Overexpression of cyp1b1 inhibited ocular fissure closure via an RA-independent pathway. The percentage of 72 hpf embryos with developmental delay and/or craniofacial abnormalities (A) due to Cyp1b1 MO (ATG or 5′ UTR) knockdown, cyp1b1 (mRNA) overexpression, Raldh2 MO knockdown, raldh2 (mRNA) overexpression compared to uninjected, mismatch (MM) MO-injected or gfp mRNA-injected controls. ([a] P < 0.0001; [b] P < 0.001; [c] P < 0.01; [d] P < 0.05). Live images of uninjected (B) and mismatch control-injected (C, G) Tg(sox10:EGFP) embryos showed that at 96 hpf, neural crest contributed to the pharyngeal arches (PA) and the ceratohyal (Ch) and Meckel's (Mk) cartilage of the jaw. Sections of 96 hpf Tg(foxd3:GFP) embryos showed that foxd3-positive neural crest cells contributed to the iris stroma in the dorsal (B', C', G', arrowhead) and ventral (double arrowhead) iridocorneal angle. In addition, differentiated retinal photoreceptors expressed foxd3, which was unrelated to neural crest cells. Injection of 5′ UTR Cyp1b1 MO had minimal effect on craniofacial neural crest (D) and overall eye (D') development. Overexpression of cyp1b1 through mRNA injection disrupted neural crest–derived Meckel's cartilage formation (E, K) and inhibited ocular fissure closure, resulting in prominent colobomas (closed arrows, E, E'). The retinal layers were properly differentiated, including the expression of foxd3. Injection of Cyp1b1 5′ UTR MO improved the ocular and neural crest phenotypes due to cyp1b1 mRNA (F, F'). As previously demonstrated, increasing (raldh2 mRNA, H, H') or decreasing (Raldh2 5′ UTR MO, I, I') RA inhibited pharyngeal arch development, disrupted ceratohyal and Meckel's cartilage formation, and decreased eye size. Injection of raldh2 mRNA rescued the ocular and neural crest phenotypes induced through Raldh2 5′ UTR MO knockdown (J, J'). Cyp1b1 MO improved eye size, retinal differentiation, and neural crest–derived pharyngeal arch and ceratohyal and Meckel's cartilage formation in embryos injected with raldh2 mRNA (K, K'). Knockdown of Raldh2 improved neural crest–derived pharyngeal arch and ceratohyal and Meckel's cartilage formation (L'), but did not rescue ocular fissure closure (arrows, L') in embryos injected with cyp1b1 mRNA.
|
