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Fig. 3. Impaired pronephros morphogenesis caused by Inversin depletion is largely independent of proliferation or apoptosis. (A) Embryos were in situ hybridized against Na-K-ATPase (inverted differential interference contrast, green) and immunostained with an anti–phospho-Histone H3 (p-Histone H3) antibody (red) and DAPI (blue). The area of the proximal pronephros was magnified (Right). (B) The percentage of mitotic (p-Histone H3-positive) cells in the pronephros was determined in five sections of 10 embryos; no significant difference was detected (P = 0.36, Student t test). (C) Section of a camptothecin-treated embryo; camptothecin almost completely abrogates mitosis. The area of the proximal pronephros was magnified (Right). (D) Positive TUNEL staining is indicated by a black arrow. The area of the proximal pronephros was magnified (Right). (E) Immunostaining with anti–Caspase-3 (green), acetylated tubulin (red), and DAPI (blue). Note that both methods did not detect increased apoptosis on the Invs-Mo–injected side. (F and G) Mitotic inhibition does not prevent ventral extension defects of pronephric loops after Invs-Mo injection. (F) Embryos were treated at stage 31 with DMSO or camptothecin and processed for in situ hybridization against Na-K-ATPase at stage 36. Brackets mark the boundaries of ventral extension of the pronephric loop. (G) Quantification of ventral extension in millimeters. Error bars represent SD (*P < 0.001). The red arrow points to a reduction of the ventral pronephros extension in Invs-Mo–injected embryos that significantly exceeds the reduction caused by camptothecin treatment alone. |
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Fig. S2. Inversin depletion reduces ventral extension of the pronephric tubule. (A–J) In situ hybridization against Na-K-ATPase was performed at different
stages. The right column shows the Invs-Mo–injected side; the left column shows the noninjected side of the same embryo. (C and D) Enlarged view of the
boxed areas in A and B, respectively. (C, E, G, and I) The primary loop extends ventrally on the uninjected side. (D, F, H, and J) In Invs-Mo–injected sides,
extension of the ventral pronephros is reduced, leading to a limited elongation of the tubular convolute. (K–L) In situ hybridization against chloride channel
ClC-Ka was performed on continuous serial sections of E17.5 mouse kidneys (8 μm) to reconstruct ClC-Ka–positive nephron segments of wild-type and Invs (−/−)
kidneys. Black bars indicate 200 μm. (M) Quantification of the average volume per tubule in millimeters cubed (P = 0.057; n = 4 kidneys, t test). |
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Fig. S6. Dishevelled knockdown mimics the Inversin phenotype. (A and B) Overexpression of mInvs did not affect pronephros formation. (C and D) Combined
knockdown of Dishevelled-1 and -3 or expression of the dominant-negative Dishevelled mutant Xdd1 (E and F) resulted in a shortened tubule with impaired
ventral extension. (G) β-Catenin was detectable at the plasma membrane but not within the nucleus. (H–K) 3D tilted projections of the confocal stacks of G.
White arrows point to cilia stained for acetylated tubulin. |
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Fig. 4. Inversin acts downstream of Frizzled-8 in convergent extension movements and pronephros development. (A–D) Embryos were unilaterally injected with Fzd-8–Mo. In situ hybridization against Na-K-ATPase shows reduced ventral extension of the intermediate tubule. (C and D) Enlarged view of the pronephros region in A and B. (E) Dorsal injection of dominant-negative Xenopus Frizzled-8 encompassing the extracellular domain of Frizzled-8 (ECD8). Axis extension defects indicative of impaired convergent extension (ICE) were scored as indicated on a scale from 1 to 3. Coexpression of Inversin mRNA partially rescued the convergent extension defects. (F–N) Staining of the pronephros with the tubule-specific antibodies 3G8 and 4A6. (H–M) Unilateral injection of Fzd-8–Mo resulted in a reduction of 3G8 and 4A6 staining ranging from mild (I) to severe (K). (L–N) Coinjection of Inversin mRNA (1 ng) rescued the defective 3G8/4A6 immunoreactivity in Fzd-8–Mo-injected embryos (*P < 0.05). |
