Fig. 6. Apoptosis and cell proliferation in the otic vesicle. (A-C) TUNEL method was used to examine apoptotic cell death in the otic vesicle of wild-type and Six1^–/– embryos at E10.5 and E11.5. The results of TUNEL analysis for E11.5 otic vesicles of the wild type (A) and Six1^–/– (B) are shown. For quantitative analysis, four to five pairs of wild-type and Six1^–/– embryos were examined. For each embryo, apoptotic cell number was measured on three transverse sections passing through the central region of the otic vesicle and converted into the apoptotic cell number per 0.01 mm². Their mean value for the three sections was adopted as the datum point for the otic vesicle of each embryo. The mean value of four or five embryos is shown with the standard deviation (C). Enhanced apoptosis was seen in the ventral and medial regions of the Six1^–/– otic vesicle in comparison with the wild type in these stages. Statistical analysis was performed by Student's t-test. (D-F) Cell proliferation in the wild-type and the Six1^–/– otic vesicle was assessed by BrdU incorporation. The results of immunohistochemistry for BrdU at E11.5 otic vesicles of wild-type (D) and Six1^–/– (E) are shown. Quantitative analysis for BrdU-incorporated cell number was performed as described in TUNEL analysis (F). BrdU incorporation was considerably reduced in the Six1^–/– ventral otic vesicle at E11.5. d, dorsal; la, lateral. Scale bars: 100 µm. *P<0.05; **P<0.005; ***P<0.01.