Zebrafish embryos exposed to the model hedgehog pathway inhibitor cyclopamine, as well as to the potential hedghog pathway disrupters SANT-2 and GANT-61.
ABSTRACT: The hedgehog (Hh) signaling pathway is important for various developmental processes during embryogenesis and for homeostasis of adult tissue in vertebrates. Aberrant signaling results in severe birth defects and malignancies, respectively. The small molecules SANT-2 and GANT-61 were identified as potential Hh-pathway inhibitors in a reporter gene assay (Shh-light II cells). We aimed to analyze the effects of these compounds on the gene expression level in zebrafish embryos in comparison with the well known Hh-pathway inhibitor cyclopamine. Zebrafish embryos were treated with Ethanol (0,1%) (control), 10 µM Cyclopamine (C), 10 µM SANT-2 (S), and 10 µM GANT-61 (G), for 24 h (0.5 - 24 hpf), respectively; all in 5 independent replicates with 100 zebrafish embryos per treatment. Total RNA was extracted and processed as recommended by Agilent. One color arrays were used and data is given as normalized log2 of the gprocessed signals.
Project description:Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of muscular activity. Whether AChE inhibitors and related neurotoxic compound also provoke specific changes in gene transcription patterns that allow to establish a mechanistic link useful for diagnostic purposes has, however, yet not been investigated. Therefore, as a proof of principle, we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive gene in response to AChE inhibitors. Comparison of expression in wild-type, ache and fixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels led to an hspb11 transcriptional up-regulation which suggest that elevated intracellular calcium levels and the resulting increase in muscular activity triggering hspb11 up-regulation. During early zebrafish development, hspb11 was specifically expressed in muscle pioneers and morpholino-knockdown resulted in defects in slow muscle myosin organization. These findings demonstrate that a combination of transcriptome and functional analyzes in the zebrafish embryo is able to correlate gene expression changes to the mechanism of action of neurotoxic compounds. Zebrafish embryos were exposed for 24 h and 48 h (26-50 and 2-50 hpf) to 5,83 µM of azinphos-methyl (APM), 509 µM of 1,4-dimethoxybenzene (DMB) and 14,2 µM 2,4-dinitrophenol (DNP), the respective LC10 of each compound, in order to determine mode of action specific responding genes, or not exposed and parallel treated as controls. 50 zebrafish embryos were used per treatment, and 4 biological independent replicates were analysed, respectively.
Project description:The morphogen Sonic H edgehog governs a wide range of developmental processes. The zebrafish genetic mutant iguana has vascular stability defects due to decreased Shh signaling. Using iguana mutant embryos and embryos treated with the Hedgehog pathway inhibitor cyclopamine, we conducted a microarray to determine genes that are specifically regulated by Shh signaling, and that might mediate vascular stability. We populate a list of 40 genes to have significantly altered expression in both conditions. Using in situ hybridization and quantitative real-time PCR, we verify the expression changes seen in a subset of genes from the list and determine their localization during embryonic development. We then assay the functional relevance of one of the array hits, the cell-cycle regulator pim1, which was upregulated on the microarray. By overexpressing pim1, we observe a loss of vascular stability, similar to that of iguana mutants. Furthermore, chemical inhibition of pim1 in iguana mutant embryos or cyclopamine treated embryos rescues vascular stability. We conclude that the microarray identified a set of genes that are differentially expressed in two distinct modes of Shh signaling interference. Furthermore, this set of genes contains a high proportion of factors potentially involved in vascular stabilization. The identification of these genes is the first step in defining the molecular mechanism by which Shh promotes vascular stability. 3 biological cyclopamine treated samples plus 3 biological DMSO treated controls, plus 3 biological replicates of iguana mutants plus 3 wild type sibling controls, all collected at 30 hpf
Project description:Investigation of whole genome expression pattern of 24 hours post fertilization Danio rerio embryos exposed to bisphenol A, 17β-estradiol, or 0.1% DMSO vehicle control This data represents 2 experiments using 2 separate microarrays. Microarray 1 [Low]: Embryos were exposed to 0.1 µM BPA, 0.1 µM E2, or control from 8 hpf to 24 hpf. Three biological replicates were collected for each treatment. 40 embryos were pooled to comprise a replicate. Microarray 2 [High]: Embryos were exposed to 80 µM BPA, 15 µM E2, or control from 8 hpf to 24 hpf. Two biological replicates were collected for each treatment. 40 embryos were pooled to comprise a replicate. These 2 microarray experiments are cross-comparable, as described in the associated publication.
Project description:To uncover the genes regulated by pharmacological activation of the Glucocorticoid Receptor, we performed microarray-based expression profiling of whole zebrafish embryos at 24 and 72 hours post fertilization (hpf) after 3-hour treatment with 25 µM of Beclomethasone, a potent glucocorticoid previously tested in zebrafish or with 0.1 % DMSO as control. Embryos at 24 and 72 hpf stages were treated with either 0.1 % DMSO or 25 µM Beclomethasone for 3 hours in triplicate experiments and then frozen for RNA extraction.
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.
Project description:We have employed the Zebrafish gene expression microarray (MZH_Zebrafish_16k_v1.0) as a discovery platform to analyze the trancriptome of 108hpf (hours post fertilization) embryos exposed from the 96hpf to 108hpf to 100µM of diethylmaleate (DEM). Four two-color microarray studies for measuring expression levels of zebrafish embryos treated with 100µM of diethylmaleate (DEM) were performed. 8 samples were analyzed in total. Four samples were treated with 0.1% DMSO as a control; four samples were treated with 100 µM of DEM and 0.1% DMSO.
Project description: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 µM of tert butyl hydroquinone or 2% DSMO for 4 hours. At 52 hpf, embryos were fixed in RNA Later and stored at -80°C. 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 Agilent’s 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) ≤10%.
Project description:Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to identify estrogen regulated genes during the first 4 days of development. Zebrafish embryos were exposed to 1 µM 17β-estradiol from 3 hours post fertilization to 4 days post fertilization, harvested daily and subjected to RNA extraction for transcriptome analysis using microarrays. Estrogen responsive genes were analyzed with hierarchical clustering followed by gene function and tissue expression analysis. Markedly distinct sets of genes were up and down-regulated by estrogen treatment at different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by estrogen were similar throughout zebrafish development. Estrogen responsive genes were enriched mainly in the liver, pancreas and brain. In conclusion, our data shows that in zebrafish distinct cohorts of E2 responsive genes are expressed in a tissue specific manner at different developmental stages. However, the biological pathways that are affected are conserved. 30 embryos were pooled as one sample and exposed to 1 μM E2 or vehicle (0.1% DMSO) at approximately 3 hours post fertilization (hpf). At different time points, 1 dpf (24 hpf), 2 dpf (48 hpf), 3 dpf (72 hpf) and 4 dpf (104 hpf), embryos were collected for total RNA extractions. Time points 1 and 2 dpf were performed in biological triplicates of independent pools of RNA while time points 3 and 4 dpf were performed in quadruplicates.
Project description:Zebrafish (Danio rerio) model system have used widespread vertebrate investigations for genetic and cell biological analyses, and is suitable for small molecular screens such as chemical, toxicity and drug in order to use for human diseases and drug discovery . Recently, These powerful zebrafish model increasingly apply to human metabolic disease such as obesity and diabetes and toxicology. Despite a lot of advantages, proteomics research at zebrafish has received little interest in comparison with genetic and biological research using histology and in situ hybridization. Protein lysine acetylation is one of the most known post-translational modifications with dynamic and reversibly controlled by lysine acetyltransferase such as histone acetyltransferases and lysine deacetylase such as histone deacetylases and sirtuins family.Also, during the past year, global lysine acetylome studies using MS-based proteomics approach was in diverse species such as human, mouse, E. coli, Yeast and plants. Based on global acetylome data, our understanding of the roles of lysine acetylation in various cellular processes has increased. . The aim of this study was to identify Lysine acetylation in zebrafish embryos and determine the homology from Human at modified site level. Here we showed the global lysine acetylation study in Zebrafish embryos using MS-based zebrafish embryos.