Figure 5.

Inversin and Nephrocystin-3 Inhibit Dishevelled-1-Induced Canonical Wnt-Signaling Activity

(A) Both inversin and NPHP3 inhibit Dvl1-induced activation of a TCF/LEF-1-dependent luciferase reporter construct (TOPFlash) in HEK293T cells. A combined expression of both NPHP proteins leads to a further suppression of the luciferase activity. A luciferase reporter construct with mutated TCF/LEF-1 binding sites (FOPFlash) showed no significant background stimulation. At least four independent experiments were done, each in triplicate. Dishevelled-induced stimulation was always above 20-fold over background. Data was normalized to β-galactosidase expression. p values were calculated with the two-tailed Student's t test.

(B–E) Morphogenetic cell movements during gastrulation and neurulation are defective upon NPHP3 knockdown in Xenopus laevis embryos and suggest a role in the PCP Wnt pathway. Gastrulation movements are delayed after NPHP3 knockdown by Morpholino-antisense oligonucleotide injected into two dorsal blastomeres (B). A clone of cells was labeled in the 64-cell stage with fluorescent dextrane, and the cell movements were followed by fluorescent microscopy at stage 12. Upper and lower panels show bright-field and fluorescent images of identical embryos at gastrulation. Incomplete closure of neural folds (C) and phenotypes of shortened body axis and dorsal bending (D) were observed at later stages, suggestive of disrupted PCP/Wnt signaling. This phenotype was dose-dependent and could be partially rescued by coinjection of NPHP3-RNA (E). The phenotypic changes were scored with the convergent extension index (CEI).