Lyons JP et al. (2009), Requirement of Wnt/beta-catenin signaling in pr...

XB-ART-38915

Mech Dev 2009 Jan 01;1263-4:142-59. doi: 10.1016/j.mod.2008.11.007.

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Requirement of Wnt/beta-catenin signaling in pronephric kidney development.

Lyons JP , Miller RK , Zhou X , Zhou X , Weidinger G , Deroo T , Denayer T , Park JI , Ji H , Hong JY , Li A , Moon RT , Jones EA , Vleminckx K , Vize PD , McCrea PD .

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The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/beta-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/beta-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/beta-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/beta-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/beta-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/beta-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.

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Species referenced: Xenopus laevis
Genes referenced: atp1a1 hnf1b lef1 lhx1 nphs1 pax8 slc12a1 slc4a4 tbk1 wnt4

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	Fig. 5. EnR-LefδN-GR755A inhibits expression of multiple pronephric glomus, epithelial tubule, and duct-specific genes. (A) Schematic showing pronephric glomus, tubule and duct segment nomenclature at stage 35. The glomus, labeled G, is shown in beige. The early and late segments within both the proximal and distal domains of tubules are shown in dark and light gray, respectively. The duct is shown in white. Injection of EnR-LefδN-GR755A (0.5 ng) with rhodamine dextran tracer was performed into single V2 blastomeres at the eight-cell stage and dexamethasone was added at stage 16. Embryos were fixed at stages 35/36 (B–C, E–F, H–I, K–L) and expression of several pronephric marker genes analyzed by whole-mount colorimetric or fluorescent in situ. Schematics represent the expression domains of four later stage pronephric markers (D, G, J, M). Shown are injected and control sides of embryos, each including insets of enlarged pronephric regions. Upon the inhibition of Wnt/β-catenin signaling, the strong late distal expression of the sodium bicarbonate cotransporter XNBC1 (slc4a4) (D) is lost on the injected (B) versus control side (C). Earlier weaker expression of this same marker is also lost (not shown). The early distal Na-K-2Cl cotransporter NKCC2 (slc12a1) (G) shows complete loss of expression on injected (E) versus control side (F). Expression of the NaK ATPase (atp1a1) in both the pronephric tubules and duct (J) is lost on the experimental side (H) as compared to the control side (I) when Wnt/β-catenin signaling is inhibited. Additionally, inhibition of Wnt/β-catenin signaling causes the normal expression of nephrin (nhps1) in the glomus (M) to be reduced in the injected side (K) as compared with the control side (L).
	Fig. 6. EnR-LefδN-GR755A reduces expression of NaK ATPase and nephrin in the pronephric region. Cross sections of embryos injected with EnR-LefδN-GR755A (left V2 blastomere at eight-cell stage), induced at stage 16, fixed at stage 35/36, and stained by in situ hybridization. (A) Fluorescent in situ hybridization of cross sections of stage 35/36 embryos shows that EnR-LefδN-GR755A inhibits NaKATPase (atp1a1) expression within the injected side (left) versus the control side (right). (B) In situ hybridization shows that expression of nephrin (nhps1), a glomus marker, is inhibited by EnR-LefδN-GR755A in injected (left) sides of stage 35/36 embryos as compared to control (right) sides.
	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.
	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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