Cyp1b1 expression in the developing eye was regulated through RA. Whole-mount in situ hybridization showed cyp1b1 expression along the dorsal (arrowheads)–ventral (arrows) axis in the developing eye at 24 and 48 hpf in uninjected (A, A') and 0.1% DMSO-treated (C, C') embryos. In sections, cyp1b1 localized to the retina and retinal pigment epithelium (B, B'). Treatment with 10 μM DEAB at 12 hpf increased cyp1b1 expression in the ventral neural crest, which subsequently forms the brachial arches (D, open arrowhead) at 24 hpf and the dorsal (arrowheads) and ventral (arrows) eye at 48 hpf (D'). Exogenous treatment with 25 nM all-trans RA at 12 hpf decreased cyp1b1 expression in the dorsal and ventral eye at 24 (E) and 48 hpf (E'). Live imaging of the Tg(RARE:mCherry) reporter line showed areas of high RA activity in the eye and olfactory placode (O) in uninjected (F) and control-injected (G) embryos. Within the eye (F', G'), RA activity was observed in the retinal pigment epithelium (arrows), retina (R), lens (L), and cornea (C). MO knockdown of Cyp1b1 (H, H') showed decreased RA activity in the medial retinal pigment epithelium (arrows) correlated with the area of cyp1b1 expression (B'). Overexpression of cyp1b1 through mRNA injection diffusely increased mCherry expression at 24 hpf throughout the craniofacial region (I). Increased RA activity in the eye was also observed in the medial retinal pigment epithelium (arrows) and ocular fissure (arrowhead).
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