Moriya N et al. (2000), In vitro organogenesis of pancreas in Xenopus l...

XB-ART-10929

Dev Growth Differ 2000 Apr 01;422:175-85. doi: 10.1046/j.1440-169x.2000.00498.x.

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In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid.

Moriya N , Komazaki S , Asashima M .

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Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10(-4) M retinoic acid for 3 h differentiated into pancreas-like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus-like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas-specific molecular markers, XIHbox8 and insulin, were induced in retinoic acid-treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas-like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.

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Species referenced: Xenopus laevis
Genes referenced: acin1 acta4 actc1 actl6a ins ncam1 pdx1

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	Fig. 1. Light micrographs of dorsal lip explants treated with or without retinoic acid. (A,B) The dorsal lip region was isolated from early gastrula (stage 10) and cultured for 10 days at 20°C in Steinberg’s solution (SS). It was fixed in Bouin’s solution, dehydrated, embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin. In the explants notochord, muscle mesenchyme and epithelium differentiated. (C,D) The dorsal lip region was isolated and then treated with retinoic acid for 10–4 M retinoic acid for 3 h. After washing they were cultured as described. In explants, notochord, an intestinal epithelium-like structure and a pancreaslike structure were differentiated. epi, epithelium; int, intestinal epithelium-like structure; mes, mesenchyme; mus, muscle; not, notochord; pan, pancreas-like structure; pha, pharyngeal epithelium-like structure. Bars, 100 μm.
	Fig. 2. Electron micrographs of the pancreas-like structure in explants. (A) An acinus-like structure formed by the cells with electron-dense granules (arrows). Lu, lumen of acinus-like structure. (B) An endocrine-like cell with small granules (arrows) in the cytoplasm. N, nucleus. (C) Large magnification of the granules shown in the endocrine-like cell. The granules were enveloped in membrane. Bar, 1 μm (A,B), 0.1 μm (C).
	Fig. 3. Expression of marker genes in dorsal lip explants treated with or without retinoic acid. Dorsal lip explants treated with or without retinoic acid were sampled after culture for 3 days at 20°C, sibling embryos were also sampled (stage 41) and they were subjected to the reverse transcription-polymerase chain reaction (RT-PCR) analysis. The expressions of neural cell adhesion molecule (NCAM), alpha skeletal muscle actin (muscle-specific actin, ms-actin), XlHbox8, insulin and EF-1a were examined using RT-PCR. EF-1a (RT+) is a loading control and EF-1a (RT–) is a negative control. Lane 1, whole embryo (stage 41); lane 2, untreated dorsal lip explants; lane 3, dorsal lip explants treated with 10–4 M retinoic acid.