Okada M, Shi YB.

             metamorphosis (sensu Amphibia)
             digestive tract development
             intestinal epithelial cell differentiation

	Fig. 1 High levels of Mad1 mRNA are present in the degenerating larval epithelial cells facing the lumen while c-Myc is expressed in the proliferating adult cells at the climax of intestinal remodeling. A Both Mad1 and c-Myc, but not their dimerization partner Max, are upregulated, during intestinal remodeling. RT-PCR was performed using whole intestine to compare the expression profiles of Mad1, c-Myc, and Max during X. laevis intestinal metamorphosis. 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). See [58] for more details. B Mad1 but not c-Myc is expressed in the larval cells undergoing apoptosis 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. Note that Mad1 expression was limited to dying larval epithelial cells facing the intestinal lumen at the climax of metamorphosis. 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. See [58] for more details
	Fig. 2 Mad1 regulates developmental cell death and Mad–Myc balance controls the expansion of adult intestinal epithelial cells. a Methyl green-pyronin Y (MGPY) staining, which stained the proliferating adult epithelial cells strongly (purple), reveals increased adult stem cell clusters in the knockout animals compared to those in the wild-type tadpoles after 3 days of T3 treatment. Premetamorphic stage 54 tadpoles were treated with 5 nM T3 for 0, 2 or 3 days and were killed 30 min after EdU injection. See [58] for more details. b Quantitative analysis of MGPY-positive areas in the epithelium and normalized by the total cellular area in epithelium. The statistical significance of the differences was determined by Student’s t test (*P < 0.05). Tadpoles of each genotype (n = 3–5) were used for counting MGPY-positive areas in the epithelium. See [58] for more details. c Cell proliferation is significantly increased in Mad1 (−/−) tadpoles treated with T3 for 3 day compared to that in the wild-type ones. Cross-sections of the intestine of the tadpoles above were stained for cell proliferation by EdU staining. EdU positive areas in epithelium of X. tropicalis intestine 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). Tadpoles of each genotype (n = 3–5) were used for counting EdU-positive areas in the epithelium. See [58] for more details. d More intestinal epithelial cell death in T3-treated wild-type animals than that in Mad1 (−/−) tadpoles. Cross-sections of the intestine of the tadpoles above were stained for apoptosis by TUNEL. 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. The statistical significance of the differences was determined by Student’s t-test (*P < 0.05). Tadpoles of each genotype (n = 3–5) were used for counting TUNEL-positive areas in the epithelium. See [58] for more details. e Summary of the changes in cell proliferation (EdU) and apoptosis (TUNEL) in the intestinal epithelium after 5 nM T3 treatment for 0, 2 or 3 days in wild type and Mad1-knockout animals. Note that Mad1 knockout not only enhances the cell proliferation but also reduces cell death in the epithelium during T3-induced metamorphosis
	Fig. 3 A balance of Mad and Myc controls cell fate determination during intestinal remodeling. a For T3-induced genes, TR normally functions as a heterodimer with RXR (9-cis retinoic acid receptor). During metamorphosis, T3 induces the expression of c-Myc directly at the transcription level through TR binding to the TRE in the c-Myc promoter. Then, c-Myc activates the expression of histone methyltransferase PRMT1 through binding to the intronic enhancer in the PRMT1 gene. PRMT1, in turn, participates in the intestinal stem cell formation/proliferation. b Both Mad1 and c-Myc can heterodimerize with Max. Thus, Mad1 competes against c-Myc to regulate target gene expression. When high levels of Mad1 are present in a cell, Mad1 causes mitotically active larval epithelial cells to exit the cell cycle to facilitate their degeneration through apoptosis. Whereas, high levels of c-Myc competes again Mad1 to activate PRMT1 expression. PRMT1, in turns, participates in the formation and/or proliferation adult intestinal stem cells. High levels of Mad1-expressing cells, undergoing apoptosis, are indicated by red dots. The c-Myc and PRMT1-expressing cells, which are proliferating adult stem cells, are indicated by yellow dots
	mxd1 (MAX dimerization protein 1) gene expression in the intestine of Xenopus laevis embryo, assayed via in situ hybridization, NF stage 61/62, in cross-section. Key: Mu= muscle, C=connective tissue. Arrow points to clusters of cells or islets in the intestinal epithelium close to the connective interface, and arrowhead points to the epithelial cells facing the lumen.
	myc (MYC proto-oncogene, bHLH transcription factor ) gene expression in the intestine of Xenopus laevis embryo, assayed via in situ hybridization, NF stage 61/62, in cross-section.

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