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Figure 4. Inhibition of Notch signaling with dibenzazepine (DBZ) inhibits intestinal metamorphosis. (A): Schematic diagram of DBZ treatment study. Premetamorphic tadpoles at stage 54 were pretreated with DMSO (control vehicle) or 5 μM DBZ for 3 days. Then, the tadpoles were treated with 10 nM T3 in the presence of DMSO or DBZ. The intestine was isolated after 0, 3, or 5 days of T3 treatment and subjected to RNA extraction or cryosectioning. (B): quantitative real-time RT-PCR revealed that pretreatment with DBZ significantly repressed the expression of Hairy1 and Hairy2b in the intestine of premetamorphic tadpoles. Error bars represent the SEM (n = 3). The results were analyzed by Student's t-test (**, p < .01). (C-D'): Gross morphology after 5 days of T3 treatment in the presence of DMSO (C, C') or DBZ (D, D'). Dorsal view of head–trunk region (C, D) and ventral view of hindlimbs (C', D') are shown. Internal hemorrhage around the nose and along the digits of hindlimb is indicated by arrowheads (D, D'). (E-N) DBZ treatment delays the down-regulation of IFABP and the formation of the adult intestinal stem cells. Cross-sections of the intestine from the tadpoles treated with T3 for 3 (K, L) or 5 (E-J, M, N) days in the presence of DMSO (E, G, I, K, M) or DBZ (F, H, J, L, N) were hybridized with antisense Hairy1 (E, F), Hairy2b (G, H), LGR5 (I, J), or IFABP (K-N) probes. The dashed-lines indicate the boundary of the epithelium and the connective tissue. Scale bars = 100 μm. Abbreviations: CT, connective tissue, DBZ, dibenzazepine, Ep, epithelium. Image published in: Hasebe T et al. (2017) © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives license Larger Image Printer Friendly View

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