remained alive after Bortezomib treatment compared to only 24.9% alive in IgG control. This protective effect could be reverted by inhibiting Notch pathway with DAPT. After cocultures of MM cells with MS5.Dll1 stromal cells, we detected an increased percentage of CD138- MM cells and a down-regulation of CD138 mRNA expression compared to cocultures with MS5 stromal cells. This could be reverted by preventing cell-cell interaction using transwells or by adding DAPT. Similar results were found with human RPMI-8226 cells. Matsui et al. (Cancer Res 2008; 68: (1) 190–197) and our lab demonstrated that CD138- MM cells were less sensitive to drugs than CD138+ MM cells. Interestingly, CD138- MM cells showed a higher level of Notch activation than CD138+ MM cells, suggesting that Notch activation is involved in the protection of CD138- MM cells from drug induced cell death. In conclusion, our data show that Dll1/Notch pathway activation could promote MM clonogenic growth and accelerate MM development. Inhibiting Notch pathway can significantly suppress MM initiation and delay the engraftment. Moreover, Dll1/Notch interaction could also induce drug resistance to Bortezomib, by altering MM cells to a more resistant CD138- phenotype. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Notch pathway inhibition targets chemoresistant insulinoma cancer stem cells

2018 ◽  
Vol 25 (2) ◽  
pp. 131-144 ◽  
Author(s):  
Y Capodanno ◽  
F O Buishand ◽  
L Y Pang ◽  
J Kirpensteijn ◽  
J A Mol ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Targeted Therapies ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Treatment Modalities ◽  
Stem Cell Markers ◽  
Gamma Secretase

Insulinomas (INS) are the most common neuroendocrine pancreatic tumours in humans and dogs. The long-term prognosis for malignant INS is still poor due to a low success rate of the current treatment modalities, particularly chemotherapy. A better understanding of the molecular processes underlying the development and progression of INS is required to develop novel targeted therapies. Cancer stem cells (CSCs) are thought to be critical for the engraftment and chemoresistance of many tumours, including INS. This study was aimed to characterise and target INS CSCs in order to develop novel targeted therapies. Highly invasive and tumourigenic human and canine INS CSC-like cells were successfully isolated. These cells expressed stem cell markers (OCT4,SOX9, SOX2, CD133 and CD34), exhibited greater resistance to 5-fluorouracil (5-FU) and demonstrated a more invasive and tumourigenic phenotypein vivocompared to bulk INS cells. Here, we demonstrated that Notch-signalling-related genes (NOTCH2andHES1)were overexpressed in INS CSC-like cells. Protein analysis showed an active NOTCH2-HES1 signalling in INS cell lines, especially in cells resistant to 5-FU. Inhibition of the Notch pathway, using a gamma secretase inhibitor (GSI), enhanced the sensitivity of INS CSC-like cells to 5-FU. When used in combination GSI and 5-FU, the clonogenicityin vitroand the tumourigenicityin vivoof INS CSC-like cells were significantly reduced. These findings suggested that the combined strategy of Notch signalling inhibition and 5-FU synergistically attenuated enriched INS CSC populations, providing a rationale for future therapeutic exploitation.

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

Development ◽  
1997 ◽  
Vol 124 (10) ◽  
pp. 1919-1928 ◽  
Author(s):  
J.F. de Celis ◽  
S. Bray ◽  
A. Garcia-Bellido
Keyword(s):  
Positive Feedback ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Notch Ligand ◽  
Asymmetrical Distribution ◽  
Drosophila Wing ◽  
Split Gene ◽  
Enhancer Of Split ◽  
Notch Activation ◽  
In Cells

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.

scholarly journals Inhibition of Notch1 promotes hedgehog signalling in a HES1-dependent manner in chondrocytes and exacerbates experimental osteoarthritis

2016 ◽  
Vol 75 (11) ◽  
pp. 2037-2044 ◽  
Author(s):  
Neng-Yu Lin ◽  
Alfiya Distler ◽  
Christian Beyer ◽  
Ariella Philipi-Schöbinger ◽  
Silvia Breda ◽  
...  
Keyword(s):  
Target Genes ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Dependent Manner ◽  
Notch 1 ◽  
Chondrocyte Hypertrophy ◽  
Hedgehog Signalling ◽  
Osteophyte Formation

ObjectivesNotch ligands and receptors have recently been shown to be differentially expressed in osteoarthritis (OA). We aim to further elucidate the functional role of Notch signalling in OA using Notch1 antisense transgenic (Notch1 AS) mice.MethodsNotch and hedgehog signalling were analysed by real-time PCR and immunohistochemistry. Notch-1 AS mice were employed as a model of impaired Notch signalling in vivo. Experimental OA was induced by destabilisation of the medial meniscus (DMM). The extent of cartilage destruction and osteophyte formation was analysed by safranin-O staining with subsequent assessment of the Osteoarthritis Research Society International (OARSI) and Mankin scores and µCT scanning. Collagen X staining was used as a marker of chondrocyte hypertrophy. The role of hairy/enhancer of split 1 (Hes-1) was investigated with knockdown and overexpression experiments.ResultsNotch signalling was activated in human and murine OA with increased expression of Jagged1, Notch-1, accumulation of the Notch intracellular domain 1 and increased transcription of Hes-1. Notch1 AS mice showed exacerbated OA with increases in OARSI scores, osteophyte formation, increased subchondral bone plate density, collagen X and osteocalcin expression and elevated levels of Epas1 and ADAM-TS5 mRNA. Inhibition of the Notch pathway induced activation of hedgehog signalling with induction of Gli-1 and Gli-2 and increased transcription of hedgehog target genes. The regulatory effects of Notch signalling on Gli-expression were mimicked by Hes-1.ConclusionsInhibition of Notch signalling activates hedgehog signalling, enhances chondrocyte hypertrophy and exacerbates experimental OA including osteophyte formation. These data suggest that the activation of the Notch pathway may limit aberrant hedgehog signalling in OA.

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