BIO treatment disturbs the morphogenesis of pharyngeal pouches. (A) Tg(TOPflash-GFP) embryos were treated with CCT036477 or BIO from bud stage to 24 hpf. Scale bar, 100 μm. CCT, CCT036477. (B) In situ hybridization analysis of nkx2.3 expression in embryos treated with DMSO or CCT036477 from the bud stage to 17 ss. (C) Heat shock-induced expression of Dkk1b in Tg(hsp70l:dkk1b-GFP) embryos. Heat shock was performed at the bud stage, and GFP fluorescence was imaged at 10 ss. Scale bar, 200 μm. (D, E) Tg(hsp70l:dkk1b-GFP) embryos were heat-shocked at the bud stage and harvested for in situ hybridization with the lef1 (D) or nkx2.3 (E) probe. (F) Comparative in situ hybridization analysis of nkx2.4b expression in wild-type embryos treated with or without BIO from the bud stage. (G, H) Wild-type embryos were exposed to different concentrations of BIO from the bud stage to 17 ss and harvested at 36 hpf for in situ hybridization with the nkx2.3 (G) or cv2 (H) probe. The ratios of affected embryos were indicated. (I) Tg(nkx2.3:mCherry) embryos were treated with DMSO or 10 μM BIO from the bud stage to 17 ss. Subsequently, these embryos were harvested at 36 hpf for in vivo confocal imaging. Scale bars, 100 μm.
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