Ishizuya-Oka A et al. (1998), Thyroid hormone-induced apoptosis of larval cel...

XB-ART-14568

Differentiation 1998 Jun 01;632:59-68.

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Thyroid hormone-induced apoptosis of larval cells and differentiation of pepsinogen-producing cells in the stomach of Xenopus laevis in vitro.

Ishizuya-Oka A , Inokuchi T , Ueda S .

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It is generally known that the anuran stomach begins to express pepsinogens (Pg) during metamorphosis. To clarify the mechanisms of differentiation of Pg-producing cells, we examined immunohistochemically the epithelial transformation from larval to adult form in Xenopus laevis stomach at the cellular level. At the beginning of metamorphic climax, concomitantly with the modification of the basement membrane, apoptotic cells labelled by TUNEL suddenly increased in number in the entire epithelium except for the primordia of adult epithelial cells in the basal region of larval glands. Subsequently, with the development of connective tissue, the adult epithelial cells actively proliferated and replaced the larval cells from the basal to the luminal region. Following the start of morphogenesis of adult glands, Pg-producing cells became differentiated in newly formed adult glands, but not in the adult surface epithelium. We then developed an organ culture system and examined effects of thyroid hormone (TH) on the differentiation of Pg-producing cells in X. laevis stomach in vitro. In the presence of TH, just as in spontaneous metamorphosis, Pg-producing cells differentiated from the adult epithelial primordia after the apoptosis of larval epithelial cells. In contrast, in the absence of TH, neither apoptotic larval cells no Pg-producing cells were detected. Therefore, we conclude that TH triggers organ-autonomously the entire process leading to the differentiation of Pg-producing cells in X. laevis stomach. In addition, the strict localization of Pg-producing cells in the adult glands both in vivo and in vitro suggests the correlation between the differentiation of Pg-producing cells and morphogenesis of the glands surrounded by the developed connective tissue.

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Species referenced: Xenopus laevis
Genes referenced: mmp11 pgc pgcl
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	Fig. 1 Immunoblotting of crude extract of the Xenopus laevis stomach. Pepsinogen (Pg) immunoreactivity was detected at stage 66 (lane 2), but not at stage 56 (lane 1). The amount of protein applied to each lane was 10 μg. The molecular weight markers are indicated on the left.:
	Fig. 2A–D Electron micrographs of the stomach at stage 51 (A, B) and at stage 61 (C, D). A The larval surface (SE) and the glandular epithelia (GE). Arrowheads indicate the boundary between them. BC Blood capillaries, L lumen. B The thin basal lamina (arrowheads) just beneath the larval glands. mit Mitochondria, CT connective tissue. C Macrophage-like cells (Mφ) in the larval epithelium (LE). One includes an apoptotic body (AB). D Vigorous folding of the basal lamina (Bl) just beneath the degenerating larval epithelium. Bars 1 μm.:
	Fig. 3A–C Immunohistochemical detection of Pg in the stomach. A At stage 57. Both the larval surface (SE) and the glandular epithelia (GE) are negative. L Lumen. B At stage 62. Positive cells are detected only in developing adult glands (arrows). CT Connective tissue. C At stage 66. The glandular epithelium is positive, while the surface epithelium remains negative. Bars 20 μm.:
	Fig. 4A–E Apoptosis labelled by nick-end labelling of fragmented DNA (TUNEL) in the stomach. A At stage 57. Both the larval surface (SE) and the glandular epithelia (GE) are negative. L Lumen. B At stage 60. Numerous apoptotic nuclei (arrows) are detected in the entire epithelium except for small adult primordia (arrowheads). C Upper region of the epithelium at stage 61. Apoptotic nuclei (arrows) are localized in the larval epithelium (LE). AE Adult epithelium. D Nuclear fragments of apoptotic bodies (arrows) in the larval epithelium at stage 61. E At stage 62. The adult epithelium is negative. Bars 20 μm.:
	Fig. 5A–F Development of the adult epithelium in the stomach stained with methyl green-pyronin Y (A, C, E) and with hematoxylin and eosin (H-E) (B, D) and hematoxylin and periodic acid-Schiff (PAS) (F) after the colcemid treatment. A, B At stage 60. Adult epithelial primordia (arrowheads) appear in the basal region of larval glands (GE) (A), where mitotic cells (arrows) are localized (B). The dotted line indicates the boundary between the larval and the adult epithelia (AE). L Lumen, SE surface epithelium. C, D At stage 61. The larval epithelium (LE) is replaced by the adult epithelium, where mitotic cells (arrows) are localized (D). The connective tissue (CT) develops. E At stage 62. The adult epithelium is forming glands. The muscularis mucosae (Mm) develops between the lamina propria (Lp) and the submucosa (S). F At stage 66. Mitotic cells (arrows) are localized in the neck region of adult glands just above PAS-positive mucous neck cells (MN). Bars 20 μm.:
	Fig. 6A–C Immunohistochemical detection of ST3 in the stomach at stage 56 (A), stage 60 (B), and stage 66 (C). Numerous positive cells (arrows) are observed in the connective tissue (CT) at stage 60 (B), but not at the other stages. L Lumen, SE surface epithelium, GE glandular epithelium, Lp lamina propria, Mm muscularis mucosae, S submucosa. Bars 20 μm.:
	Fig. 7 Schematic illustration of the stomach remodelling of X. laevis. Around stage 60 both the larval surface (L-SE) and the glandular epithelia (L-GE) undergo apoptosis, while the adult epithelium actively proliferates. The basal lamina (Bl) becomes thick, and the connective tissue (CT) develops. By stage 63 the adult epithelium becomes differentiated into the surface (A-SE) and the glandular epithelia (A-GE) expressing Pg. Lp Lamina propria, Mm muscularis mucosae, S submucosa, ML muscular layer.: