Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

0

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. 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%.

ORGANISM(S): Danio rerio

SUBMITTER: Andrew McArthur 

PROVIDER: E-GEOD-51298 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

altmetric image

Publications

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

O'Shields Britton B   McArthur Andrew G AG   Holowiecki Andrew A   Kamper Martin M   Tapley Jeffrey J   Jenny Matthew J MJ  

Biochimica et biophysica acta 20140418 9


The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel  ...[more]

Similar Datasets

2014-04-25 | GSE51298 | GEO
2010-05-22 | E-GEOD-12516 | biostudies-arrayexpress
2020-10-20 | E-MTAB-9698 | biostudies-arrayexpress
2016-05-01 | GSE74944 | GEO
2013-08-28 | E-GEOD-50376 | biostudies-arrayexpress
2023-02-22 | GSE212929 | GEO
2021-04-26 | E-MTAB-10085 | biostudies-arrayexpress
2020-06-23 | GSE151293 | GEO
2011-08-20 | E-MEXP-2948 | biostudies-arrayexpress
2022-08-22 | GSE201834 | GEO