Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio)

We developed an inducible in vivo reporter system to examine expression of the aryl hydrocarbon receptor (AhR) during development in zebrafish (Danio rerio). AhR is a ligand-activated transcription factor that mediates the to xic actions of environmental contaminants such as 2,3,7,8-tetrachlorodibenz o-p-dioxin (TCDD). Induction of cytochrome P4501A1 (CYP1A1) is an early bio marker of AhR activation. A 1905 base pair region of the human CYP1A1 promo ter/enhancer region was regulated by AhR in zebrafish liver cells after exp osure to TCDD (10 nM) in a transient transfection assay. This regulatory re gion was fused to the cDNA sequence encoding green fluorescent protein (GFP ) of jellyfish (Aequorea victoria). Transgenic zebrafish were generated to express this AhR-regulated GFP construct. Injected fish exposed to TCDD exh ibited induction of GFP in the eye, nose, and vertebrae of zebrafish embryo s (48 and 72 hr after fertilization) compared to vehicle controls (DMSO), w hich did not express GFP. To investigate whether AhR-regulated GFP expressi on correlated with sites of TCDD toxicity, we exposed wild-type zebrafish t o DMSO or TCDD and examined them for morphologic abnormalities. By 5 days a fter fertilization, TCDD-exposed fish exhibited gross dysmorphogenesis in c ranio-facial and vertebral development.