Morphological Effects of Phenanthrene on Somitogenesis in Zebrafish via Perturbation of the Wnt/β-catenin Signaling Pathway

Polycyclic aromatic hydrocarbons (PAHs) are a class of widespread organic toxins released into the environment from a variety of anthropogenic and natural sources. Concern continues to grow for the increasing concentration of PAHs found in marine environments and their toxic effects on the organisms therein. Phenanthrene, a PAH composed of three fused benzene rings, has been reported to cause numerous teratogenic effects in a number of developing organisms including zebrafish. The canonical Wnt/β-catenin signaling pathway has been implicated as a target of phenanthrene as this evolutionarily conserved pathway is an important mediator of normal development and plays a critical role in somite patterning and subsequent differentiation in a number of vertebrate systems. In this study, I examined the extent of the morphological abnormalities, specifically relating to somitogenesis (development of precursor vertebral structures), when zebrafish embryos were exposed to phenanthrene during early development. Sublethal concentrations of phenanthrene led to spinal curvature and incomplete somitogenesis. Dose-dependent and stage-specific effects of the environmental toxin were determined by exposing embryos to 6-30 μM phenanthrene at either the blastula or 6-somite stage through 72 hours post fertilization (hpf). Phenanthrene had the greatest affect when embryos were exposed to 30 μM phenanthrene before the onset of somitogenesis, at the blastula stage. Furthermore, two kinases within the Wnt/β-catenin pathway, glycogen synthase kinase-3β (GSK-3β) and casein kinase 1 (CK1) were explored as potential targets to explain the mechanism by which these phenanthrene-induced spinal abnormalities occurred. Embryos were exposed at the blastula stage through 72 hpf to 50 μM indirubin (a GSK-3β inhibitor), 200 μM A3 hydrochloride (a CK1 inhibitor), or 30 μM phenanthrene. Indirubin and phenanthrene led to similar morphological defects. The effect of A3 hydrochloride was mild compared to the effects of indirubin and phenanthrene. Using western blot analysis, the effect of phenanthrene and indirubin on GSK-3β inactivation through phosphorylation at serine 9 was examined. Embryos exposed to phenanthrene or indirubin had higher levels of the inactive form of GSK-3β compared to the control group. GSK-3β is a known antagonist of the canonical Wnt/β-catenin signaling pathway, therefore this data establishes a link between phenanthrene and the Wnt/β-catenin pathway. Given the similarities in spinal deformities after exposure to phenanthrene or indirubin, this research suggests that phenanthrene leads to abnormal somitogenesis via disruption of the Wnt/β-catenin pathway through untimely inhibition of GSK-3β.