繊毛病マウス変異蝸牛における平面セル・-極性表現型の評価

The overall goal of this protocol is to examine ciliary mutant mouse cochlea for planar-cell-polarity phenotypes. These techniques allow for an extensive detailing of the cochlear phenotype, which will lead to a more precise understanding of the ciliary involvement in establishing vertebrate PCP signaling. The main advantage of this protocol is that once mastered, the dissected cochlear epithelia can be used for a variety of different types of phenotypic analysis.

After preparing reagents and tissue from experimental animals, at developmental stage E16.5 to p3, use a scalpel blade or a small pair of scissors to dissect the head along the sagittal midline beginning at the nose and extending cotyle. Remove the brain from each half of the skull with a pair of forceps. Then, identify the temporal bones which contain the developing bony labyrinths of the inner ear.

While working with the sample under a dissection microscope, use a pair of forceps to prize the bony labyrinths away from the skull by running the forceps gently underneath the bony labyrinths. Following dissection, use the tip of the dissecting forceps to clear the oval and round windows and make a small hole in the apex of the cochlear spiral. Next, fix the bony labyrinths in 1.5 milliliters of 4%paraformaldehyde for five minutes on ice to allow for easy removal of the tectorial membrane.

Now, place the bony labyrinths in a black silicone elastomer-coated dissecting dish containing PBS to facilitate dissection. Use number five forceps to remove the outer cartilage, exposing the cochlear duct. Start at the oval window, insert the bottom tip of the forceps into the oval window, and gently pry open the cartilage, slowly moving upwards towards the apex.

Next, use a fine pair of forceps to pinch the Reissner's membrane at the base of the cochlear duct, and peel it off in an upward motion. Visualize the dorsal aspect of the cochlear duct, including the sensory epithelium. Then, use number 55 forceps to pinch the tectorial membrane at the base of the cochlea, and peel upwards towards the apex.

Complete removal of the tectorial membrane is required to obtain the best images with immunohistochemistry, and is essential for scanning electron microscopy. After exposing the organ of Corti, further fix the tissue and perform immunohistochemistry, or prepare for scanning electron microscopy, as detailed in the written portion of the protocol. After the tissue has been stained, place the sample in a black silicone elastomer dish, containing PBS.

Using fine forceps, remove the vestibular region from the cochlear spiral. Then, very carefully remove the underlying cartilage and mesenchyme from the cochlear spiral. The remaining cochlear spiral contains the inner sulcus, organ of Corti, and outer sulcus.

Transfer the cochlear spiral into a drop of PBS placed on a microscope slide. Wick away the PBS with an absorbent tissue or piece of filter paper, and gently re-position the cochlear spiral if the epithelia has shifted and overlaps onto itself. Add a drop of mounting media directly onto the cochlea sample, and gently place a cover slip directly onto the sample, taking care to avoid air bubbles.

Use an epifluorescent or laser scanning confocal microscope equipped with high magnification and high numerical aperture objectives, to capture images of the apical surface of the cochlear hair cell. Use lower objectives to take overlapping images of the cochlear spiral to quantify the length of the cochlear duct. The following images show immunofluorescent staining of basal turn, post-natal day one type cochlea.

Phalloidin labels filamentous actin in stereocilia, and cortical actin at the cell periphery. Myosin VIIA is a marker for inner and outer hair cells. ZO_1 labels the tight junctions in between cells, making it a great marker to distinguish cell boundaries.

Acetylated alpha-tubulin is used as a marker for the kinocilium at the vertex of the bundle, as indicated by the white arrow. Internal microtubules are also labeled by acetylated alpha-tubulin, which are particularly abundant in pillar cells, as indicated by the white asterisks. This image of dissected cochlear ducts at E18.5, stained with acetylated tubulin, demonstrates marked shortening of IFT20 conditional knockout cochlear ducts compared to control.

The following image is a whole-mount preparation of basal cochlear turn, in a BBS8 knockout mice, at post natal day zero. Stereociliary bundles in BBS8 knockout cochleae are variably rotated, as indicated by the right arrow, or flattened and mis-localized, as indicated the middle arrow. The kinocilia are mis-localized, or axonemes are missing, as indicated by the left arrow.

When preparing cochlear phenotype for analysis, it's important to remember that differences in genetic background can modify the cochlear phenotype. Therefore, it's important to use littermate controls for analysis. Further analyses include audiometric testing and culturing of cochlear explants, which allows for treatment with various signaling activators or inhibitors, thereby contributing to mechanistic understanding of developmental processes involved.

After watching this video, you should have a good understanding of how to prepare cochlear tissue for analyzing the role of ciliary components and their distinct effects on PCP signaling.