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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.
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 Steinbergâs solution (SS). It was fixed in Bouinâs 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 10â4 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 (RTâ) is a negative control. Lane 1, whole embryo
(stage 41); lane 2, untreated dorsal lip explants; lane 3, dorsal
lip explants treated with 10â4 M retinoic acid.
