Proc Natl Acad Sci U S A
November 23, 2010;
Mutations of inversin
cause type II nephronophthisis, an infantile autosomal recessive disease characterized by cystic kidney
disease and developmental defects. Inversin
regulates Wnt signaling and is required for convergent extension movements during early embryogenesis. We now show that Inversin
is essential for Xenopus pronephros
formation, involving two distinct and opposing forms of cell movements. Knockdown of Inversin
abrogated both proximal pronephros
extension and distal
tubule differentiation, phenotypes similar to that of Xenopus deficient in Frizzled-8. Exogenous Inversin
rescued the pronephric defects caused by lack of Frizzled-8, indicating that Inversin
acts downstream of Frizzled-8 in pronephros
morphogenesis. Depletion of Inversin
prevents the recruitment of Dishevelled
in response to Frizzled-8 and impeded the accumulation of Dishevelled
at the apical membrane of tubular epithelial cells in vivo. Thus, defective tubule morphogenesis seems to contribute to the renal pathology observed in patients with nephronophthisis type II.
Proc Natl Acad Sci U S A
pronephric nephron tubule morphogenesis
Disease Ontology terms:
NEPHRONOPHTHISIS 2; NPHP2
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References [+] :
Fig. 1. Inversin is required for pronephric tubule development in X. laevis. (A and B) Bilateral injection of Invs-Mo at the four-cell stage resulted in severe edema. (C) The pronephros-specific antibodies 3G8 and 4A6 stain the entire pronephric tubule on the uninjected side. (D) On the Invs-Mo–injected side of the same embryo, staining is absent in the distal and intermediate tubule segment but is maintained in the proximal segment. (E and F) Enlarged view of the pronephros in C and D, respectively. (G) Percentages of embryos with unobstructed tubular excretion on both sides. (H) Fluorescent dextran (70 kDa) excretion shows tubule patency in Invs-Mo–injected embryos. (I–L) Enlarged views of the pronephros at the phase of maximal dextran excretion. Note the reduction in tubular coiling on the Invs-Mo–injected side (J) compared with the uninjected side (I). (K and L) Injection of control-Mo (CTL-Mo) did not interfere with tubule patency or length. (M) Tubule-area size was measured as depicted. d.v., dorso-ventral; a.p., anterior-posterior. (N) Ratios of injected vs. uninjected tubule areas of the same embryos.
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.
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).
An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.