Figure 5

Downregulation of Pax-2 by BMP4. (A-C) Control group after seven days in culture. Similar to the in vivo situation [5], (B) islet-1-positive cells (green) appear in the incipient Pax-2-expressing sensory epithelia (red); (C) myosin VIIA-positive early hair cells (red) that express islet-1 (green) occur within islet-1-positive epithelial patches. (D-F) In otocysts treated with 10 ng/ml BMP4 from the beginning of the culture period, Pax-2 expression was clearly reduced, whereas islet-1-expression and hair cell generation were not affected. (G) In a comparative Western blot analysis, Pax-2-protein levels were clearly reduced in otocysts treated for 48 h with BMP4. The amount of protein loaded per lane was the total protein of 5 otocysts. (H) Similar experiment to the one shown in (G), but the duration of the BMP4-treatment was reduced to 24 h and the total protein load per lane was equalized. (I-K) In cryosections of stage 25 chicken inner ear, BMP4 mRNA is expressed in the basilar papilla sensory epithelium primordium (dark precipitate in (I)). (J)Pax-2 protein expression (depicted in red) becomes noticeably reduced in the BMP4-expressing area simultaneously when epithelial islet-1 expression (shown in green) occurs. Panel (K) is the merged image of (I) with (J).