Figure 2. Cell proliferation was attenuated by selectively inhibiting the MAP kinase cascade and the Rb–Raf-1 interaction during the early development of the inner ear.

Otocysts were explanted at HH16–18 and kept in culture medium with DMSO (A1–A4, E1–E4), RRD-251 10 µM (B1–B4), RRD-251 20 µM (C1–C4), RRD-251 40 µM (D1–D4), U0126 30 µM (F1–F4), U0126 50 µM (G1–G4), or a combination of RRD-251 20 µM and U0126 30 µM (H1–H4) for 24 h and then exposed to 10 mM EdU for 30 min. Cell proliferation is indicated by EdU (green) staining and the nuclei are counterstained with DAPI (blue). Based on the morphological changes that occur in culture that mimic the normal development of the inner ear and the expression of Tuj1 (red) that serves as a marker of neural processes, the cultured otocysts were divided into the otic vesicle area (OV, Tuj1-negative) and the acoustic-vestibular ganglia area (AVG, Tuj1-positive), which would develop into sensory epithelium and the spiral ganglion, respectively. The OV and AVG areas were measured with the ImageJ software, and the data are presented as means ± SEM relative to control values (I, J). High magnification pictures were taken from the center of the otocysts (A4–H4), and the EdU-positive cells in each 63×high power field (HPF) were counted under the different conditions (K). * = P<0.05 compared to control, # = P<0.05 compared to RRD-251 10 µM, $ = P<0.05 compared to RRD-251 20 µM, and & = P<0.05 compared to U0126 30 µM (I–K). The changes in the ratios between the OV area and AVG area after treatment with U0126 and RRD-251 are shown in (L). * = P<0.05 and # = P<0.01 compared with controls (L). At least six otic vesicles per condition from three different experiments were evaluated, and statistical significance was estimated using one-way ANOVA. Scale bar = 50 µm.