Abstract
Dysregulation of microRNA (miRNA, miR) by environmental stressors influences the transcription of mRNA which may impair organism development and/or lead to adverse physiological outcomes. Early studies evaluating the effects of oil on developmental toxicity in early life stages of fish showed that reductions in expression of miR-203a were associated with enhanced expression of downstream mRNAs that predicted altered eye development, cardiovascular disease, and improper fin development. To better understand the effects of miR-203a inhibition as an outcome of oil-induced toxicity in early life stage (ELS) fish, embryonic zebrafish were injected with an miR-203a inhibitor or treated with 3.5 µM phenanthrene (Phe) as a positive control for morphological alterations of cardiovascular and eye development caused by oil. Embryos treated with Phe had diminished levels of miR-203a at 7 and 72 h after injection. Embryos treated with the miR-203a inhibitor and Phe exhibited a reduced heart rate by 48 h post fertilization (hpf), with an increased incidence of developmental deformities (including pericardial edema, altered eye development, and spinal deformities) and reduced caudal fin length by 72 hpf. There were significant reductions in lens and eye diameters in 120 hpf miR-203a-inhibitor and Phe-treated fish, as well as a significantly reduced number of eye saccades, determined by an optokinetic response (OKR) behavioral assay. The expression of vegfa, which is an important activator during neovascularization, was significantly upregulated in embryos receiving miR-203a inhibitor injections by 7 and 72 hpf with increased trends in vegfa expression in 72 hpf larvae treated with Phe. There were decreasing trends in crx, neurod1, and pde6h expression by 72 hpf in miR-203a inhibitor and Phe treatments, which are involved in photoreceptor function in developing eyes and regulated by miR-203a. These results suggest that an inhibition of miR-203a in ELS fish exhibits an oil-induced toxic response that is consistent with Phe treatment and specifically impacts retinal, cardiac, and fin development in ELS fish.