Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo
Figure 10
Kif7 protein localization is modulated by Hh pathway activity.
(A–C) Parasagittal optical sections of the neural tube in 20ss embryos, showing the distribution of the endogenous Kif7 protein. In wild-type (A) and smo (B) embryos, Kif7 accumulates in puncta throughout the cytoplasm as well as in the primary cilium. In ptch1; ptch2 double mutant embryos (C) by contrast, the cytoplasmic puncta are completely absent with Kif7 remaining only at the tips of the cilia. Scale bar: 10 µm. (D–I) similar distributions of Kif7 are seen in the otic vesicle (D–F) and the myotome (G–I) of wild-type and mutant embryos. Insets show a magnified view of a part of each image; note that Kif7 accumulates at the tips of some primary cilia in wild-type (D,G), smo mutant (E,H) but at elevated levels in all cilia in ptch1;ptch2 double mutant (F,I) embryos. Scale bars: 10 µm; Inset scale bar: 1 µm. (J) Quantification of fluorescence intensity of Kif7 from the otic vesicle at 20ss from wild-type (WT), smo mutants and ptch1;2 double mutants, revealing a decrease in Kif7 levels detected by immunofluorescence. Error bars represent standard deviation in spot intensity in pre-processed confocal stacks. (K) Western blot analysis of endogenous Gli2a and Kif7 protein from wild-type (WT), cyclopamine exposed wild-type (WT), Shh RNA injected (Shh) and cyclopamine treated Shh RNA injected (Shh CycA) wild-type embryos exposed to cyclopamine. Note the increase in full-length Gli2a levels following Shh overexpression. Relative levels of Kif7 protein normalized to wild-type are indicated in (L).