Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of zebrafish embryos reveals AHR Activation by TCDD Downregulates Sox9b expression producing jaw malformation

ABSTRACT: Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species and its effects are especially prominent in early life stages of fish. TCDD activates the aryl hydrocarbon receptor (AHR), a transcription factor that mediates most if not all TCDD responses. We investigated the transcriptional response in the developing zebrafish jaw following TCDD exposure using DNA microarrays. Zebrafish larvae were exposed to TCDD at 96 h postfertilization (hpf) and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4 and 12 h postexposure (hpe). Numerous chondrogenic transcripts were misregulated by TCDD in the jaw. Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different. Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw. We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting formation of the embryonic jaw. Morpholino knock down of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype. Heterozygous sox9b deletion mutant embryos were sensitized to TCDD. Lastly, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos. These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation. Experiment Overall Design: Three independent replicate microarray time course experiments were performed comparing transcript levels between TCDD-exposed and control zebrafish. For each experiment, zebrafish were exposed to TCDD for 1 h starting at 96 hpf as described above. For each time point (97, 98, 100 and 108 hpf) and treatment jaw samples were pooled from 10 dissections for RNA isolation and hybridization with Affymetrix zebrafish arrays (Affymetrix, Santa Clara, CA). Each microarray contains roughly 14,900 probes corresponding to approximately 30% of the zebrafish genome. For each array, total RNA (1 Âµg) was isolated from 10 jaw microdissections with the QIAGEN RNeasy Mini kit following the manufacturerâs protocol (QIAGEN, Valencia, CA). The One-Cycle Target Labeling and Control Reagents kit was used to synthesize cDNA and biotinylated cRNA following the manufacturerâs protocol (Affymetrix, Santa Clara, CA). Biotin-labeled cRNA (15 Âµg) was fragmented and hybridized onto Affymetrix Zebrafish Genechip Arrays following the protocol in the Affymetrix Genechip Expression Analysis Technical Manual. Following hybdrization, the arrays were washed and stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 400 using the protocol EukGE WS2v4. Arrays were scanned with an Agilent Gene Array Scanner.

ORGANISM(S): Danio rerio

SUBMITTER: Kong Xiong

PROVIDER: E-GEOD-11893 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. microRNAs (miRNAs), single-stranded RNA molecules of ~22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. We hypothesized that exposure to xenobiotics can alter miRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. To test this hypothesis for one well-known teratogen, we exposed zebrafish embryos to DMSO (0.1%) or TCDD (5 nM) for 1 hr at 30 hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60 hpf. TCDD caused strong induction of CYP1A at 36 hpf (62-fold) and 60 hpf (135-fold) as determined by qPCR, verifying the effectiveness of the exposure. microRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD) and real time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60 hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 miRNAs as potentially differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only miRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and qPCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular development. MicroRNA profiling was performed (N=3 biological replicates per group) using custom designed Agilent miRNA microarrays (8x15K) and exiqon miRCURY LNA microarrays. Custom-made Agilent oligonucleotide miRNA microarrays were designed based on zebrafish mature miRNA sequences from miRbase v.16. Each individual array contained a total of 548 unique probes representing 259 mature miRNAs from zebrafish (245), Fugu rubripes (11) and Tetraodon nigriviridis (3). Exiqon miRNA probes were based on zebrafish miRNA sequences from miRbase v. 12.

Project description:Purpose: This study aimed to identify differentially expressed genes and transcripts in zebrafish embryos and larvae following benzo[a]pyrene (BaP) exposure. Methods: Adult zebrafish (2 males × 4 females, N=6 replicate tanks for each treatment) were acclimated for 7 days in an 818 Low Temp Illuminated Incubator (Precision Scientific, Chennai, India) at 28.5°C. Next, adult fish were waterborne exposed to control or 50 μg/L (ppb) BaP for 7 days; ethanol was used as vehicle solvent, and final ethanol concentration was 0.1 mL/L (100 ppm) in all treatment groups. This dose of ethanol is not teratogenic to zebrafish. Water was changed and/or re-dosed daily. From day 7 to 11 of the parental exposure, eggs were collected, counted, and raised in normal conditions (control) or continuously exposed to 50 μg/L BaP until 3.3 and 96 hours post fertilization (hpf). At 3.3 or 96 hpf, embryos (200/pool) or larvae (10/pool) were collected and pooled. Total RNA was isolated for transcriptomic RNA sequencing with Illumina HiSeq2000 (2X100bp). RNA-seq reads were uploaded to the galaxy platform https://main.g2.bx.psu.edu/. RNA-seq reads were trimmed, filtered, and aligned to the zebrafish genome (Danio_rerio.Zv9.68) with the Tophat for Illumina tool. Counting and annotation of RNA-seq reads were performed with Partek Genomics Suite version 6.11. Refseq Transcripts (2013-04-10) and Ensembl Transcripts release 70 databases were used for gene and transcript annotation. Differential expression of gene and transcript reads between treatments was analyzed with R package EdgeR. Genes/transcripts with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Differentially expressed genes were defined as genes with altered expression at either gene or transcript level. Results: Differential expression analysis with EdgeR revealed that gene expression was vastly different between 3.3 hpf zebrafish embryos and 96 hpf larvae. Using Refseq annotation, we found that 10644 out of 13950 transcribed zebrafish genes were differentially expressed between the two developmental time-points, with 5961 up-regulated genes and 4683 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. Similarly, using Ensembl annotation, 16529 out of 19886 transcribed zebrafish genes were differentially expressed, with 9318 up-regulated genes and 7211 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. In 3.3 hpf embryos, four genes and seven transcripts were differentially expressed after BaP exposure. In 96 hpf larvae, 447 and 484 zebrafish genes were significantly up- and down-regulated, respectively, by BaP exposure. Conclusions: Parental and developmental BaP exposure caused gene expression changes in zebrafish embryos and larvae. Illumina HiSeq2000 deep sequencing was used to generate transcriptomic profiles for BaP-exposed 3.3 hpf zebrafish embryos (n=3 for control, n=3 for BaP) and 96 hpf larvae (n=2 for control, n=2 for BaP).

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Transcription profiling of zebrafish embryos reveals AHR Activation by TCDD Downregulates Sox9b expression producing jaw malformation

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