Okada M , Miller TC , Wen L .

	Figure 1. Mad1 is highly conserved in vertebrates. Amino-acid sequence alignment of X. laevis (NP_001090200), X. tropicalis (NP_001072228), H. sapiens (NP_002348, NP_001189442, NP_001189443) and M. musculus (NP_034881) Mad1. The shared amino acids are indicated with yellow boxes. The highly conserved HLH DNA-binding domain of Mad1 is boxed
	Figure 2. Mad1 is strongly upregulated specifically in the epithelium of the intestine at the climax of metamorphosis when epithelial apoptosis and adult stem cell development take place. (a) The expression of Mad1 was analyzed by RT-PCR on total RNA from the isolated epithelium and non-epithelium (the rest of the intestine) of X. laevis tadpoles at indicated stages during development. (b) Both Mad1 and c-Myc are upregulated, whereas Max expression changes little during intestinal remodeling. The expression of Mad1, c-Myc, and Max was analyzed by RT-PCR on total RNA isolated from whole intestine of X. laevis tadpoles at indicated stages during development. The mRNA level for Mad1, c-Myc and Max was normalized against that of EF1α RNA. The data are shown in arbitrary unit as the mean±S.E. (n=3). The statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (*P<0.05)
	Figure 3. T3 induces Mad1 expression in the intestine, before high levels of c-Myc expression. RT-PCR analysis of Mad1 and c-Myc expression was performed by using total RNA isolated from the intestine of X. laevis stage 54 tadpoles treated with 10 nM T3 for indicated numbers of days. The mRNA level for Mad1 and c-Myc gene was normalized against that of EF1α mRNA. The data are shown as the mean±S.E. (n=3). The statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (*P<0.05)
	Figure 4. In situ hybridization analysis indicates that Mad1 is expressed in the epithelial cells at the climax of intestinal remodeling. Cross-sections of X. laevis intestine at premetamorphic stage 54, metamorphic climax stages 61/62 and the end of metamorphosis (stage 66) were hybridized with Mad1 (a) and c-Myc (b) antisense probe. The boxed region in a and b at stage 61/62 were enlarged and shown in (a′ and b′), respectively, at the bottom. Note that Mad1 expression was limited to dying larval epithelial cells facing the intestinal lumen in the epithelium at the climax of metamorphosis, consistent with the expression data in Figure 2. The expression of c-Myc was also high at the climax of metamorphosis but was in the epithelial layer close to the connective tissue. Arrows point to clusters of cells or islets in the epithelium close to the connective interface and expressing c-Myc, whereas arrowheads point to the epithelial cells facing the lumen, expressing Mad1. The approximate epithelium–mesenchyme boundary was drawn based on morphological differences between epithelial cells and mesenchyme cells in the photographs, under enhanced contrast and/or brightness by using Photoshop, if needed (dotted lines). Scale bar, 50 μm. CT, connective tissue; Ep, epithelium; Mu, muscle; Lu, lumen; Ty, typhlosole
	Figure 5. High levels of Mad1 mRNA are expressed in apoptotic but not proliferating epithelial cells. Double staining of PCNA immunohistochemistry (green: cell proliferation) and Mad1 (a) or c-Myc (b) in situ hybridization (purple) was performed on cross-sections of X. laevis intestine at metamorphic climax stage 61/62. Note that Mad1 expression was limited to the epithelial cells facing the lumen, which are known to be dying larval epithelial cells at the climax of metamorphosis.21 These cells did not have PCNA. The PCNA positive, proliferating cells lacked Mad1 mRNA and were localized in between the dying epithelial cells and the connective tissue. In contrast, c-Myc-expressing cells were found to colocalize with the proliferating cells expressing PCNA (b). Higher magnification of boxed areas in the merged panel in (a and b) are shown in a′ and b′, respectively. The approximate epithelium–mesenchyme boundary was drawn based on morphological differences between epithelial cells and mesenchyme cells in the photographs, under enhanced contrast and/or brightness by using Photoshop, if needed (dotted lines). Note that for unknown reason, we observed that the PCNA signals in the connective tissue in (b) were much stronger than in (a). It is possible that it was in part because of weaker staining in (a) and in part that the section in (b) happened to have more proliferating cells. Nonetheless, the colocalization of PCNA with c-Myc but not Mad1 was consistently observed in different intestinal sections. Scale bar, 50 μm
	Figure 6. Mad1 knockout tadpoles have no growth defects. (a) X. tropicalis Mad1 is similarly upregulated during intestinal metamorphosis as in X. laevis. The expression of Mad1 was determined by RT-PCR analysis of total RNA isolated from whole intestine of X. tropicalis tadpoles at indicated stages during development. Mad1 mRNA level was normalized against that of EF1α mRNA. The statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (*P<0.05). (b) T3 induces Mad1 expression in X. tropicalis premetamorphic tadpole intestine. Stage 54 X. tropicalis tadpoles were treated with or without 10 nM T3 for 2 days. Total RNA was isolated from the intestine and analyzed by RT-PCR for Mad1 expression. Mad1 mRNA level was normalized against that of EF1α mRNA. The data are shown as the mean±S.E. (n=3). The statistical significance of differences was determined by Student’s t-test (*P<0.05). (c) Mad1 genomic structure and target sequences of the TALEN against X. tropicalis Mad1 locus. There are three exons in X. tropicalis Mad1 and Mad1-specific TALEN left (L) and right (R) arms were generated to target exon 1. The left and right TALEN-binding sequences are shown in red and blue, respectively, and the spacer region is in black. (d) Sequence of homozygous (Δ10/Δ10, Δ10/Δ7) and heterozygous (+/Δ10, +/Δ7) tadpoles found in F2 generation tadpoles. Deletions are indicated by dashes. (e-g) No gross defect because of Mad1 knockout is found in animals at the end of metamorphosis. Tadpoles were reared identically to stage 66 and then genotyped. The time to reach stage 66 (e), the body weight (f) and intestinal length (g) at stage 66 for each animal was determined and plotted with the mean, marked as a line, and standard error. Note that no significant difference was observed for any of the parameters
	Figure 7. Mad1 knockout enhances intestinal epithelial cell proliferation during T3-induced metamorphosis. Premetamorphic stage 54 tadpoles were treated with 5 nM T3 for 0, 2 or 3 days and were killed 30 min after EdU injection. (a) Cross-sections of the intestine from the resulting tadpoles were stained for MGPY. Note there were more epithelial cells in clusters strongly stained by MGPY in the cross-sections of the intestine from Mad1 (−/−) tadpoles treated with T3 for 3 day compared with wild-type ones. Arrowheads indicate the clusters of epithelial cells. (b) Cross-sections of the intestine from the tadpoles were double-stained for EdU (cell proliferation) and with Hoechst (DNA). Again, there are more EdU-positive epithelial cells in clusters in the T3-treated Mad1 (−/−) tadpole intestinal cross-sections compared with the wild-type ones. The dotted lines depict the epithelium–mesenchyme boundary. Scale bar, 50 μm. (c) Cell proliferation is significantly increased in Mad1 (−/−) tadpoles treated with T3 for 3 day compared with wild-type ones. Red colored EdU positive areas in epithelium were measured and normalized against the total cellular area in epithelium determined by Hoechst staining. The statistical significance of the differences was determined by Student’s t-test (*P<0.05)
	Figure 8. Mad1 knockout inhibits cell death in the epithelium during T3-induced metamorphosis. (a) Cross-sections of the intestine of premetamorphic stage 54 tadpoles treated with 5 nM T3 for 0, 2 or 3 days were stained for apoptosis by TUNEL. Note that apoptosis in the epithelium peaked after 2 days of T3 treatment and that there were more intestinal epithelial cells in wild-type animals strongly stained by TUNEL compared with the ones in Mad1 (−/−) tadpoles. The dotted lines depict the epithelium–mesenchyme boundary. Scale bar, 50 μm. (b) Quantitative analysis of apoptosis by counting TUNEL-positive areas in the epithelium and normalized by the total cellular area in epithelium determined by Hoechst staining

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