Identification and characterization of androgen responsive biomarker genes in zebrafish embryos
Ontology highlight
ABSTRACT: Exposure experiments with the non-aromatizable fish androgen, 11-ketotestosterone in the range of 0.05-5000 nM were conducted in order to identify potential androgen-responsive genes in zebrafish embryos by microarray analysis. Zebrafish embryos were treated from 96 hpf to 120 hpf in a single experiment with three controls (embryonic medium). Microarray studies were performed using a Custom 8x60k Gene Expression Microarrays (Amadid G4102A, based on the ensembl zebrafish genome version 3) from total RNA with low input labelling and hybridization kit (Agilent Technologies) and one-color design according to the manufacturer instructions. Fluorescent intensities of individual microarray spots were extracted using the Agilent Feature Extraction software. Raw microarray data were converted to log2 values and quantile-normalized. For further statistical analysis, fold changes in relation to the mean of the controls were calculated for each treatment. Zebrafish embryos were treated from 96 hpf to 120 hpf in a single experiment with three controls and 11 concentrations of 11-ketotestosterone ranging from 0.05-5000 nM.
Project description:Exposure experiments with the non-aromatizable fish androgen, 11-ketotestosterone in the range of 0.05-5000 nM were conducted in order to identify potential androgen-responsive genes in zebrafish embryos by microarray analysis. Zebrafish embryos were treated from 96 hpf to 120 hpf in a single experiment with three controls (embryonic medium). Microarray studies were performed using a Custom 8x60k Gene Expression Microarrays (Amadid G4102A, based on the ensembl zebrafish genome version 3) from total RNA with low input labelling and hybridization kit (Agilent Technologies) and one-color design according to the manufacturer instructions. Fluorescent intensities of individual microarray spots were extracted using the Agilent Feature Extraction software. Raw microarray data were converted to log2 values and quantile-normalized. For further statistical analysis, fold changes in relation to the mean of the controls were calculated for each treatment.
Project description:Aiming to identify insulin-independent modulators of glucose homeostasis, we performed a drug screen on zebrafish insulin (ins) mutants and identified androgen receptor (AR) antagonists. To investigate how AR antagonism mediates glucose level reduction in ins mutants, we evaluated the effects of antagonist treatment using transcriptomic studies. RNA-Seq analyses were performed on 120 hours post fertilization (hpf) ins mutants treated with Flutamide or Cyproterone starting at 84 hpf compared to vehicle (DMSO) treated mutants.
Project description:In order to discover the targets of Foxj1, we made transgenic zebrafish in which Foxj1 is ubiquitously overexpressed in response to heat [Tg(hsp70::foxj1a)]. Transgenic embryos and wild type control embryos were collected, given two heat shocks (at 18 hours post fertilization (hpf) and 20 hpf), then analyzed at 22 hpf. Gene expression profiles of embryos overexpressing Foxj1a were compared to gene expression profiles of wild type embryos using Nimblegen whole transcriptome zebrafish microarrays.
Project description:Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the Aryl Hydrocarbon Receptor (AHR) in a structurally-dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at a concentration that induces developmental malformations by 120 hours post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burden was analyzed at these time points using GC-MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the smallest number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure and may provide a path for unraveling the toxicity of complex PAH mixtures. Gene expression was measured in zebrafish embryos after exposure to PAHs. Embryos were batch-exposed in groups of 40 to 25 μM BAA, 25 μM DBT, 25 μM PYR or 1% DMSO vehicle control starting at 6 hpf and collected at 24 or 48 hpf. Four independent biological replicates were prepared for each treatment. The reference was a pool of zebrafish embryos exposed to DMSO control until 24 and 48 hpf.
Project description:Forebrain and optic cups were microdissected from zebrafish embryos at 21 hours post-fertilization (hpf) after heat-shock at 15 hpf to induce overexpression of BMP in heterozygous tg(hsp70l:bmp4) embryos vs wild type controls.
Project description:In zebrafish, vitellogenic oocytes can incorporate significant amounts of 17β-estradiol released from nearby granulosa cells according to a first-order kinetics, since the steroid low polarity ensures high permeability and affinity for yolk lipids. Estrogen bioactivity is likely, because the maternal mRNA for the estrogen receptor-β2 (ers2a) is highly expressed in ovulated oocytes. This transcript is available for translation in the embryo until its sharp decline from 4 to 8 hours post-fertilization (hpf), being replaced by low levels of zygotic ers2a mRNA from 24 hpf to hatching at 48 hpf, as determined by qRT-PCR. Estrogen receptors-α and -β1 are only expressed zygotically at low levels from 24 hpf onwards. To test the functional role of maternal ers2a mRNA, 1- or 2-cell embryos were injected with 10.3 ng each of morpholino to knockdown translation (MO2-ers2a) of both maternal and zygotic ers2a transcripts, missplicing morpholino (MO3-ers2a) to block post-transcriptionally the zygotic transcript alone, and a nonspecific morpholino (MO-control) as a control. Treatment with MO2-ers2a caused severe malformations in 63% of 1-5 dpf larvae, as compared to 10-11% in those treated with MO3-ers2a and MO2-control. Defects included body growth delay and curved shape, abnormal brain and splanchnocranium development, enlarged and hemorrhagic pericardial cavity, uninflated swim bladder and rudimentary caudal fin with aberrant circular motion. Affected larvae could survive for only 12-14 days. Co-injection of an anti-p53 MO failed to rescue the MO2-ers2a-phenotypes, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Ers2a protein deficiency in 8-hpf MO2-ers2a-embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. Whole-mount in situ hybridization revealed an intensified expression of the genes six3.1 and emx1 in MO2-ers2a-embryos at 24-48 hpf, as compared to controls. These findings suggest the involvement of maternal ers2a mRNA in the epigenetic programming of zebrafish development. MO2-ers2a morphants were compared with MO-control at 8 hpf and 48 hpf. MO2-ers2a is a morpholinos selected to knockdown translation of ers2a mRNA
Project description:We use the zebrafish embryo model to study the innate immune response against Staphylococcus epidermidis. Therefore, we injected S. epidermidis (and three controls groups) into the yolk at 2 hpf and samples at mutiple timepoints. Gene expression profiles were obtained at 6, 30, 54, 78, 102 and 126 hpi by microarrays. The results show that the gram-positive bacterium S. epidermidis induces a late immune response with a strong response at 102 hpi. This microarray study was designed to determine the gene expression profile during infection with Staphylococcus epidermidis. RNA was isolated from groups of embryos (20) at 6 timepoints during the infection. Wildtypes zebrafish embryos were micro-injected into the yolk (2hpf) with (1) 20 CFU of S. epidermdis O-47 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), (2) mock-injected with PVP as a control, (3) Needle insertion as control, (4) Non-injected as a control. After injections embryos were transferred into fresh egg water and incubated at 28M-BM-0C. At 8 hpf (6 h post infection), 32 hpf (30 h post infection), 56 hpf (54 h post infection), 80 hpf (78 h post infection), 104 hpf (102 h post infection) or 128 hpf (126 h post infection) twenty embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent. All treatment groups were analyzed using a common reference approach.
Project description:Congenital malformations are a prevalent cause of infant mortality in the United States and their induction has been linked to a variety of factors, including exposure to teratogens. However, the molecular mechanisms of teratogenicity are not fully understood. MicroRNAs are an important group of small, non-coding RNAs that regulate mRNA expression. MicroRNA roles in early embryonic development are well established, and their disruption during development can cause abnormalities. We hypothesized that developmental exposure to teratogens such as valproic acid alters microRNA expression profiles in developing embryos. Valproic acid is an anticonvulsant and mood-stabilizing drug used to treat epilepsy, bipolar disorder and migraines. To examine the effects of valproic acid on microRNA expression during development, we used zebrafish embryos as a model vertebrate developmental system. Zebrafish embryos were continuously exposed to valproic acid (1 mM) or vehicle control (ethanol) starting from 4 hours post-fertilization (hpf) and sampled at 48 and 96 hpf to determine the miRNA expression profiles prior to and after the onset of developmental defects. At 96 hpf, 95% of the larvae showed skeletal deformities, abnormal swimming behavior, and pericardial effusion. Microarray expression profiling was done using Agilent zebrafish miRNA microarrays. Microarray results revealed changes in miRNA expression at both the time points. Thirteen miRNAs were differentially expressed at 48 hpf and 22 miRNAs were altered at 96 hpf. Among them, six miRNAs (miR-16a, 18c, 122, 132, 457b, and 724) were common to both time points. Bioinformatic target prediction and examination of published literature revealed that these miRNAs target several genes involved in the normal functioning of the central nervous system. These results suggest that the teratogenic effects of valproic acid could involve altered miRNA expression. Small RNA profiles were deteremined in valproic acid exposed zebrafish embryos using Agilent miRNA microarrays
Project description:The zebrafish (Danio rerio) is a popular animal model in studies of vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and of 84% in human disease-causing genes, specifically. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well-known post-translational modification (PTM), which performs various biological functions. Recent mass spectrometry (MS) developments have enabled the study of global phosphorylation patterns by using MS-based proteomics coupled with TiO2 phosphopeptide enrichment. In the present study, we identified 3,500 non-redundant phosphorylation sites on 2,166 phosphoproteins and 1,564 quantified phosphoproteins in zebrafish embryos.