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Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development

ABSTRACT: 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.

ORGANISM(S): Danio rerio

PROVIDER: GSE51298 | GEO | 2014/04/25

SECONDARY ACCESSION(S): PRJNA222382

REPOSITORIES: GEO

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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: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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Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development

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