An Organ Culture System to Model Early Degenerative Changes of the Intervertebral Disc: II Profiling Global Gene Expression Changes
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ABSTRACT: To mimic a degenerative insult, rat intervertebral discs were cultured in the presence of TNF-alpha, IL-1beta and serum limiting conditions. Gene expression analysis was performed using a microarray to identify differential gene expression between experimental and control groups. The discs were collected and the annulus fibrosus (AF) was separated from the nucleus pulposus (NP). Total RNA from NP was used for RNA extraction and hybridization on Affymetrix microarrays: Control group - Ctr-1, Ctr-2, Ctr-3, Ctr-4 and Experimental group-Exp-1, Exp-2, Exp-3, Exp-4.
Project description:Research on disc degeneration has been heterogeneous in their use of control discs used for comparison with diseased discs. Discs from scoliosis, cadavers and voluntary organ donors are the common controls used in intervertebral disc research. In order to find out the ideal control among these discs, the characters of scoliotic discs and discs from MRI normal voluntary organ donors controls used in disc research has been analysed using proteomics and to establish 'True Controls' that can be utilized for future Intervertebral disc (IVD) research.
Project description:To evaluate the effect of Hypoxia-inducible factor 1-alpha inhibitor (HIF-1) in nucleus pulposus (NP) cells, human NP cells were lentivirally transduced with either control or FIH-1 targeted shRNA. Gene expression changes between samples from control and FIH-1 silenced cells were evaluated using a microarray. Primary NP cells were isolated from human discs and grown in culture. Cells were transduced with either control or FIH-1 targeting shRNA constructs. Silencing of FIH-1 was confirmed for each set using qRT-PCR and Western blot. Total RNA from NP cells was used for RNA extraction and hybridization on the Affymetrix microarray. Control samples: C1, C2, and C3 and FIH-1-silenced samples: E1, E2, and E3.
Project description:Intervertebral disc degeneration is accompanied by a loss of Extra-cellular matrix (ECM) content due to an imbalance in anabolic and catabolic pathways. Identifying ECM proteins with anabolic and/or regenerative potential could be the key to developing regenerative therapies. Since human fetal discs grow and develop very rapidly, studying these discs may provide valuable insights on proteins with regenerative potential. This study compares core matrisome of 9 fetal and 7 healthy adult (age 22-79) nucleus pulposus (NP), using a proteomic and bioinformatic approach. In order to assess whether the protein that decrease from fetus to healthy adult NP’s further decrease in severely degenerated discs, expression levels of all proteins of interest that were identified in regenerative pathways were used for an additional analysis
Project description:We have used microarrays to identify genes expressed and required for the second mitotic wave (SMW) during eye development. Eye discs expressing Spitz under the control of GMR Gal4 have no SMW as Spitz promotes G1 arrest, ectopic differentiation also occures. To control for the ectopic differentiation, Spi expressing eye antennal discs were compared to eye antennal discs expressing activated RasV12. In discs expresseding RasV12 under the control of GMRGal4 the SMW takes place normally prior to any ectopic differentiation. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. Experiment Overall Design: Drosophila eye antennal imaginal discs expressing either UAS RasV12 or UAS Spi under the control of GMRGal4 were dissected from 3rd instar larvae for RNA extraction and hybridization on Affymetrix microarrays.
Project description:RNA-seq on 120hr L3 larval wing imaginal discs. 3 replicates of dis3L2 null mutant, and 3 replicates of control wing discs. rRNA depleted, Illumina TruSeq libraries. Paired-end sequencing on a HiSeq 3000.
Project description:Aim of the project: Genome wide gene expression analysis for cytokinin (100nM 2ip, 20nM NaPi buffer) fast (1h) response genes from 3 months old Hybrid aspen(Populus tremula x tremuloides)apical part (30 cm from tip; diameter:4-5mm) of stem. Stems of two individuals (trees 15 and 16) was cut into 50-100um thick (free hand)cross sections randomly selected stem discs are set to two pools: cytokinin treatment and control treatment. Samples are collected noon time (11:00-11:30). Cytokinin treatment: stem discs (several hundred) are submerged with 100nM 2ip, 20nM NaPi buffer, with modest shaking for 60 min +/- 2 min (time to collect about 30mg stem discs (several dozens) for RNA sample. Control treatment: identical to cytokinin treatment, only without 100nM 2ip.
Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs
Project description:Apoptosis is an important process to eliminate cells from tissue which have incurred irreparable DNA damage. While dE2F1/dDP complexes respond to such damage by transcriptionally activating apoptotic genes, previous data suggests that activation of the previously characterized apoptotic target genes of dE2F1/dDP alone may not be the only gene regulation important for gamma irradiation-induced apoptosis. Here we report that following irradiation in dDP mutant 3rd instar larval eye imaginal discs, many genes important for oxidative phosphorylation are down-regulated, which are not down-regulated following irradiation in wild type eye discs. Biological triplicates of wild type, dDP mutant and de2f1, deleted in the posterior, eye discs were untreated or irradiated with 40Gy of gamma radiation. Total RNA was extracted by Trizol from untreated eye discs and from irradiated eye discs 4h after irradiation. RNA was column purified. PCR amplified RNAs were hybridized on Affymetrix Drosophila Genome 2.0 Array.
Project description:Low back pain continues to be a major public health problem worldwide. In this study, we used single-cell transcriptomic analysis to identify new specific biomarkers for nucleus pulposus (NP) and annulus fibrosis (AF) cells and to define cell populations within non-degenerating and degenerating human intervertebral discs (IVD). Freshly isolated human NP and AF cells were separately isolated from non-degenerating (nD) and degenerating (D) discs of the same individual. Isolated cells were subjected to droplet-based single-cell RNA sequencing (scRNA-Seq) using 10X Genomics platform. A total of 3134 (AFD), 3182 (AFnD), 3665 (NPD) and 3918 (NPnD) individual cells were profiled from nD and D discs respectively.
Project description:The transcription cofactor Yki drives growth and proliferation in part by controlling mitochondrial network formation. To determine if Yki and Sd are directly bound to DNA corresponding to mitochondrial genes, we used chromatin immunoprecipitation and whole genome tiling arrays (ChIP-chip) to identify regions bound by these factors in eye-antenna and wing imaginal discs. The supplementary .bed files contain all Yki or Sd binding sites (called at 5% FDR) in wing or eye-antenna imaginal discs, as well as shared Sd+Yki sites and associated target genes. Wing or eye-antenna imaginal discs ChIPped for Yki or Sd-GFP vs. input DNA from corresponding imaginal discs.