The zebrafish embryo as an alternative model for the evaluation of liver toxicity using histopathology and expression profiling
ABSTRACT: The zebrafish is as a powerful vertebrate model system for modeling human disease including liver pathology. In ZFE, hepatic responses can be expected after exposure to hepatotoxicants, because hepatocytes are present from 36-hpf and at 72-hpf the liver is fully functioning. These characteristics make the whole ZFE an attractive alternative model for compound-induced hepatotoxicity screening. Therefore, the main objective of this study is to further strengthen the applicability of whole ZFE as an alternative model for hepatotoxicity testing, with a special focus on the ability to identify gene expression responses in whole ZFE that are suggestive of hepatotoxicity. Deep sequence technology is applied to assess whether hepatotoxicity-specific transcripts that are identified in livers of hepatotoxicant treated adult zebrafish can be detected in whole ZFE as well. Whole zebrafish embryo and adult zebrafish are exposed for 48 hours to human hepatotoxicants. Afterwards, the adult liver is dissected and snap frozen untill processed for RNA isolation. The whole zebrafish embryos are pooled, one sample consists of 15 embryos, and are also snap frozen untill processed for RNA isolation. For every sample RNA is isolated according to the protocol of de Jong et al (2009). and then equal amounts of every sample is pooled. The two pools (one for the adult zebrafish liver, the other for the whole zebrafish embryo) are sequenced.
Project description:Zebrafish embryos have been proposed as an attractive alternative model system for hepatotoxicity testing. In this study we determined gene expression responses after exposure to reference hepatotoxicants and controls to identify biomarkers for hepatotoxicity. 188 samples; hepatotoxic compounds at 3 concentrations and with multiple replicates; negative controls at single concentration and with multiple replicates; water (DSW) and 0.2% DMSO vehicle control.
Project description:To identify genes regulated by Rx3 during optic vesicle morphogenesis, adult zebrafish carriers of a null rx3 mutation were mated. Before 13 hours post fertilization (hpf), the earliest time point at which optic vesicle evagination phenotypes could be reliably detected, offspring were phenotypically separated into pools comprising of mutants with an absence of optic vesicles or siblings exhibiting a wild-type phenotype. Three replicates of pooled RNA samples from 13 hpf eyeless mutants (rx3-/-) or phenotypically wild-type siblings (rx3+/+ or rx3+/-), and one replicate of 13 hpf wild-type zebrafish larva were collected for whole transcriptome sequencing. Whole transcriptome sequencing (RNA-seq) was performed on zebrafish rx3-/- mutants, wild-type siblings and wild-type AB strains at 13 hpf
Project description:Full title: Mercury-Induced Hepatotoxicity in Zebrafish: In Vivo Mechanistic Insights from Transcriptome Analysis, Phenotype Anchoring and Targeted Gene Expression Validation In this study, we performed microarray-based expression profiling on liver of zebrafish exposed to 200 µg/L of mercuric chloride for 8-96 h, to identify global transcriptional programs and biological pathways involved in mercury-induced adaptive responses under in vivo environment. We analyzed 12 arrays for mercuric chloride treated zebrafish liver and 12 arrays for control liver.
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:We designed 4C-Seq experiments for hoxa13a, hoxa13b and hoxd13a promoters in three developmental stages in zebrafish embryos: 24, 48 and 60 hpf. 4C-seq samples for three different hox13 genes promoters in three developmental stages in zebrafish embryos: 24, 48 and 60 hpf.
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:In our previous study, we found zebrafish embryos treated with 5uM 11,12-EET (epoxyeicosatrienoic acid) had increased stem cell marker, runx1, expression in the AGM. EET also induced ectopic runx1 expression in the tail. To systematically study how EET regulates gene expression, we performed microarray analysis on EET-treated embryos. Zebrafish whole embryos were synchronized at fertilization. Embryos were grown at 28 degree overnight. 25 embryos per group were treated with DMSO or 5uM 11,12-EET starting from 24 hpf (hour post fertilization) until 36 hpf at 28 degree. The triplicates were from three different clutches of embryos, and split into DMSO v.s. EET for each clutch. EET vs. DMSO
Project description:Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days post-amputation) time-point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.
Project description:We performed microarray-based expression profiling on liver of male zebrafish exposed to 15ppm (~ 192 µM) of arsenic [As(V)] for 8-96 h, to identify global transcriptional programs and biological pathways involved in chloroaniline-induced adaptive responses under in vivo environment. We analyzed 12 arrays of chloroaniline-treated adult female zebrafish liver and 12 arrays of control fish.
Project description:We performed microarray-based expression profiling on liver of male zebrafish exposed to 30 µg/L of cadmium (II) chloride for 8-96 h, to identify global transcriptional programs and biological pathways involved in cadmium-induced adaptive responses under in vivo environment. We analyzed 10 arrays of Cadmium-treated adult male zebrafish liver and 12 arrays of control fish.