Xenopus Nphp4 is essential for building functional cilia. (A and B) Confocal microscopy of multiciliated cells of Xenopus epidermal skin. Cilia and apical cell surface are labeled with anti–acetylated tubulin (Ac-tub; red in merge) and membrane-targeted GFP (mGFP; green in merge). (A) Maximum intensity projection (top), and 3D reconstruction projected in the x-z plane (lateral) for the confocal datasets are shown. The apical surface is indicated by a solid white line. Embryos injected with 20 ng control morpholino (ctl MO) showed normal ciliogenesis, whereas 20 ng nphp4 ATG MO–injected embryos showed ciliogenesis defects. (B) Single optical sections comparing the signals for anti–acetylated tubulin at levels 1 and 2 in ctl MO– and nphp4 ATG MO–treated Xenopus embryos (see dashed white lines in the lateral views of A) revealed that ciliary microtubules are nucleated below the apical cell surface in nphp4-deficient, but not ctl MO–treated, Xenopus embryos. (C) Scanning EM of the Xenopus epidermis confirmed the ciliogenesis defect in 16 ng nphp4 ATG MO– and 8 ng nphp4 SB MO–injected Xenopus embryos. (D) Labeling of basal bodies and the apical cell surface with RFP-Centrin and mGFP revealed that basal bodies are aligned along the apical cell surface and distributed uniformly across the cell surface in ctl MO–injected Xenopus embryos. In contrast, basal bodies in nphp4 MO–injected Xenopus embryos failed to migrate to the apical cell surface and remained within the cytoplasm. (Top) Maximum intensity projection of confocal datasets. (Bottom) Serial confocal images projected in the x-z plane with the apical cell surface indicated by a dashed white line. (E) The multiciliated cells of the Xenopus epidermis formed a dense actin cytoskeleton (phalloidin, green) at the apical cortex. Apically localized basal bodies (RFP-Centrin, red) were embedded in this actin web (left). In nphp4-deficient Xenopus embryos, basal bodies remained within the cytoplasm below the apical actin web, which is also thinner than in control cells (right). Confocal 3D datasets were processed with Imaris software, and the lateral (top), top (middle), and cytoplasmic (bottom) views are shown. (F) Depletion of nphp4 resulted in a decreased rate of cilia-driven fluid flow as revealed by the movement of polypropylene beads across the epidermis. Shown is a summary of four independent experiments (n ≥ 25). Error bars, SEM; t test; *, P < 0.002. (G) nphp4-deficient Xenopus embryos showed a mild perturbation of basal body polarization, revealed by the relative position of the basal bodies (RFP-Centrin) and the ciliary rootlets (GFP-Clamp). A moderate dose of 6.8 ng nphp4 SB MO was injected to allow apical docking of basal bodies, a prerequisite for polarization. The right panels show the magnified images for the regions enclosed by white boxes. Only a small number of basal bodies were incorrectly polarized (asterisk) in nphp4-deficient cells. The polarization was quantified by angular measurements of Clamp/Centrin pairs. Depletion of nphp4 resulted in a moderate increase of circular standard deviation as compared with the control. Error bars, SEM. (H) Transmission electron microscopy detected occasional ciliary axonemes (white arrowhead, box 1), and a large number of basal bodies (black arrowheads, box 2) in the cytoplasm of nphp4-depleted Xenopus multiciliated cells. Bars: (A–E) 5 µm; (G) 1 µm; (H) 200 nm.