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Molecular and cytological analyses reveal distinct transformations of intestinal epithelial cells during Xenopus metamorphosis.

Okada M, Wen L, Miller TC, Su D, Shi YB - Cell Biosci (2015)

Bottom Line: Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis.Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5.Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

View Article: PubMed Central - PubMed

Affiliation: Section on Molecular Morphogenesis, Program in Cellular Regulation and Metabolism (PCRM), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 18 Library Dr., Bethesda, MD 20892 USA.

ABSTRACT

Background: The thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells.

Results: Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis.

Conclusions: Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

No MeSH data available.


Related in: MedlinePlus

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′)
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Fig1: 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′)

Mentions: The remodeling of the intestine leads to distinct changes in the morphology of the intestinal cross-section. This can be easily detected by staining the tissue sections with a mixture (MPGY) of methyl green, which stains DNA, and pyronin Y, which stains RNA [20–22]. Earlier studies have shown that MGPY stains strongly clusters of epithelial cells formed at the climax of metamorphosis or after T3 treatment, while the surrounding cells that are poor stained. As the epithelial cell clusters or islets at the climax of metamorphosis can be labeled with DNA synthesis markers [16, 20], it has been assumed that the clusters with active DNA synthesis are the same as those stained strongly with MGPY. To demonstrate this directly, we treated premetamorphic tadpoles at stage 54 with T3 for 0–6 days to induce metamorphosis. One hour prior to being sacrificed, the tadpoles were injected with 5-ethynyl-2′-deoxyuridine (EdU) to label the newly synthesized cellular DNA. The intestinal cross-sections from the resulting tadpoles were double-stained with MGPY and for EdU. As shown in Fig. 1, in premetamorphic intestine, the epithelium was uniformly stained with MGPY and some of the epithelial cells were EdU positive (Fig. 1A, a″). T3 treatment for 3 days had little effect on either intestinal morphology or staining (Fig. 1B, b″). However, after 6 days of T3 treatment, clusters of cells in the epithelium appeared and were more strongly stained by MGPY than the surrounding cells (Fig. 1C). Furthermore, these clusters were labeled by EdU (Fig. 1C, c″), indicating that the cell clusters strongly stained by MGPY are indeed the proliferating cell clusters.Fig. 1


Molecular and cytological analyses reveal distinct transformations of intestinal epithelial cells during Xenopus metamorphosis.

Okada M, Wen L, Miller TC, Su D, Shi YB - Cell Biosci (2015)

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′)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696227&req=5

Fig1: 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′)
Mentions: The remodeling of the intestine leads to distinct changes in the morphology of the intestinal cross-section. This can be easily detected by staining the tissue sections with a mixture (MPGY) of methyl green, which stains DNA, and pyronin Y, which stains RNA [20–22]. Earlier studies have shown that MGPY stains strongly clusters of epithelial cells formed at the climax of metamorphosis or after T3 treatment, while the surrounding cells that are poor stained. As the epithelial cell clusters or islets at the climax of metamorphosis can be labeled with DNA synthesis markers [16, 20], it has been assumed that the clusters with active DNA synthesis are the same as those stained strongly with MGPY. To demonstrate this directly, we treated premetamorphic tadpoles at stage 54 with T3 for 0–6 days to induce metamorphosis. One hour prior to being sacrificed, the tadpoles were injected with 5-ethynyl-2′-deoxyuridine (EdU) to label the newly synthesized cellular DNA. The intestinal cross-sections from the resulting tadpoles were double-stained with MGPY and for EdU. As shown in Fig. 1, in premetamorphic intestine, the epithelium was uniformly stained with MGPY and some of the epithelial cells were EdU positive (Fig. 1A, a″). T3 treatment for 3 days had little effect on either intestinal morphology or staining (Fig. 1B, b″). However, after 6 days of T3 treatment, clusters of cells in the epithelium appeared and were more strongly stained by MGPY than the surrounding cells (Fig. 1C). Furthermore, these clusters were labeled by EdU (Fig. 1C, c″), indicating that the cell clusters strongly stained by MGPY are indeed the proliferating cell clusters.Fig. 1

Bottom Line: Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis.Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5.Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

View Article: PubMed Central - PubMed

Affiliation: Section on Molecular Morphogenesis, Program in Cellular Regulation and Metabolism (PCRM), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 18 Library Dr., Bethesda, MD 20892 USA.

ABSTRACT

Background: The thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells.

Results: Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis.

Conclusions: Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

No MeSH data available.


Related in: MedlinePlus