Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways

Abstract

Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.

Figure 1

Inversin inhibits canonical Wnt signaling. (a) Expression of inversin reduced endogenous levels of cytoplasmic, but not membrane-bound, β-catenin in HEK 293T cells transiently transfected with Dvl1 and either inversin or a control plasmid (CD2AP). Western blots were reprobed for γ-tubulin as a loading control. (b) Inversin blocked Dvl1-induced, but not β-catenin-induced, activation of a TCF/LEF-1–dependent luciferase reporter construct (TOPFLASH) in HEK 293T cells. Experiments were done in triplicate, and data were normalized for β-galactosidase activity. (c) Inversin inhibited secondary body axes in X. laevis embryos. Embryos were injected with RNA (Inv, inversin) into one ventral blastomere and scored at tadpole stage. The Dsh-induced secondary axes (arrows) were inhibited in the presence of inversin. Scale bars, 1 mm. See also Table 1.

Figure 2

Inversin interacts and colocalizes with Dvl1. (a) Mouse Dvl1-HA was coexpressed with FLAG-tagged inversin (FLAG-inv) or a control protein (FLAG-CD2AP) in HEK 293T cells. After immunoprecipitation with anti-FLAG, Dvl1-HA was present in immunoprecipitates formed by inversin but not by CD2AP. Equal expression of Dvl1-HA in cellular lysates was confirmed by immunoblotting with antibody to HA (top). In the reverse experiment, FLAG-inv coprecipitated with Dvl1-HA but not with the control protein HA-Akt (middle). To demonstrate endogenous interactions, MDCK cell lysates were immunoprecipitated with antiserum to either HA (control) or Dvl2. Immobilized inversin was detected with an inversin-specific antiserum (bottom). (b) The C-terminal domain of inversin interacted with a GST fusion protein in vitro, containing the basic region and the PDZ domain of Dsh. ³⁵S-methionine–labeled full-length inversin (left), N-terminal inversin (amino acids 1–553; middle) or C-terminal inversin (amino acids 554–1,062; right) was incubated with GST (middle lane) or a GST fusion protein containing the basic region and PDZ domain of Dsh (amino acids 152–394; right lane). The left lane of each panel shows 10% of the input material of the in vitro–translated inversin. Full-length inversin and C-terminal inversin bound to the GST-Dsh fusion protein, whereas N-terminal inversin did not interact with Dsh. Equal amounts of the GST proteins were confirmed by Coomassie staining. (c) MDCK cells overexpressing Dsh-GFP were colabeled with a rabbit polyclonal antiserum to inversin at subconfluent (upper row) and confluent stages (lower row). Both inversin and Dsh-GFP translocate to the lateral plasma membrane during differentiation of MDCK cells.

Figure 3

Inversin facilitates the degradation of Dvl1. (a) Levels of both transiently expressed (top left) and endogenous Dvl1 (middle) were reduced in HEK 293T cells expressing inversin. The amount of transfected FLAG-tagged inversin (top left) was 0, 0.1, 1 and 5 µg in lanes 1–4, respectively. The half-life of Dvl1 decreased in the presence of inversin (lower left). Twenty hours after transfection, 40 µg ml⁻¹ of cycloheximide (Cx) was added for 0, 4 or 8 h to HEK 293T cells to block protein synthesis; levels of Dvl1 were monitored by western-blot analysis. The reduction of Dvl1 levels in cells expressing inversin was blocked by the proteasome inhibitor ALLN (upper right). Increased ubiquitination of Dvl1 in the presence of inversin in Wnt3a-treated HEK 293T cells (lower right). (b) The mutation L493S destroys D-box 1 of inversin. The mutant protein was expressed (middle) but did not block Dvl1-induced activation of the TOPFLASH promoter construct or reduce Dvl1 levels. (c) Inversin targets cytoplasmic Dvl1 for degradation. HEK 293T cells were transfected with Dvl1-HA and either FLAG-inversin or a control plasmid (FLAG-CD2AP; upper right). Western-blot analysis of the cytoplasmic and membrane fractions showed that only cytoplasmic Dvl1 was targeted by inversin. Equal protein loading was confirmed by γ-tubulin. Wild-type Dvl1 (Dvl1-HA) assumes a vesicular cytoplasmic staining pattern in transiently transfected HeLa cells, whereas Dvl1 containing a myristylation-palmitoylation consensus site (M/P-Dvl1-HA) assumes a diffuse cytoplasmic and plasma membrane localization (left). Inversin reduced wild-type Dvl1 (lower right) but had only a modest effect on Dvl1 containing a myristylation-palmitoylation consensus site. Reprobing with γ-tubulin (lower right) confirmed comparable protein loading.

Figure 4

Inversin is required for convergent extension movements in X. laevis embryos. (a) Knock-down of inversin by inversin morpholino antisense oligonucleotides (inv-MO) or overexpression of mouse inversin (mInv) impaired convergent extension movements of dorsal-mesodermal derived tissues during axis extension. Upper, reduced axis extension and dorsal flexure of embryos injected with 16 ng of inv-MO; these changes were rescued by mouse inversin RNA. Scale bars, 1 mm. Results are summarized in the lower panel (number of experiments per group is in parenthesis). The defects in convergent extension movements were scored as mild (ICE 1), moderate (ICE 2) or severe (ICE 3). (b) The dorsal blastomers of four-cell–stage embryos were injected with inv-MO and the lineage tracer β-galactosidase; embryos at late neurula stage were assayed for β-galactosidase activity in situ. Cells that inherited the inv-MO failed to converge in the midline and did not extend sufficiently in an anterior-posterior direction during neurulation. (c) inv-MO blocked elongation of activin-induced animal cap explants. Inhibition of activin (Act)-induced elongation by inv-MO was completely reversed by coinjection of mouse inversin RNA (mInv) not targeted by the morpholino oligonucleotide. Right, summary of the data; the numbers of animal caps of four independent experiments are shown above the bars.

Figure 5

Diversin rescues the renal cysts caused by inversin knockdown in zebrafish. (a) Embryos injected with a control morpholino oligonucleotide (MO) had normal morphology and development (173 of 176 injected embryos). (b,c) Histological sections of the pronephric kidneys at 4 d.p.f. showed the midline glomerulus (Glm) and pronephric tubule (Pt) of control animals (b), whereas embryos injected with invs-MO had ventral axis curvature (258 of 272 injected embryos; 95%; c). (d) The cystic glomerulus in invs-MO–injected 4-d.p.f. embryos had a flattened septum (asterisk) at the midline and a fluid-filled cyst lined with completely flattened epithelia. (e) 50 pg of mouse diversin mRNA alone had no marked effect on zebrafish development (282 of 312 injected embryos), whereas higher amounts resulted in progressive ventralization (ref. 15 and data not shown). (f) Histological section of 4-d.p.f. embryos injected with mouse diversin mRNA showed a normal pronephric kidney. (g–i) Molecular analysis of morpholino-targeted invs splicing defects. RT-PCR analysis of invs expression in 4-d.p.f. embryos injected with control morpholino generated a 746-bp invs fragment encoding the C-terminal domain of inversin (g; lane C, nucleotides 2,233–2,979; lane M, 1 kb plus markers, Invitrogen). Embryos injected with invs-MO (g; lane invs-MO; 4 d.p.f.) or invs-MO plus mouse diversin mRNA (g; lane invs-MO + diversin; 4 d.p.f.) analyzed with the same RT-PCR primers generated a 189-bp RT-PCR product, representing a C-terminal invs deletion allele. Some recovery of wild-type mRNA was observed at 4 d.p.f. (h) RT-PCR of mouse diversin mRNA in the same RNA samples as in g showed mouse diversin gene expression in animals coinjected with invs-MO and 50 pg of mouse diversin mRNA. (i) RT-PCR of β-actin mRNA in the same RNA samples as in g. (j) Coinjection of 50 pg mouse diversin mRNA with invs-MO rescued the cystic defects caused by inversin knockdown (261 of 284; 92%) but not the ventral axis curvature. (k) Histology section of 4-d.p.f. zebrafish, coinjected with invs-MO and mouse diversin mRNA, showed that diversin ameliorated cyst formation and partially rescued the glomerulus (Glm) and pronephric tubule structure (Pt). The fluid-filled cyst of the pronephric tubules is marked with an asterisk. (l–o) Knock-down of invs enhances canonical β-catenin signaling in vivo. (l,m) The expression of the Wnt target gene boz in control zebrafish embryos at 60% epiboly stage is restricted to the dorsal margin of the yolk syncytial layer. (n,o) Invs MO knockdown induces ectopic boz expression in the entire zebrafish yolk syncytial layer. (p) Inversin accumulates in response to flow. Using antiserum to inversin, the expression of inversin was determined by western blotting after 2 h of flow or without flow. Representative blots including the loading control 14-3-3 are shown. The mean value ± s.d. of seven independent experiments was determined using densitometry. Flow slightly decreased cytoplasmic β-catenin levels. Control cells were not exposed to flow. Both changes were statistically significant (P < 0.05).