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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). |
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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. |
