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MicroRNA profiling in Atlantic killifish (Fundulus heteroclitus)

ABSTRACT: The Atlantic killifish (Fundulus heteroclitus) is an ideal model species to study physiological and toxicological adaptations to stressors. Killifish inhabiting the PCB-contaminated Superfund site in New Bedford Harbor, MA (NBH) have evolved resistance to toxicity and activation of the aryl hydrocarbon receptor (AHR) signaling pathway after exposure to PCBs and other AHR agonists. Until recently, a lack of genomic information has limited efforts to understand the molecular mechanisms underlying environmental adaptation to stressors. The advent of high throughput sequencing has facilitated an unbiased assessment of coding as well as non-coding RNAs in any species of interest. Among non-coding RNAs, microRNAs (miRNAs) are important regulators of gene expression and play crucial roles in development and physiology. The objective of this study is to catalog the miRNAs in killifish and determine their expression patterns in the embryos from contaminated (NBH) and pristine (Scorton Creek, MA (SC)) sites. Embryos from NBH and SC were collected daily from 1 to 15 days post-fertilization and RNA from pooled samples from each site was sequenced using SOLiD sequencing. We obtained 7.5 and 11 million raw reads from pooled SC and NBH samples, respectively. Analysis of the sequencing data identified 216 conserved mature miRNA sequences that are expressed during development. Using the draft killifish genome, we retrieved the miRNA precursor sequences. Based on the capacity of these putative precursor sequences to form the characteristic hairpin loop (assessed using RNAfold), we identified 197 conserved miRNA sequences in the genome.

ORGANISM(S): Fundulus heteroclitus

PROVIDER: GSE70953 | GEO | 2015/07/20

SECONDARY ACCESSION(S): PRJNA290009

REPOSITORIES: GEO

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Project description:Hypoxia in coastal waters is an increasing concern, with the frequency of hypoxic events rising because of climate change and human-driven impacts such as eutrophication. The Atlantic killifish (Fundulus heteroclitus), an estuarine species known for its resilience to environmental stressors, provides a valuable model to investigate the physiological, transcriptional, and epigenetic mechanisms underlying adaptation. Some killifish populations, like those in New Bedford Harbor (NBH), Massachusetts, have evolved resistance to dioxin-like polychlorinated biphenyls (PCBs) due to chronic exposure. These populations offer a unique opportunity to investigate how toxicant resistance might influence responses to secondary stressors like hypoxia. The objective of this study is to characterize the impacts of evolved resistance to toxicants on the ability to cope with acute hypoxia. We compared hepatic gene expression and DNA methylation patterns in response to two levels of hypoxia in killifish from NBH and Scorton Creek (SC), a reference population from a relatively pristine environment. We hypothesized that NBH fish would exhibit altered molecular responses to hypoxia due to trade-offs associated with toxicant resistance. Our results revealed significant differences between the two populations. SC fish demonstrated a dose-dependent increase in gene expression in response to hypoxia, while NBH fish exhibited a muted transcriptional response to severe hypoxia, indicating potential impairment in their ability to cope with hypoxic stress. Interestingly, NBH fish showed significant DNA methylation changes in response to hypoxia, while SC fish did not exhibit notable epigenetic alterations. These findings suggest that toxicant-adapted killifish may face trade-offs in their response to additional environmental stressors, with potential consequences for their ability to survive in increasingly hypoxic coastal habitats. Further research is needed to elucidate the functional implications of these epigenetic modifications and their role in adaptive stress responses.

Project description:This study presents statistical analyses of gene expression in 5, 10 and 15 day post-fertilization (dpf) embryos of the teleost Fundulus heteroclitus treated with control vehicle (DMSO) or a potent non-ortho-PCB (PCB-126; 3,3’,4,4’,5-pentachlorobiphenyl). The embryos were from two populations: a clean, reference population (SC, Scorton Creek, MA USA) and a polluted Superfund population (N, New Bedford Harbor, MA USA). For each site, eggs from 8 females (~1100 total) were fertilized using minced testes from 5 males. After non-fertile eggs were culled, embryos were exposed to vehicle (DMSO; 0.1%) or PCB-126 (50 nM) in filtered seawater (salinity 25 part per thousand, 65 embros per 20 ml in glass petri dish) for 4 hr at 20º C. After exposure, the embryos were washed in filtered seawater and incubated at 20º C under a 14-h light, 10-h dark cycle. At 5-, 10-, and 15-dpf, embryos were collected as three pools of 20 embryos from each treatment group and flash frozen in liquid nitrogen and stored at -80º C until used for RNA isolation. A loop design was used for the microarray hybridizations where each sample is hybridized to 2 arrays using both Cy3 and Cy5 labeled fluorophores. We used three loops in which each loop consisted of Cy3- and Cy5-labeled embryo aRNAs from 12 samples: one sample from each population-treatment-time combination. Within a population-treatment-time, embryos were randomly assigned to one of the three loops. In total, 36 embryos were hybridized to 36 microarrays. The loops formed were SC5C→ SC5P → NBH5C→ NBH5P →SC10C→ SC10P→ NBH10C→ NBH10P → SC15C→ SC15P → NBH15C → NBH15P → SC5C, where each arrow represents a separate hybridization (array) with the biological sample at the base of the arrow labeled with Cy3 and the biological sample at the head of the arrow labeled with Cy5. SC represents the Scorton Creek, Sandwich, MA population (reference), NBH represents the New Bedford Harbor, MA population, C represents control dose (DMSO), P represents the PCB-126 dose, 5 represents 5 dpf, 10 represents 10 dpf and 15 represents 15 dpf.

Project description:Dioxin-like chemicals are well-known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We conducted a thorough investigation to address the following questions: (1) does AHR genotype itself affect constitutive expression of microRNAs? (2) does TCDD affect microRNA levels and, if so, is this response dependent on the AHR? (3) does TCDD affect microRNA levels differently in animals that are sensitive to dioxin toxicity versus those that are dioxin-resistant? We used the Exiqon miRNA array platform as well as quantitative RT-PCR to measure miRNA levels in wildtype vs. Ahr-null mice. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in mouse livers and those changes that were statistically significant were of modest magnitude. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD M-bM-^@M-^S only a few miRNAs differed in expression between Ahr-null mice compared to mice with wildtype AHR. It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult mouse liver. Manuscript Submitted: Moffat ID, Boutros PC, Celius T, Pohjanvirta R & Okey AB. Micro-RNAs in rodent liver are refractory to dioxin treatment. Toxicological Sciences May, 2007. Keywords: miRNA expression, gene knockout, response to xenobiotics, genetic modification This was a two-factor, two-level design, the factors being genotype Ahr+/+ (WT) or Ahr-/- (KO) and treatment TCDD-treated (T) or vehicle control (C). Each total-RNA sample was separately labeled either with a Hy3- or a Hy5-fluorophore (Exiqon). Hy3- and Hy5-labeled samples were co-hybridized to an array. Dye-reversal was performed to eliminate dye bias. We tested 3 TCDD-treated and 3 control mice in the Ahr-/- groups and 4 TCDD-treated and 4 control mice in the Ahr+/+ groups. Technical replication of 1 TCDD-treated and 1 control mice in the Ahr-/- groups was performed. A loop design was used.

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MicroRNA profiling in Atlantic killifish (Fundulus heteroclitus)

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