Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Nrf2b: a novel zebrafish paralog of the oxidant-responsive transcription factor Nrf2.

ABSTRACT: We identified a novel paralog of the antioxidant response element transcription factor, Nrf2 in zebrafish. To determine whether the paralogs Nrf2a and Nrf2b regulate different sets of genes, we performed loss-of-function experiments followed by microarray-based gene expression profiling, performed on embryos in which expression of Nrf2a, Nrf2b, or both paralogs was knocked down. Embryos at the 1-4 cell stage were injected with 3-5 nl of a control morpholino (MO) or gene-specific MO to knockdown Nrf2a, Nrf2b, or a Nrf2a+b combination. At 48 hpf, triplicate groups of 5 embryos were exposed to 2 M-BM-5M of tert butyl hydroquinone or 2% DSMO for 4 hours. At 52 hpf, embryos were fixed in RNA Later and stored at -80M-BM-0C. RNA was extracted, bioanalyzed, labeled with Cy3 and hybridized to the Agilent V3 4x44K zebrafish microarray (cat. #G2519F-026437) at the Genome Technology Core of the Whitehead Institute (Cambridge, MA). Raw array data obtained from the Whitehead Institute were extracted using Agilent's feature extraction software using background detrending (spatial and multiplicative).Prior to normalization, Cy3 values below 5 were set to 5. The data were then normalized using the non-linear scaling method based on rank invariant probes. After normalization but before statistical analyses, probes not significantly above background in all microarrays were removed (3147 probes in all; based on AgilentM-bM-^@M-^Ys 2.6 standard deviation method). None of the probes were saturated for Cy3 signal on any microarray, so no further filtering was applied. There were a total of 40,456 probes for statistical analyses. Statistical tests were performed using MeV v4. Data were log transformed and median centered for each probe. An ANOVA was run for morpholino injection, with p-value based on 1000 permutations of the data and alpha of 0.01. The probes found significant in the ANOVA were subsequently examined using Rank Product (RP) analysis to identify probes up- and down-regulated by the MO injection For each RP test, a two-class unpaired RP analysis was performed using 100 permutations of the data with a false discovery rate (FDR) M-bM-^IM-$10%.

ORGANISM(S): Danio rerio

SUBMITTER: Andrew McArthur

PROVIDER: E-GEOD-32594 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Nrf2b, novel zebrafish paralog of oxidant-responsive transcription factor NF-E2-related factor 2 (NRF2).

Timme-Laragy Alicia R AR Karchner Sibel I SI Franks Diana G DG Jenny Matthew J MJ Harbeitner Rachel C RC Goldstone Jared V JV McArthur Andrew G AG Hahn Mark E ME

The Journal of biological chemistry 20111215 7

NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares seve ...[more]

PMID: 22174413

Similar Datasets

Project description:The MTF-1 transcription factor is considered to be a master regulator of zinc homoestasis. We previously characterized a constitutively nuclear, dominant-negative zebrafish MTF-1/eGFP fusion protein (dnMTF-1). In this study, in vitro transcribed dnMTF-1 mRNA was microinjected into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. The role of MTF-1 signaling in zebrafish embryos was examined by injection of in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. A Narishige IM-300 microinjector was used to microinject 1-2 cell embryos with ~100 pg of dnMTF-1 IVT mRNA (~2.1 nL microinjection volume with a concentration 50 ng/M-BM-5L IVT mRNA), or ~100 pg of enhanced green fluorescent protein (eGFP) IVT mRNA. Immediately after microinjection, embryos from each treatment were divided into 4 replicates of 30 pooled embryos and held in 25 mL of 0.3X DanieauM-bM-^@M-^Ys at 28.5M-BM-0C under a 14 hour light/10 hour dark cycle. eGFP positive embryos (dnMTF-1 and eGFP) were collected at 28 and 36 hpf and flash frozen in liquid nitrogen for future RNA isolation and microarray hybridization. Total RNA was prepared using the RNA STAT60 protocol (Tel-Test, Inc., TX). RNA was quantified using a Nanodrop 2000C spectrophotometer and assessed for quality using an Agilent 2100 Bioanalyzer Lab-on-chip system. Only samples with RNA integrity number (RIN) between 9.8-10 were used for microarray analysis. For each RNA sample, a single microarray was hybridized with 750 ng Cy3 labeled cDNA using AgilentM-bM-^@M-^Ys standard conditions for single-color microarrays. The samples were hybridized to a the Agilent 4x44K feature zebrafish microarray using the Agilent In situ Hybridization Kit Plus by the Genome Technology Core at the Whitehead Institute for Biomedical Research (Cambridge, MA). Post-hybridization, microarray slides were washed as per the Agilent In situ Hybridization Kit Plus and scanned with an Agilent DNA Microarray Scanner. Extraction of raw microarray results was performed using AgilentM-bM-^@M-^Ys Feature Extraction software with background detrending (spatial and multiplicative). The data were normalized using a non-linear scaling method based on rank invariant probes. Cy3 signal saturated probes and probes not above background in all replicated were then removed prior to statistical analysis. Statistical tests were performed using the MeV microarray analysis suite. All data were log transformed and median centered for each probe. A two-factor ANOVA was performed for time (28 vs. 36 hpf), treatment (dnMTF-1 vs. eGPF), and their interaction with p-value based on 1000 permutations of the data and alpha of 0.01. For post-hoc analyses to find biologically relevant results, the probes significant for treatment and time-treatment interaction were secondarily examined using Rank Product (RP) analysis. Rank product analysis was performed to provide lists of probes up- and down-regulated between GFP control and MTF-1 for each time point. For each test, a two-class unpaired RP analysis was performed using 1000 permutation of the data with a false discovery rate (FDR) not exceeding 5%.

Project description:The aryl hydrocarbon receptor repressor (AHRR) is a bHLH-PAS protein closely related to the aryl hydrocarbon receptor (AHR). AHRR is a transcriptional repressor of AHR and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, while AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, very little is known about the endogenous roles of AHRRs in development and physiology. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by microarray analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides (MO) against AHRRa or AHRRb to disrupt endogenous signaling during development. At 72 hours post-fertilization (hpf), embryos were sampled for microarray analysis. Agilent microarray analysis revealed altered expression of 279 and 116 genes with knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the differentially expressed genes were a large number related to photoreceptor development, including cone-specific genes such as short-wave, medium-wave and long-wave sensitive opsins (opn1sw1, opn1sw2, opn1mw1, opn1lw2), phosphodiesterases (pde6H, pde6C), retinol binding protein (rbp4l), phosducin, and arrestin. These results suggest that AHRRa may be involved in photoreceptor development. In addition, AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment of genes related to any specific biological process. Overall, these results suggest that AHRRs play multiple roles during development. Three different treatment conditions (Control-MO, AHRRa-MO and AHRRb-MO) and two replicates per treatment

Project description:Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrocyte differentiation; however, how these transcription factors functionally interact and collaborate to regulate chondrogenesis remains unclear. Here we show that the protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 by Sox9. Knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest formation. We also find that Sox6 is specifically induced during craniofacial chondrogenesis and this induction is inhibited by MLTK knockdown. Remarkably, Sox6-knockdown embryos display essentially the same phenotype as the MLTK-knockdown embryos; the drastic loss of cartilages and the marked down-regulation of genes involved in chondrogenesis. Microarray analysis reveals a remarkable similarity between Sox6-depleted and MLTK-depleted embryos in their gene expression pattern. Moreover, we find that ectopic expression of MLTK can induce Sox6 expression in a Sox9-dependent manner. These results identify a novel, key regulator for chondrogenesis. We used microarrays to describe the genome-wide gene expression profiles of xMLTK-depleted and xSox6-depleted embryos. CoMO, xMLTK-MO, or xSox6-MO was injected into the animal pole of all blastomeres (10 ng/cell) at the four-cell stage. The heads of embryos were cut out and harvested at 72 hours after fertilization (corresponding to about stage 41). Total RNA was extracted using TRIsol reagent, treated with DNase (TURBO DNase, Ambion), and then purified using RNeasy Mini Kit (QIAGEN) according to the manufacturerM-bM-^@M-^Ys instruction. The quality of total RNA was assessed using the Agilent 2100 BioAnalyzer. Reverse transcription to synthesize first-strand cDNA, second-strand cDNA synthesis, in vitro transcription to synthesize Biotin-modified aRNA, aRNA purification and fragmentation of the labeled aRNA were performed using the GeneChip 3M-bM-^@M-^Y IVT Express Kit and hybridization to the Affymetrix GeneChip Xenopus laevis Genome 2.0 Array was performed using the GeneChip Hybridization Wash and Stain Kit according to the manufacturerM-bM-^@M-^Ys instruction. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned Chip images were analyzed with GeneChip operating Software v.1.4 (GCOS) and GeneSpring GX 11.0.2. (Agilent technologies).

Dataset Information

Nrf2b: a novel zebrafish paralog of the oxidant-responsive transcription factor Nrf2.

Publications

Nrf2b, novel zebrafish paralog of oxidant-responsive transcription factor NF-E2-related factor 2 (NRF2).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets