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Dev Growth Differ
2002 Apr 01;442:161-7. doi: 10.1046/j.1440-169x.2002.00631.x.
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In vitro induction of the pronephric duct in Xenopus explants.
Osafune K
,
Nishinakamura R
,
Komazaki S
,
Asashima M
.
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The earliest form of embryonic kidney, the pronephros, consists of three components: glomus, tubule and duct. Treatment of the undifferentiated animal pole ectoderm of Xenopus laevis with activin A and retinoic acid (RA) induces formation of the pronephric tubule and glomus. In this study, the rate of induction of the pronephric duct, the third component of the pronephros, was investigated in animal caps treated with activin A and RA. Immunohistochemistry using pronephric duct-specific antibody 4A6 revealed that a high proportion of the treated explants contained 4A6-positive tubular structures. Electron microscopy showed that the tubules in the explants were similar to the pronephric ducts of normal larvae, and they also expressed Gremlin and c-ret, molecular markers for pronephric ducts. These results suggest that the treatment of Xenopus ectoderm with activin A and RA induces a high rate of differentiation of pronephric ducts, in addition to the differentiation of the pronephric tubule and glomus, and that this in vitro system can serve as a simple and effective model for analysis of the mechanism of pronephros differentiation.
Fig. 1. Double immunostaining of a stage 42 equivalent Xenopus explant (A,B) and stage 40 larvae (C,D) using pronephric
duct-specific antibody 4A6 (blue) and pronephric tubule-specific antibody 3G8 (red). Whole-mount immunostaining patterns (B,D)
and photomicrographs of sections (A,C) are shown. (A,B) Explants treated with activin A and retinoic acid contain both 3G8-positive
(pronephric tubule, arrows) and 4A6-positive tubular structures (pronephric duct, arrowheads). Bar, 1 mm.
Fig. 2. Electron micrographs of the pronephros of a stage 42tadpole and Xenopus explants treated with activin A and retinoic acid.
Three types of tubular structure are observed in the explants (A–C). (A) Cuboidal cells with scant microvilli on their luminal surface
(arrow) resemble the pronephric duct cells of normal larvae (D). (B) Tubular cells with abundant microvilli on their luminal surface
(arrow) resemble the pronephric tubule cells of normal larvae (E). (C) Cells with cilia consisting of microfilaments (arrow) correspond
to the cells of normal larvae located in the intermediate segment connecting the proximal and distal tubules or in the neck segment
between the pronephric glomus and proximal tubule (F).
Fig. 3. Whole-mount in situ hybridization of Gremlin and c-ret in treated Xenopus explants cultured until stage 32 equivalence.
Expression of Gremlin and c-ret mRNA is seen in explants treated with activin A and retinoic acid (AâE). (A) Antisense Gremlin probe;
(B) sense Gremlin probe; (C) Antisense c-ret probe; (D) sense c-ret probe; (E) higher magnification of (C). Gremlin antisense (F) and
c-ret antisense (G) patterns in stage 32 larvae (arrow) are also shown.
