Unknown,Transcriptomics,Genomics,Proteomics

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Translational profiling through biotinylation of tagged ribosomes in zebrafish

ABSTRACT: Phenotypic heterogeneity within a population of cells of the same cell type is a common theme in metazoan development. Apprehending complex developmental and physiological processes critically relies on our ability to probe the expression profile of these genetically defined cell sub-populations. Current strategies rely on cell enrichment based on tandem or simultaneous evaluation of multiple intersecting markers starting from a heterogeneous cellular suspension. The extensive tissue manipulations required for single-cell suspension generation, as well as the complexity of the required equipment, inherently complicates these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a and the bacterial biotin ligase, BirA. We have applied this technique to skeletal muscle development, identifying genes with novel regulation and have developed tools for highly specific gene expression profiling during vertebrate development. Translational profiling of zebrafish skeletal muscle during 4 time points (21, 24, 27, 34 hours post fertilization)

ORGANISM(S): Danio rerio

SUBMITTER: Michael Housley

PROVIDER: E-GEOD-59355 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Prenatal exposure to ethanol leads to a myriad of developmental disorders known as fetal alcohol spectrum disorder, often characterized by growth and mental retardation, central nervous system damage and specific craniofacial dysmorphic features. Although the exact mechanisms of ethanol toxicity are not well understood it is known that ethanol exposure during development affects the expression of several genes involved in cell cycle control, apoptosis and transcription. MicroRNAs (miRNAs) are implicated in some of these processes however it is unclear if they are involved in ethanol-induced toxicity. Here we tested whether ethanol deregulates miRNA expression in zebrafish embryos and if a miRNA deregulation signature could be inferred. For this, zebrafish embryos were exposed to two different ethanol concentrations (1% and 1.5%) from 4 hours post-fertilization (hpf) to 24hpf. MicroRNA expression profiles revealed that ethanol exposure induces deregulation of miRNA expression significantly. Seven miRNAs are commonly up-regulated after both ethanol treatments, namely miR-153a, miR-725, miR-30d, let-7k, miR-100, miR-738 and miR-732, whereas downregulation of miR-23a, miR-203, let-7c, miR-128 and miR-193b is detected after 1% ethanol exposure only. Target prediction of deregulated miRNAs shows that putative targets are involved in cell cycle control, apoptosis and transcription, which are the main processes affected by ethanol toxicity. The overall study shows that the effects of ethanol on miRNA deregulation are dose-dependent and that miRNAs are relevant in the context of alcohol toxicity. Moreover, a miRNA toxicity signature for embryonic ethanol exposure was obtained. Zebrafish embryos were obtained from spawning adults in groups of about 10 males and 10 females. Zebrafish embryos were collected and Petri dishes with approximately 250 eggs each were incubated at 28M-BM-:C to allow normal zebrafish development until 4hpf, when blastula is reached. At this stage, embryos were examined under a dissecting microscope and those that had developed normally were selected for EtOH exposure (approximately 200 eggs). Briefly, 200 embryos were randomly distributed into plastic Petri dishes containing 20 mL of EtOH test solutions (1% EtOH, 1.5% EtOH). All solutions were made by dilution of absolute EtOH in system water. Exposure was from 4hpf to 24hpf. At this stage, solutions were changed by system water and embryos were allowed to grow until 24hpf. The control group was allowed to grow in plain system water. Zebrafish embryos were collected at 24hpf for microarray analysis. Two biological replicates were performed for each assay.

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Translational profiling through biotinylation of tagged ribosomes in zebrafish

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