Unknown,Transcriptomics,Genomics,Proteomics

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Linking Notch signaling to ischemic stroke

ABSTRACT: Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype. Microarray Studies. Biotinylated cRNA samples from freshly sorted brain SMCs (four Notch 3 +/- mice and five Notch 3 -/- mice) were fragmented before hybridization (15 ug each) onto mouse 430 2.0 Affymetrix chips. The chips were washed, stained by using strepavidin-phycoerytrin, and scanned the next day as described in ref. 24. For data normalization, all probe sets were scaled to a target intensity of 150. Microarray data analysis was performed by using Rosetta Resolver. All cells were from 10- to 12-week-old male mice. Gene Ontology Analyses. PANTHER software was used to define over- and underrepresented functions in the list of signature genes found by microarray analysis (25). P values were calculated by using binomial statistics.

ORGANISM(S): Mus musculus

SUBMITTER: Joseph Arboleda-Velasquez

PROVIDER: E-GEOD-58368 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Notch signaling functions in retinal pericyte survival.

Arboleda-Velasquez Joseph F JF Primo Vincent V Graham Mark M James Alexandra A Manent Jan J D'Amore Patricia A PA

Investigative ophthalmology & visual science 20140711 8

<h4>Purpose</h4>Pericytes, the vascular cells that constitute the outer layer of capillaries, have been shown to have a crucial role in vascular development and stability. Loss of pericytes precedes endothelial cell dysfunction and vascular degeneration in small-vessel diseases, including diabetic retinopathy. Despite their clinical relevance, the cellular pathways controlling survival of retinal pericytes remain largely uncharacterized. Therefore, we investigated the role of Notch signaling, a ...[more]

PMID: 25015359

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Project description:Gene expression profiling on IL-10-secreting and non-secreting murine Th1 cells, stimulated in the presence or absence of the Notch ligand Delta-like 4 (Dll4), was performed to identify transcription factors co-expressed with IL-10. Primary naM-CM-/ve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions. For activation, 0.25E06 naM-CM-/ve T cells were co-cultured with 0.75E06 MACSi Beads in 96-well flat bottom plates. MACSi Beads were coated with anti-CD3 and anti-CD28 (30 M-BM-5g of total primary IgG antibody per 1.0E08 beads) prior to seeding. Notch activation via Dll4 was induced by additional co-culture with MACSi Beads covalently coated with recombinant mouse Dll4. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to separate IL-10-secreting and non-secreting cells. Using a BD Aria or DIVA cell sorter (Becton Dickinson), living CD4+ IL-10-secreting and non-secreting cells, with (co-culture with Dll4; 'TH1Notch') or without ('TH1Control') activation of the Notch signaling pathway, were isolated. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The preparation for the hybridization to the chip was done using the GeneChip 3' IVT Express Kit. Fifteen micrograms of fragmented cRNA of each sample were hybridized to a total of 4 mouse genome 430 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.1.1., both Affymetrix. The data was analyzed using the original GCOS CHP-file Signals, Excel and AmiGO website.

Project description:Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. Here we have analyzed the temporal changes in transcription following a short Notch activation treatment and have related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were cultured in Schneiders medium (Invitrogen) supplemented with 10% FBS (Sigma), 5ug/ml insulin (Sigma) and 5% penicillin / streptomycin (Sigma) according to standard protocols. Notch signalling was initiated by replacing cell media with 2mM EDTA in PBS. The cells were stimulated for 5 minutes before washing out EDTA using normal culture media. The cells were then collected at 18 time points at 0M-bM-^@M-^Y, 5M-bM-^@M-^Y, 10M-bM-^@M-^Y, 15M-bM-^@M-^Y, 20M-bM-^@M-^Y, 25M-bM-^@M-^Y, 30M-bM-^@M-^Y, 35M-bM-^@M-^Y, 40M-bM-^@M-^Y, 50M-bM-^@M-^Y, 60M-bM-^@M-^Y, 70M-bM-^@M-^Y, 80M-bM-^@M-^Y, 90M-bM-^@M-^Y, 100M-bM-^@M-^Y, 110M-bM-^@M-^Y, 120M-bM-^@M-^Y and 150M-bM-^@M-^Y after stimulation in 4 independent time trials. For control samples, for each time trial, media was replaced with fresh media to mimic the addition and removal of EDTA in corresponding experimental samples and then collected at equivalent times. For each trial 50 M-BM-5g of the untreated control RNA sample of each time point was pooled to create a trial specific reference sample. Samples from trials #1 and #3 were labelled with Cy5 and trails #2 and #4 as dye-swap with Cy3.

Dataset Information

Linking Notch signaling to ischemic stroke

Publications

Notch signaling functions in retinal pericyte survival.

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