Fig. 5.

Knockdown of Rreb1 prevents palatal fusion in organ culture. (A) Knockdown of Rreb1 by siRNA was performed on dissected shelves 48 h prior to the paired palatal shelf organ culture. Then, the paired palatal shelf was cultured for 24 h. Histological sections of horizontal palatal shelves showed the fusion defects of the palatal shelves and the remaining MEE in the Rreb1 knockdown group. Histological sections of horizontal palatal shelves were stained with HE. The incidence of fusion of palatal shelves in each treatment group is shown in the lower panel. (B) Persistent expression of E-cadherin and p63 was observed at the contacting palatal shelves in the Rreb1 knockdown group. (C) Knockdown of Rreb1 by siRNA was performed on dissected shelves for 48 h. RNA was extracted from palatal shelves in each group. The expression of Rreb1 was evaluated by qPCR. Gapdh was used as an internal control for normalization. Means±s.d. (n=3) are shown as horizontal bars. **P<0.01 (two-way ANOVA). (D) Palatal shelves from control culture and Rreb1 knockdown culture were lysed in RIPA buffer. Cell lysates were immunoblotted with antibodies against RREB1, p63, pERK, ERK and α-tubulin. This experiment was performed three times with similar results. (E,F) Analysis of cell death and cell proliferation in palatal shelf organ cultures. Control and Rreb1 knockdown palatal shelves were cultured for 24 h. (E) The number of TUNEL-positive cells in remaining MEE cells (arrowheads) is markedly reduced in Rreb1 knockdown cultures. (F) The proliferation activity is maintained in the remaining MEE cells (arrowheads) in Rreb1 knockdown cultures. Scale bars: 200 μm (A,B); 100 μm (E,F).