Okada M, Wen L, Miller TC, Su D, Shi YB.

	MGPY stains strongly the clusters (islets) of proliferating adult intestinal epithelial cells during T3-induced intestinal metamorphosis. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and with MGPY. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a″–c″). The approximate epithelium-mesenchyme boundary was drawn based on morphological differences between epithelial cells and mesenchyme cells in the pictures of the double-stained tissues, under enhanced contrast and/or brightness by using Photoshop, if needed (dotted lines). Note that the clusters (islets) of EdU labeled cells in the epithelium after 6 days of T3 treatment were strongly stained by MGPY (C, c″). Arrowheads indicate the clusters of proliferating cells or islets (c′)
	MGPY and EdU co-stain the clusters (islets) of proliferating adult intestinal epithelial cells at the climax of natural metamorphosis. Tadpoles at premetamorphic stage 54 (A), climax (B stage 62), and end of metamorphosis (C stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and with MGPY. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a″–c″). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Arrowhead indicates the clusters of proliferating cells or islets (b′)
	The newly formed proliferating adult intestinal epithelial cells during T3-induced metamorphosis have little or no expression of IFABP. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for IFABP by immunohistochemistry and for EdU. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a′–c′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that the EdU labeling revealed profound cell proliferation after T3 treatment. The proliferating cells in the epithelium after 6 days of T3 treatment were present mainly in clusters where IFABP staining was weak or absent (C, c′)
	Clusters of proliferating adult intestinal epithelial cells at the climax of natural metamorphosis lack IFABP. Tadpoles at premetamorphic stage 54 (A), climax (B stage 62), and end of metamorphosis (C, stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and IFABP by immunohistochemistry. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a″–c″). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that the EdU-labeled proliferating cells in the epithelium were few and expressed IFABP at premetamorphosis (A) and increased in form of clustered cells that lacked IFABP at the climax of metamorphosis (B, b″). At the end of metamorphosis, EdU-labeled proliferating cells were localized mainly in the troughs of the epithelial folds where IFABP expression was low (C, c″)
	The EdU-labeled clusters (islets) of proliferating adult intestinal epithelial cells during T3-induced intestinal metamorphosis express the adult intestinal stem cell marker Lgr5. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for Lgr5 by in situ hybridization and for EdU. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that the clusters (islets) of EdU labeled cells in the epithelium after 6 days of T3 treatment had high levels of Lgr5 mRNA (c, c′)
	Lgr5 and EdU co-stain the clusters (islets) of proliferating adult intestinal epithelial cells at the climax of natural metamorphosis. Tadpoles at premetamorphic stage 54 (A), climax (B stage 62), and end of metamorphosis (C stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and Lgr5. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1)
	EdU and TUNEL-labeling reveals that apoptotic and proliferating cells are non-overlapping epithelial cells during T3-induced intestinal metamorphosis. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for apoptosis by TUNEL and for EdU. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that apoptosis in the epithelium occurred prior to the appearance of the clusters (islets) of EdU labeled cells and in distinct epithelial cells during T3 treatment (C, c′)
	Epithelial apoptosis takes place prior to the appearance of clusters (islets) of proliferating adult intestinal stem cells during natural metamorphosis. Tadpoles at premetamorphic stage 54 (A), climax (B stage 60, C stage 62), and end of metamorphosis (D stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for apoptosis by TUNEL and for EdU. Higher magnifications of boxed areas in (A–D) are shown in (a′–d′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1)
	Fig. 1. MGPY stains strongly the clusters (islets) of proliferating adult intestinal epithelial cells during T3-induced intestinal metamorphosis. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and with MGPY. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a″–c″). The approximate epithelium-mesenchyme boundary was drawn based on morphological differences between epithelial cells and mesenchyme cells in the pictures of the double-stained tissues, under enhanced contrast and/or brightness by using Photoshop, if needed (dotted lines). Note that the clusters (islets) of EdU labeled cells in the epithelium after 6 days of T3 treatment were strongly stained by MGPY (C, c″). Arrowheads indicate the clusters of proliferating cells or islets (c′)
	Fig. 2. MGPY and EdU co-stain the clusters (islets) of proliferating adult intestinal epithelial cells at the climax of natural metamorphosis. Tadpoles at premetamorphic stage 54 (A), climax (B stage 62), and end of metamorphosis (C stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and with MGPY. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a″–c″). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Arrowhead indicates the clusters of proliferating cells or islets (b′)
	Fig. 3. The newly formed proliferating adult intestinal epithelial cells during T3-induced metamorphosis have little or no expression of IFABP. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for IFABP by immunohistochemistry and for EdU. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a″–c″). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that the EdU labeling revealed profound cell proliferation after T3 treatment. The proliferating cells in the epithelium after 6 days of T3 treatment were present mainly in clusters where IFABP staining was weak or absent (C, c″)
	Fig. 4. Clusters of proliferating adult intestinal epithelial cells at the climax of natural metamorphosis lack IFABP. Tadpoles at premetamorphic stage 54 (A), climax (B stage 62), and end of metamorphosis (C, stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and IFABP by immunohistochemistry. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′) and (a″–c″). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that the EdU-labeled proliferating cells in the epithelium were few and expressed IFABP at premetamorphosis (A) and increased in form of clustered cells that lacked IFABP at the climax of metamorphosis (B, b″). At the end of metamorphosis, EdU-labeled proliferating cells were localized mainly in the troughs of the epithelial folds where IFABP expression was low (C, c″)
	Fig. 5. The EdU-labeled clusters (islets) of proliferating adult intestinal epithelial cells during T3-induced intestinal metamorphosis express the adult intestinal stem cell marker Lgr5. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for Lgr5 by in situ hybridization and for EdU. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that the clusters (islets) of EdU labeled cells in the epithelium after 6 days of T3 treatment had high levels of Lgr5 mRNA (c, c′)
	Fig. 6. Lgr5 and EdU co-stain the clusters (islets) of proliferating adult intestinal epithelial cells at the climax of natural metamorphosis. Tadpoles at premetamorphic stage 54 (A), climax (B stage 62), and end of metamorphosis (C stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU and Lgr5. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1)
	Fig. 7. EdU and TUNEL-labeling reveals that apoptotic and proliferating cells are non-overlapping epithelial cells during T3-induced intestinal metamorphosis. Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0 (A), 3 (B), or 6 days (C) and were sacrificed 1 h after injection with EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for apoptosis by TUNEL and for EdU. Higher magnifications of boxed areas in (A–C) are shown in (a′–c′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1). Note that apoptosis in the epithelium occurred prior to the appearance of the clusters (islets) of EdU labeled cells and in distinct epithelial cells during T3 treatment (C, c′)
	Fig. 8. Epithelial apoptosis takes place prior to the appearance of clusters (islets) of proliferating adult intestinal stem cells during natural metamorphosis. Tadpoles at premetamorphic stage 54 (A), climax (B stage 60, C stage 62), and end of metamorphosis (D stage 66) were injected with EdU 1 h before being sacrificed. Cross-sections of the intestine from the resulting tadpoles were double-stained for apoptosis by TUNEL and for EdU. Higher magnifications of boxed areas in (A–D) are shown in (a′–d′). The dotted lines depict the epithelium-mesenchyme boundary (see Fig. 1)

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