Identification of Notch ligand useage by splenic B cells using Dll1 and Dll4 Notch ligand blockade.
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PROVIDER: PRJNA1148919 | ENA |
REPOSITORIES: ENA
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Project description:Marginal zone (MZ) B cells leverage Notch2 signals to attain rapid differentiation responses and positional cues with in the spenic environment.To determine the likely molecular source of Notch2 signals we sought to block the Notch ligands delta-like ligand 1 (Dll1) and delta-like ligand 4 (Dll4) in wildtype mice using in-vivo treatment with monoclonal blocking antibodies. Splenic populations of MZ B cells and follicular B cells were sort purified after 12, 24 and 48 hours of blockade and analyzed by RNA-seq.
2024-10-18 | GSE274991 | GEO
Project description:Objective DLL1 is a mammalian Notch ligand with essential functions during embryonic development. We tagged endogenous DLL1 in one homologous recombination step such that AcGFP-HA tagged DLL1 could be converted by Cre-mediated site-specific recombination into StrepFlag tagged DLL1. We anticipated that this should allow us to visualise DLL1 in living cells as well as allow for sorting and enrichment of DLL1-expressing cells and efficient purification of DLL1 protein complex. Results We generated constructs to express a DLL1 variant that carried C-terminal in frame an AcGFPHA tag flanked by loxP sites followed by a StrepFlag tag out of frame. Cre-mediated recombination removed AcGFP-HA and added StrepFlag in frame to DLL1. The AcGFPHAstopStrepFlag tag was added to the Dll1 cDNA to allow for tests in cultured cells in vitro and was introduced into endogenous DLL1 in mice using ES cells modified by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cell and embryo lysates. In CHO cells the AcGFP protein fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the StrepFlag tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or StrepFlag-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.
2021-11-03 | PXD024680 | Pride
Project description:Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in the embryonic aorta. In most vertebrates, hemogenic cells protrude into the aortic lumen forming clusters where the hematopoietic features are acquired. Although much is known about the molecular characteristics of the hematopoietic cells, we still lack basic understanding on the origin and 3D-organization of intraortic hematopoietic clusters (IAHC) in the mouse embryo and how they evolve over time. Here we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modelling to show that IAHC formation is a two-step process. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the cluster. Next, cells from the surrounding hemogenic endothelium are recruited into the IAHCs, increasing their size and heterogeneity. We found that the recruitment phase of IAHC formation is negatively regulated by Notch signalling via the Delta-like-4 (Dll4) Notch ligand. We show that blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modelling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions. Single cell transcriptomic analysis indicates that cell identities are preserved after Dll4 blockage. Our data provides for the first time a detailed characterization of the aortic hematopoietic stem cell niche and its maturation dynamics, identifying the Notch ligand Dll4 as a major molecular player governing dynamics of cluster formation and HSCs production.
2020-02-03 | GSE124477 | GEO
Project description:Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in the embryonic aorta. In most vertebrates, hemogenic cells protrude into the aortic lumen forming clusters where the hematopoietic features are acquired. Although much is known about the molecular characteristics of the hematopoietic cells, we still lack basic understanding on the origin and 3D-organization of intraortic hematopoietic clusters (IAHC) in the mouse embryo and how they evolve over time. Here we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modelling to show that IAHC formation is a two-step process. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the cluster. Next, cells from the surrounding hemogenic endothelium are recruited into the IAHCs, increasing their size and heterogeneity. We found that the recruitment phase of IAHC formation is negatively regulated by Notch signalling via the Delta-like-4 (Dll4) Notch ligand. We show that blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modelling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions. Single cell transcriptomic analysis indicates that cell identities are preserved after Dll4 blockage. Our data provides for the first time a detailed characterization of the aortic hematopoietic stem cell niche and its maturation dynamics, identifying the Notch ligand Dll4 as a major molecular player governing dynamics of cluster formation and HSCs production.
2020-01-10 | GSE124875 | GEO
Project description:Dll4-Notch signaling is required for cell fate decisions and neoplasias. However, emerging evidence suggests a role for Dll4-Notch signaling in metabolic and immune diseases. To date, there is no evidence of a direct effect of Dll4-Notch signaling inhibition on pancreatic islet function and insulin secretion.
2018-08-13 | PXD005333 | Pride
Project description:The lactate receptor GPR81 contributes to cancer development, yet the mechanisms remain unclear. Here, we show that GPR81 is upregulated in human cancers and further upregulated by extracellular lactate and three-dimensional growth of breast cancer spheroids. GPR81 knockdown (KD) increased spheroid necrosis and reduced invasion and in vivo tumor growth. GPR81 KD altered GO terms relating to extracellular matrix (ECM) and cell adhesion, including upregulation of Protocadherin-7 (PCDH7), which exhibited a striking, clustered expression in spheroids. Single cell analysis revealed that PCDH7 was inversely related with Ki-67 and co-clustered with other GPR81 KD-upregulated genes including Ephrin-type-A receptor-7 (EPHA7). The matricellular protein SPARC and the Notch ligand Delta-like-4 (DLL4), were downregulated upon GPR81 KD, and their KD elicited spheroid necrosis (DLL4) and inhibited invasion. We conclude that GPR81 supports breast cancer aggressiveness at least in part via SPARC and DLL4. Our findings substantiate GPR81 as a promising target in breast cancer treatment.
2022-05-23 | GSE203441 | GEO
Project description:In hESCs, expression of the Notch ligand DLL4 parallels the emergence of bipotent hematoendothelial progenitors (HEPs) and promotes their hematopoietic differentiation. During differentiation, DLL4 is only expressed in a subpopulation of HEPs. To study the developmental fate of the two subpopulations of HEPs identified by DLL4 expression, we FACS-isolated DLL4high and DLL4low/- HEPs at day 15 of differentiation and performed gene expression analysis using microarrays
2014-05-01 | GSE56881 | GEO
Project description:In hESCs, expression of the Notch ligand DLL4 parallels the emergence of bipotent hematoendothelial progenitors (HEPs) and promotes their hematopoietic differentiation. During differentiation, DLL4 is only expressed in a subpopulation of HEPs. To study the developmental fate of the two subpopulations of HEPs identified by DLL4 expression, we FACS-isolated DLL4high and DLL4low/- HEPs at day 15 of differentiation and performed gene expression analysis using microarrays 10^5 DLL4high and DLL4low/- HEPs purified by FACS from H9 and AND1 hEBs at day 15 of hematopoietic differentiation were used for gene expression analysis using Whole Human Genome Oligo Microarray chips (Agilent Technologies).
2014-05-01 | E-GEOD-56881 | biostudies-arrayexpress
Project description:Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase MFng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.
2019-12-03 | GSE110614 | GEO
Project description:Hypoxia (low oxygen) and Notch signaling are two important regulators of vascular development, but how they interact in controlling the choice between arterial and venous fates for endothelial cells during vasculogenesis is less well understood. In this report, we show that hypoxia and Notch signaling intersect in promotion of arterial differentiation. Hypoxia upregulated expression of the Notch ligand Dll4 and increases Notch signaling, in a process requiring the vasoactive hormone adrenomedullin but not endogenous VEGF. Notch signaling also upregulated Dll4 expression, leading to a positive feedback loop sustaining Dll4 expression and Notch signaling. In addition, functional Notch signaling was required for hypoxia to upregulate the arterial marker genes Depp, connexin40 (Gja5), Cxcr4 and Hey1. In conclusion, the data reveal an intricate interaction between hypoxia and Notch signaling in the control of endothelial cell differentiation, including a hypoxia/adrenomedullin/Dll4 axis that initiates Notch signaling and a requirement for Notch signaling to effectuate hypoxiamediated induction of the arterial differentiation program.
2013-02-08 | GSE35894 | GEO