XB-IMG-26027
Xenbase Image ID: 26027
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Fig. 9. Characterization and pronephric phenotypic analysis of LefδN-βCTA-GR755A, an inducible fusion construct promoting Wnt/β-catenin signaling. (A) LefδN-βCTA-GR755A schematic including the mouse Lef1 DNA-binding HMG box (aa 265–391), a double HA epitope-tag, the mouse β-catenin transactivation domain, and the human Glucocorticoid receptor hormone-binding domain (aa 512–777). Xenopus embryos were injected with 0.1 ng LefδN-βCTA-GR755A into a single ventral-vegetal blastomere at the four-cell stage and grown to stages 33–35 with or without dexamethasone. In the presence of dexamethasone, embryos showed a robust level of secondary axis phenotypes (B), while embryos not treated with dexamethasone developed normally (C). Fluorescent (FITC) immunohistochemistry of animal caps excised from stage 9/10 embryos show nuclear localization of LefδN-βCTA-GR755A in the presence of dexamethasone (D) and cytoplasmic localization in the absence of dexamethasone (E) (prior to animal cap excision, embryos were injected with 1.0 ng LefδN-βCTA-GR755A into single animal-dorsal blastomeres at the 2-cell stage). (F) Most all embryos noted in B (presence of dexamethasone) exhibited one or more features reflecting an ectopic dorsal axis, while none of those noted in C (absence of dexamethasone) exhibited duplicate axes. (G) To assess LefδN-βCTA-GR755A protein stability following dexamethasone addition, a time course was undertaken of embryos injected with 0.5 ng LefδN-βCTA-GR755A into single vegetal-ventral (V2) blastomeres at the eight-cell stage. HA epitope-tag Western blotting shows the inducible chimera (like that of EnR-LefδN-GR755A and EnR-GR) is stably present until addition of dexamethasone at stage 16, at which time each experiences increased metabolic turnover. (H) Xenopus embryos injected with 0.1 ng LefδN-βCTA-GR755A into single vegetal-ventral (V2) blastomeres at the eight-cell stage (+dexamethasone at stage 16) showed decreased expression of an early pronephric tubule epithelial marker hnf1β (in situ at stage 26), and variable decreased expression of a more mature tubule epithelial marker 3G8 (immunostaining at stage 35). The left panels show whole tadpoles along with enlargements of observed phenotypes on injected sides, while right panels depict uninjected/control sides with corresponding enlargements. Image published in: Lyons JP et al. (2009) Copyright © 2009. Image reproduced with permission of the Publisher, Elsevier B. V.
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