Correlation between activin and retinoic acid (RA), both of which affect early amphibian development, was studied using Xenopus laevis embryos. In the first set of experiments, two isomers of RA, all‐trans RA and 13‐cis RA, were compared in terms of stability of biological activity against light. Xenopus blastulae were dipped in RA solutions which had either been kept away from light, or had been exposed to light for a few hours. At doses ranging from 10–4to 10–6M, RA elicited head deformity. All‐trans RA, under both dark and light conditions, had similarly potent effects. On the other hand, 13‐cis RA under dark conditions had much weaker effects than it did under light conditions. In the second set of experiments, activin was mixed with all‐trans RA, and the inducing effects on the animal cap explants were investigated. Activin at a concentration of 10 ng/ml induced notochord. In combination with 10–6M RA, muscle was well induced instead of notochord. In combination with 10–5M RA, pronephric tubules were markedly induced. Pronephric tubules were never induced by activin alone, at any of the various concentrations employed. This is the first report on the very high frequency of induction of pronephric tubules by the combination of activin A and all‐trans RA in the Xenopus ectoderm.
In the present study, isolated presumptive ectoderm from Xenopus blastula was treated with activin and retinoic acid to induce differentiation into pancreas. The presumptive ectoderm region of the blastula consists of undifferentiated cells and is fated to become epidermis and neural tissue in normal development. When the region is isolated and cultured in vitro, it develops into atypical epidermis. Isolated presumptive ectoderm was treated with activin and retinoic acid. The ectoderm frequently differentiated into pancreas-like structures accompanied by an intestinal epithelium-like structure. Sections of the explants viewed using light and electron microscopy showed some cells clustered and forming an acinus-like structure, including secretory granules. The pancreas-specific molecular markers insulin and XIHbox8 were also expressed in the treated explants. The pancreatic hormones, insulin and glucagon, were detected in the explants using immunohistochemistry. Therefore, sequential treatment with activin and retinoic acid can induce presumptive ectoderm to differentiate into a morphological and functional pancreas in vitro. When ectoderm was immediately treated with retinoic acid after treatment with activin, well-differentiated pronephric tubules were seen in a few of the differentiated pancreases. Treatment with retinoic acid 3-5 h after activin treatment induced frequent pancreatic differentiation. When the time lag was longer than 15h, the explants developed into axial mesoderm and pharynx. The present study provides an effective system for analyzing pancreas differentiation in vertebrate development.
Human recombinant activin A, which is identical with erythroid differentiation factor (EDF), was tested for its mesoderm-inducing activity in concentrations from 0.3-50 ng/ml, using ectoderm of Xenopus late blastula (Stage 9) as the responding tissue. At a low concentration of activin A, blood-like cells, mesenchyme, and coelomic epithelium were induced; at a moderate concentration muscle and neural tissue, and at a high concentration notochord. Activin A thus induced all mesodermal tissues in a dose-dependent manner, such that a low dose induced ventral structures and a high dose induced dorsal structures. Activin may act as an intrinsic inducing molecule responsible for establishing the dorso-ventral axis in early Xenopus development.
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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