daam1-deficient multiciliated cells recapitulate the ciliary phenotypes of nphp4-deficient cells. (A) GFP-tagged Daam1 (green in merge) preferentially localized to a region distal to the basal body (RFP-Centrin, red in merge) at the apical surface. Maximum intensity projection (top) or 3D reconstruction (bottom) of confocal images is shown. (B) Confocal imaging of cilia (Ac-tub, red). As compared with the control, reduced number of cilia formed above the apical cell surface (membrane-associated GFP [mGFP], green) after injection of 12 ng daam1 MO. The apical cell surface is depicted by dashed white lines on the right panels. (C) The ciliogenesis defect was confirmed by scanning electron microscopy. (D) daam1-deficient Xenopus embryos show a decreased rate of cilia-driven fluid flow across the epidermis (four independent experiments with n > 45; error bars, SEM; t test; *, P = 0.0014). Depletion of daam1 was also associated with a polarity defect and an increase in circular SD (t test; *, P < 0.001). (E) Basal bodies in daam1-deficient multiciliated cells failed to migrate to the apical cell surface and remained in the cytoplasm. Basal bodies and ciliary rootlets were labeled with RFP-Centrin and GFP-Clamp. Maximum intensity projection (top) or projection in the x-z plane (bottom) of serial confocal images are shown. The apical cell surface is indicated by dashed white line. (F) The subapical actin layer displayed irregularities in daam1–deficient multiciliated cells. Ciliary rootlets were labeled with GFP-Clamp (green in merge) and actin stained with phalloidin (red in merge). (Top) Maximum intensity projection of serial confocal images. Single optical sections at the level of apical and subapical actin layer of the boxed area are shown magnified in the middle and bottom panels (4× magnification). Bars, 5 µm.