Izutsu Y et al. (2000), Loss of reactivity to pan-cadherin antibody in ...

XB-ART-10390

Dev Growth Differ 2000 Aug 01;424:377-83. doi: 10.1046/j.1440-169x.2000.00527.x.

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Loss of reactivity to pan-cadherin antibody in epidermal cells as a marker for metamorphic alteration of Xenopus skin.

Izutsu Y , Tochinai S , Onoé K .

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Pan-cadherin antibodies recognize the conserved C-terminal region of the family of cell-cell adhesion molecules, cadherins, and have a broad spectrum of reactivity to the molecules. In the present study, by immunohistochemistry using an anti-pan cadherin monoclonal antibody (mAb), expression dynamics of cadherins in epidermal tissues were analyzed during metamorphosis of Xenopus laevis. At early stages of development, the anti-pan cadherin mAb detected signals at cell-cell boundaries and in the cytoplasm of both trunk and tail epidermal cells. During metamorphosis, the immunoreactivity decreased in the trunk skin tissue but remained in the tail. At the climax stage, immunoreactivity was observed only in the regressing tail epidermis. The signals disappeared completely from the trunk epidermis, which had already transformed into adult-type tissue. This observation was confirmed by western blot analysis. A specific band was detected in the larval skin, but not in the adult lysate, at approximately 135 kDa in molecular size, corresponding to the molecular mass of cadherins. This different immunoreactivity in larvae and adults was observed in the epidermis of the skin, but not in any other tissues examined, that is, brain, kidney and liver. The immunoreactivity seen in larval epidermal cells was drastically downregulated by thyroid hormone treatment in vitro. These changes of immunoreactivity were specific for the C-terminal region of cadherins, suggesting intracellular alteration of the molecules during metamorphosis, and the anti-pan cadherin mAb can be a marker for larval-type epidermal cells that is applicable to analysis of Xenopus metamorphosis.

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	Fig. 1. Developmental changes in the expression of the antigen molecules detected by anti-pan cadherin mAb in the epidermis during metamorphosis. Unfixed frozen sections were subjected to immunohistochemical staining using anti-pan cadherin mAb (red signals) and were counterstained with quinacrine dihydrochloride (green). The thickness of the epidermis is shown by the white line on the right side of each panel. Left panels show the back epidermis of a stage 57 tadpole (A), a stage 59 tadpole (C) and a 2–3-month-old froglet (E). Right panels show the tail epidermis of stage 57 (B) and stage 59 (D). Letter G in panels C and E indicates the developing gland. Bar, 100 μm.
	Fig. 2. Immunohistochemistry of the border area between the trunk and tail epidermis at the metamorphic climax stage using the anti-pan cadherin mAb. Unfixed frozen sections obtained from a stage 64 tadpole were stained with the anti-pan cadherin mAb and counterstained with quinacrine dihydrochloride. The dotted line shows the locations of the basement membrane and the trunk and tail areas of the epidermis are also indicated in the figure. Letter G marks the developing gland, which is a characteristic feature of the adult-type skin. Bar, 100 μm.
	Fig. 3. Western blot analysis of larval and adult skin lysates detected with anti-pan cadherin mAb. Skin lysates were obtained from the whole skin of tadpoles at stage 55/56 (larva) and from the whole adult skin of 2–3-month-old froglets (adult). The lysates were subjected to 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis at a concentration of 50 μg/lane. Note that a strong band of approximately 135 kDa indicated by an arrow (a) is seen in the larval lane but only faintly in the adult. Larval specific bands at 53 and 59 kDa, and an adult specific band at 40 kDa are indicated by arrows (b), (c) and (d), respectively.
	Fig. 4. Disappearance of the molecules detected by anti-pan cadherin mAb in larval epidermal cells after in vitro culture in the presence of thyroid hormone (T3). Epidermal cells were obtained from the trunk skin of tadpoles at stage 55/56 and cultured in 96-well dishes (1.23105/well) at 28°C for 3 days in the absence (A) or presence (B) of 100 nM T3. The cells are stained with the antipan-cadherin mAb (red signals). Bar, 50 μm.