A Zeiss LSM 700 microscope was used for imaging of IF samples. The pinhole was set to 1 Airy unit at maximal optical resolution before gain and offset were calibrated. Fluorescent signal intensities were within the linear range of detection. For all IF image analyses, ImageJ software was used. For high resolution IF, a 40x silicone immersion objective (numerical aperture: 1.25) was used at the Nikon AX confocal microscope. Pixel size was set to 100 nm and z sections were obtained at the ventral rim of the embryo to reduce background signal. Images were deconvolved using Nis-Elements 3D Deconvolution. Quantification of the ciliogenesis defects in X. tropicalis MCC cells was performed by immunostaining against acetylated tubulin and phalloidin to identify the ciliary axoneme and MCC border, respectively. MCC motile cilia were classified as ‘Normal’ or ‘Defect’ (showing lower cilia density and shorter cilia) as well as ‘Mild’ (>half the length compared to control), ‘Severe’ (X. tropicalis embryos were examined by calculating the ratio of MCC or Ionocytes to total cells. pLrp6 stainings as well as LRP6 WT, LRP6VA and LRP6(VA)P(PA) stainings were quantified by measuring their IF-positive areas and then normalizing to the AcTub+ area for each MCC using Fiji (ImageJ). Each data point represents one ratio. For the GSK3 ciliary biosensor live cell imaging, the mean signal intensity was measured for one MCC per experiment and time-point. The mean signal intensity for each MCC was normalized to that of the first time point (t = 0) in the representative graph. In fixed ciliary GSK3 biosensor-injected embryos, one full image per embryo was measured using Fiji (ImageJ). Ciliary GSK3 mean intensity was normalized to Arl13b-mKate2 mean intensity in the latter.
Images for phenotypical analyses were taken with the AxioCam MRc 5 microscope (Zeiss). Embryos were scored blind and data are representative images from 2 or more independent experiments. Representative embryos were selected using Magnetic Lasso tool of Adobe Photoshop CS6. Background was adjusted uniformly for presentation. Phenotypical features were analyzed by comparison to control embryos and classified and ‘normal’ and ‘defect’ (showing DV/AP patterning defects).
Images for phenotypical analyses were taken with the AxioCam MRc 5 microscope (Zeiss). Embryos were scored blind and data are representative images from 2 or more independent experiments. Representative embryos were selected using Magnetic Lasso tool of Adobe Photoshop CS6. Background was adjusted uniformly for presentation. Phenotypical features were analyzed by comparison to control embryos and classified and ‘normal’ and ‘defect’ (showing DV/AP patterning defects).
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