Eyelid epithelial AHR is required for TCDD toxicity in embryonic eyelid closure.A, immunofluorescent staining using anti-CYP1A1 (green) and Hoechst (blue) for nucleus of the eyelids in WT E15.5 embryos with or without TCDD exposure (50 μg/kg body weight) at 11.5 days. Dash lines mark basement membrane that separated the eyelid epithelium and the underneath dermis; white arrows point at CYP1A1 positive staining. B, Hoechst (blue) staining for nucleus of eyes of the E15.5 mTmG/Le-Cre embryos. Cre expressing, i.e., GFP positive, cells were located at the ocular surface epithelium (OSE), but not stroma of eyelid (EL), cornea (Co), and lens. The dotted lines mark the basement membrane that separated the epithelium and the stroma. Pups exposed at E11.5 to TCDD (250 μg/kg body weight) were collected at E17.5, and their eyes (C) were photographed, red arrow, eyelid leading edge, and (D) quantification of eyes displaying open eye phenotype in Map3k1+/−AhrF/F (N = 18) and Map3k1+/−AhrΔOSE/ΔOSE (N = 8) fetuses. N= number of pups of the indicated genotype. The scale bars represent 50 μm in A, 100 μm in B, and 500 μm in C. AHR, aryl hydrocarbon receptor; MAP3K1, mitogen-activated protein kinase kinase kinase 1; OSE, ocular surface epithelium; TCDD, 2,3,7,8-tetrachlorodibenzo-para-dioxin.