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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. |
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Fig. 7. EnR-LefδN-GR755A generates reduced expression of pronephric mesenchyme markers. EnR-LefδN-GR755A (0.5 ng) with rhodamine dextran tracer was injected into single V2 blastomeres at the eight-cell stage and dexamethasone was added at stage 16. Embryos were fixed at stage 23 (A and B), stage 25 (C and D), and stage 27 (E and F). The injected side of embryos shows reduced expression of pronephric mesenchyme markers XPax8 (pax8) (A), XLim1 (lhx1) (C), and vHNF1 (hnf1β) (E), shown via whole-mount colorimetric or fluorescent in situ. The control side of embryos shows expected normal distribution of pronephric mesenchyme markers (B, D, F). Injected and control side images shown are from the same embryo and insets are enlargements of the pronephric region. |
