Fig. 6

Kif7 modulates Gli2a localization and its association with Sufu.

(A) Western blot analysis of anti-Gli2a immune-precipitates of WT and MZkif7 (MZ) embryos, showing association of Kif7 with Gli2a in wild-type embryos and increased association of Sufu with Gli2a in MZkif7 embryos, as indicated by the ratio of the normalized intensities of the MZ to WT signals. (B) Western blot analysis of anti-Sufu immuno-precipitates of WT, Shh RNA injected (Shh), cyclopamine (cycA) exposed and MZkif7 (MZ) embryos; the Gli2aFL:Sufu ratios normalized to wild-type are shown below each lane. Error bars represent standard deviation obtained from three independent biological replicates. Note the increase in levels of Gli2aFL that co-precipitates with Sufu in MZkif7 embryos. (C,D) Localization of a functional eGFP tagged Gli2a protein (green) to primary cilia in wild-type (C) and MZkif7 (D) embryos. The axonemes of the primary cilia are marked by acetylated α-tubulin (red) and the basal bodies by γ-tubulin (blue) staining. In wild-type, Gli2a localizes to the tip of the cilia whereas in MZkif7, localization is restricted to the base of the cilia; the lower two panels in (D) are the same images as in the upper panels but with the red channel removed to show the juxtaposed GFP-Gli2a and γ-tubulin signals more clearly. Scale bar: 5 μm. (E) Western blot of wild-type (WT) and sufu morpholino-injected (sufu MO) embryo extracts probed with anti-Sufu and anti γ-tubulin, showing significant depletion of Sufu levels in the morphants relative to wild-type (MO/WT). (F–K) Parasagittal optical sections of 30 hpf wild-type (WT) or MZkif7 embryos showing the effect on eng2a:eGFP expression of morpholino mediated knockdown of gli1 (H,I) or gli1 and sufu (J,K). Depletion of gli1 in WT embryos (H) has no discernible effect, whereas it causes a drastic suppression of the ectopic expression in MZkif7 (I). This suppression is abrogated by simultaneous removal of Sufu and Gli1 from MZkif7 embryos (K). Scale bar: 50 μm.