Zen software

For light sheet imaging, embryos were anaesthetised using tricaine in E3 medium and mounted in 1% agarose. A light sheet Z.1 system was used and images were acquired using ZEN software (Zeiss). For confocal microscopy, still images were taken using an Ultraview VOX confocal spinning disc system (Perkin Elmer), Zeiss LSM880 with Airyscan and Leica SP5. Images were analysed using Volocity®V5.3.2, ZEN software (Zeiss) and Leica LCS Confocal software. In cases where samples displayed drift during time lapses, these were corrected using the translation algorithm in ZEN (Blue Edition).

For histological analysis, mice were perfused with 1% or 4% PFA/PBS under deep anesthesia and the organs were postfixed with 1% or 4% PFA/PBS overnight at 4 °C. On 6- to 7-μm sections of the paraffin-embedded organs, hematoxylin-eosin staining was performed. Images were taken with an AxioCam MRc5 (Zeiss) on an Axiophat microscope (Zeiss) with an ACHROSTIGMAT 5x/0.12, Plan-NEOFLUAR 10x/0.30, or Plan-NEOFLUAR 40x/0.75 objective using the ZEN software (Zeiss). Image processing was done using Image Composite Editor (Microsoft) and ZEN software (Zeiss).

Live images of neurospheres as well as NPC scratch assay were captured with a Zeiss Primovert inverted cell culture microscope, Zeiss Axiocam ERc5s camera, and Zen software (Zeiss). Primary neurospheres or NPCs that underwent scratch assays (migration assays) were imaged at 100 × magnification. Secondary neurospheres were swirled into the center of the well and imaged at 40 × magnification.
Immunostained brain sections and cultures were imaged with a Zeiss Axio Imager M2 fluorescence microscope, ORCA-Flash LT sCMOS Camera, and Zen software (Zeiss). Four to six sections in every sample encompassing medial and lateral planes were imaged at 100 × magnification in a single plane. Representative images show tiled images encompassing OB in a sagittal plane or cropped images encompassing OB neuronal layers. Images from each set of immunostaining were acquired using identical settings.

Angles of proliferation, migration, orientation, and localization were calculated using Axiovision Angle3 software, and cilium length was measured using Zen software (Zeiss, Oberkochen, Germany). Scratch assays were carried out in wildtype and Talpid3^−/− MEFs grown to confluence and serum starved (DMEM + 0.5% FCS) for 48 hr with a p10 pipette tip. Medium was then renewed and MEFs incubated for four hours before fixation in ice-cold methanol prior to immunofluorescence. Angles of orientation were then taken as a measurement of the angle from the center of the nucleus, through the center of the leading edge (towards the wound, identified by phalloidin staining for F-actin) and again through the center of the Golgi apparatus (identified by TGN46 antibody staining). Tiled Z stacks of the scratch/wound were analyzed for greater accuracy.
The expected orientation of the stereocilia of the basilar papilla hair cells were taken as being at 90° to the abneural edge of the basilar papilla. The angle of orientation was taken by drawing a line through the cell perpendicular to the abneural edge and a second from the center of the cell, intersecting with both the perpendicular line and center of the actin bundle. The internal angle was taken to be the angle by which cell orientation deviated from the expected. Cilium length was measured using Zen software (Zeiss, Oberkochen, Germany).