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.

Project description:Gene expression was measured using microarrays in 8 hour postfertilization embryos, comparing control versus ethanol-treated (2 to 8 hours postfertilization) embryos. This experiment was performed to determine the gene expression changes that occur in response to ethanol treatment as a model of fetal alcohol spectrum disorder. Fetal alcohol spectrum disorder (FASD) occurs when pregnant mothers consume alcohol, causing embryonic ethanol exposure and characteristic birth defects that include craniofacial, neural and cardiac defects. Gastrulation is a particularly sensitive developmental stage for teratogen exposure, and zebrafish is an outstanding model to study gastrulation and FASD. Epiboly (spreading blastomere cells over the yolk cell), prechordal plate migration and convergence/extension cell movements are sensitive to early ethanol exposure. Here, experiments are presented to characterize mechanisms of ethanol toxicity on epiboly and gastrulation. Epiboly mechanisms include blastomere radial intercalation cell movements and yolk cell microtubule cytoskeleton pulling the embryo to the vegetal pole. Both of these processes were disrupted by ethanol exposure. Ethanol effects on cell migration also indicated that cell adhesion was affected, which was confirmed by cell aggregation assays. E-cadherin cell adhesion molecule expression and distribution, which control epiboly and gastrulation, were not affected. Gene expression microarray analysis was used to identify potential causative factors for early development defects, and expression of the cell adhesion molecule protocadherin-18a (pcdh18a), which controls epiboly, was significantly reduced in ethanol-exposed embryos. Injecting pcdh18a synthetic mRNA in ethanol-treated embryos partially rescued epiboly cell movements, including enveloping layer cell shape changes. Together, data show that epiboly and gastrulation defects induced by ethanol are multifactorial, and include yolk cell (extraembryonic tissue) microtubule cytoskeleton disruption and blastomere adhesion defects, in part caused by reduced pcdh18a expression. Control vs. ethanol-treated zebrafish embryos that were treated from 2 to 8 hours postfertilization. Total RNA from control and ethanol-treated embryos were harvested at 8 hours postfertilization.

Project description:Gene expression was measured using microarrays in 8 hour postfertilization embryos, comparing control versus ethanol-treated (2 to 8 hours postfertilization) embryos. This experiment was performed to determine the gene expression changes that occur in response to ethanol treatment as a model of fetal alcohol spectrum disorder. Fetal alcohol spectrum disorder (FASD) occurs when pregnant mothers consume alcohol, causing embryonic ethanol exposure and characteristic birth defects that include craniofacial, neural and cardiac defects. Gastrulation is a particularly sensitive developmental stage for teratogen exposure, and zebrafish is an outstanding model to study gastrulation and FASD. Epiboly (spreading blastomere cells over the yolk cell), prechordal plate migration and convergence/extension cell movements are sensitive to early ethanol exposure. Here, experiments are presented to characterize mechanisms of ethanol toxicity on epiboly and gastrulation. Epiboly mechanisms include blastomere radial intercalation cell movements and yolk cell microtubule cytoskeleton pulling the embryo to the vegetal pole. Both of these processes were disrupted by ethanol exposure. Ethanol effects on cell migration also indicated that cell adhesion was affected, which was confirmed by cell aggregation assays. E-cadherin cell adhesion molecule expression and distribution, which control epiboly and gastrulation, were not affected. Gene expression microarray analysis was used to identify potential causative factors for early development defects, and expression of the cell adhesion molecule protocadherin-18a (pcdh18a), which controls epiboly, was significantly reduced in ethanol-exposed embryos. Injecting pcdh18a synthetic mRNA in ethanol-treated embryos partially rescued epiboly cell movements, including enveloping layer cell shape changes. Together, data show that epiboly and gastrulation defects induced by ethanol are multifactorial, and include yolk cell (extraembryonic tissue) microtubule cytoskeleton disruption and blastomere adhesion defects, in part caused by reduced pcdh18a expression.

Project description:Congenital malformations are a prevalent cause of infant mortality in the United States and their induction has been linked to a variety of factors, including exposure to teratogens. However, the molecular mechanisms of teratogenicity are not fully understood. MicroRNAs are an important group of small, non-coding RNAs that regulate mRNA expression. MicroRNA roles in early embryonic development are well established, and their disruption during development can cause abnormalities. We hypothesized that developmental exposure to teratogens such as valproic acid alters microRNA expression profiles in developing embryos. Valproic acid is an anticonvulsant and mood-stabilizing drug used to treat epilepsy, bipolar disorder and migraines. To examine the effects of valproic acid on microRNA expression during development, we used zebrafish embryos as a model vertebrate developmental system. Zebrafish embryos were continuously exposed to valproic acid (1 mM) or vehicle control (ethanol) starting from 4 hours post-fertilization (hpf) and sampled at 48 and 96 hpf to determine the miRNA expression profiles prior to and after the onset of developmental defects. At 96 hpf, 95% of the larvae showed skeletal deformities, abnormal swimming behavior, and pericardial effusion. Microarray expression profiling was done using Agilent zebrafish miRNA microarrays. Microarray results revealed changes in miRNA expression at both the time points. Thirteen miRNAs were differentially expressed at 48 hpf and 22 miRNAs were altered at 96 hpf. Among them, six miRNAs (miR-16a, 18c, 122, 132, 457b, and 724) were common to both time points. Bioinformatic target prediction and examination of published literature revealed that these miRNAs target several genes involved in the normal functioning of the central nervous system. These results suggest that the teratogenic effects of valproic acid could involve altered miRNA expression. Small RNA profiles were deteremined in valproic acid exposed zebrafish embryos using Agilent miRNA microarrays

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Ethanol alters microNA expression in zebrafish embryos

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