Depletion of sept7b reduces the length of cilia in the pronephric tubule and Kupffer′s vesicle, and causes misorientation of basal bodies and cilia. (A) Acetylated tubulin (red) stains cilia in 30-hpf wt1b::GFP embryos. (B) Staining of 30-hpf sept7b-TBMO morphants for acetylated tubulin (red) reveals shortened cilia, indicating that sept7b is essential for ciliogenesis. (C) The length of pronephric cilia is reduced in sept7b morphants (TBMO). Ctrl, control. (D,E) Kupffer′s vesicle of control (D) and sept7b morphant (E) embryos at 12–13 hpf that have been stained for acetylated tubulin to visualize cilia. (F) The length of cilia of Kupffer′s vesicle is reduced in sept7b morphants compared with controls. (G) Immunostaining of cilia for acetylated α-tubulin (red) and of basal bodies for γ-tubulin (green) in the multi-ciliated region of the pronephric tubule at 30 hpf in control embryos shows well-organized basal bodies that localized at the base of cilia in the tubular epithelial cells. (H) In sept7b-TBMO-injected embryos, the basal bodies are disorganized (arrowheads), the tubule diameter is increased (asterisk) and cilia appear misoriented. (I) Electron microscopy reveals apically docked basal bodies with outgrowths of ciliary axonemes in 4-dpf control larvae. (J) In sept7b-TBMO-injected larvae, the basal bodies docked at the apical cell membrane but appeared disorganized (indicated by the lines showing the orientation of the basal bodies). (K,L) The ciliary angle in control (20.9°; K) and sept7b-TBMO-injected larvae (41.1°; L) differed substantially, indicating that cilia are misoriented in sept7b-depleted larvae. The lines are drawn through the central pairs of the microtubules to indicate the orientation of the cilia. Scale bars: 20μm (A,B,D,E,G,H); 1μm (I–L).
|
