Fig. 5. Differentiation of sensory patches with the production of hair cells is inhibited by DAPT treatment initiated at an early stage. Organ cultures established at stage HH16-17 were maintained for 2-5 days in either control medium or in medium containing 20 µM DAPT. (A-F) Bmp4 expression analysed after 2 or 5 days in vitro. In control specimens (A,D), typically two or three patches of strong Bmp4 expression are observed (arrowheads); in DAPT-treated specimens (B,E), the size and mean number of Bmp4-positive patches are reduced and the intensity of Bmp4 expression within them is greatly diminished. (C,F) Quantitative analysis of the mean number of patches of Bmp4 expression in control versus DAPT-treated otocyst after 2 (C) or 5 (F) days in vitro. (G,H) Serrate1 and hair-cell antigen (HCA) immunostaining in control (G) and DAPT-treated (H) otocysts after 5 days in vitro. The micrographs are z-projections of confocal optical sections encompassing the entire thickness of the dissected otocysts. Serrate1 is expressed in several patches containing differentiated hair cells (arrows in G) in control otocysts. In DAPT-treated otocysts, Serrate1 expression is abolished or greatly reduced and hair cells are few, although densely clustered (arrow and inset in H). (I,J) Quantitation of the number of patches of Serrate1 expression (I) and of the number of hair cells (J) after 5 days of culture in control versus DAPT-supplemented medium. Error bars represent s.e.m. All the differences between control and DAPT-treated groups were statistically significant (t-test; ^*P<10^-6).