Ectopic supporting cells associated with ectopic hair cells in Hey2 mutants. Cochlear whole mount preparations from E18.5 wild-type control (A-C) and Hes5-/-;Hey2-/- (E-G) animals were stained for F-actin (phalloidin, green) and Prox1 (red). Confocal Z-scans were taken to capture F-actin labeling from the apical surface (A,E) to Prox1 labeling at the layer of supporting cell nuclei (B,F). The Z-scans were stacked to project both labels in the organ of Corti (C,G). Orthogonal cross sections of the projections of the organ of Corti stained with F-actin and Prox1 were selected to view the relative positions of hair cells (D,H), in which stereocilia and cellular cortex were labeled strongly by F-actin, and the nuclei of supporting cells were labeled by Prox1. The positions where the orthogonal sections were taken are indicated by double green lines (B,C) or double red lines (F,G). Note that precise patterning 4 rows of hair cells and 5 rows of Prox1+ supporting cells is seen in the control preparation (A-D), while ectopic hair cells in the Hes5-/-;Hey2-/- preparation are associated with ectopic Prox1+ supporting cells (E-H). (I-N) Cross sections from E18.5 control and Hes1-/-;Hey2+/- embryos were stained for Myosin6 (I,L, Myo6), Prox1 (J,M), and p27/Kip1 (J, M). K and N are overlays of the triple stains for each sample. The asterisks mark hair cells (A,I,L), supporting cells (B,D,F,H), or hair cells and supporting cells (K,N). OP: outer pillar cell; OHC: outer hair cells; IHC: inner hair cell. Scale bars: 10 μm for A-H, I-N.