Figure 4.

The relative distribution of Pk2 does not change across the line of reversal. A, D, The line of reversal (dashed yellow line) in P0 saccule A and utricle D is visualized easily by the position of the kinocilium void within the cuticular plate of hair cells that have been labeled with an antibody against α-spectrin (red). The polarities of pairs of cells on opposite sides of the line of reversal from both epithelia are indicated by arrows. Despite the change in hair cell polarity, the relative distribution of Pk2 (green) remains constant. B, C, Higher-magnification image containing two hair cells from saccule B and utricle C located on opposite sides of the line of reversal. In each the Pk2 (green) is located on the medial edge of the cell; however, there is a relative change in the amount of Pk2 protein between the two cells. D′, Gray scale image of Pk2 across the line of reversal in utricle reveals a correlation between the position of the kinocilium and relative levels of Pk2. An asterisk (D, D′) indicates a hair cell that does not share the polarity of its neighbors and has a corresponding decrease in Pk2 protein. E, The line of reversal is first apparent at E15.5 and can be visualized by the position of the kinocilium (acetylated tubulin; red) relative to the stereocilia bundle (phalloidin; blue). At this time, Pk2 (green) already is forming crescents along the medial edges of both mature and newly differentiated hair cells (asterisks). The polarity of developing hair cells is indicated by arrows. F, G, Low-magnification images of Pk2 immunofluorescence across the entire saccular (F) and utricular (G) epithelia emphasize the correlation between levels of Pk2 protein and the location of the line of reversal (dashed yellow line). Scale bars: A–E, 5 μm; F–G, 100 μm.