Development of the morpholino gene knockdown technique in Fundulus heteroclitus: A tool for studying molecular mechanisms in an established environmental model

Abstract

a b s t r a c t A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in ␤-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish

“…Embryos exposed to ERSE at 24 hpf were scored for cardiac deformities at 144 hpf. The embryos were screened in a double blinded fashion using a semi-quantitative scale previously used and shown in detail (Matson et al 2008; Clark et al 2010). The scale is used to categorize cardiac structural alteration as normal (0), mild misalignment of cardiac chambers (1), severe cardiac misalignment/tube heart (2).…”

Adaptation of the Estuarine Fish Fundulus heteroclitus (Atlantic Killifish) to Polychlorinated Biphenyls (PCBs)

“…Ongoing studies using traditional quantitative trait locus (e.g., Ellegren and Sheldon 2008) approaches and high-density genomic approaches (e.g., Miller et al 2007) may contribute to our understanding of the mechanisms of killifish tolerance. Additional genomic resources for this species would certainly speed our progress (e.g., Matson et al 2008).…”

When evolution is the solution to pollution: Key principles, and lessons from rapid repeated adaptation of killifish (Fundulus heteroclitus) populations

“…The fact that the primary contaminants at the southern site are PAHs, which can be bioactivated by CYP1A, may have mitigated the benefits of duplication at this locus. However, both chemical and morpholino blockade of CYP1A actually increase the dioxin‐like toxicity of PAHs, suggesting significant benefit to CYP activity for that population (Matson et al., 2008; Wassenberg & Di Giulio, 2004). Thus, the different chemical pollutants acting as selective agents may require finely tuned compensatory changes (Reid et al., 2016).…”