Figure 2.

Increased production of hair cells with exogenous BMP4 and decrease in hair cell numbers in response to blockade of BMP signaling. (A) Hair cell antigen (red) and myosin VIIA-positive hair cells (green) in a cryosectioned otocyst after seven days culture. Filamentous actin is visualized with phalloidin-labeling (blue fluorescence). We routinely observed both hair cells organized in epithelia (see also higher magnification in (F)) and scattered hair cells (arrowhead). (B) BMP4 at 5 ng/ml, applied on the third day in culture, leads to substantial increase in the number of hair cells in epithelial cells and also in the scattered population of isolated hair cells. (C) Noggin at 0.5 μg/ml diminishes the number of hair cells. (D) The effect of noggin-treatment (0.5 μg/ml)) can be rescued by addition of 5 ng/ml exogenous BMP4. (E) Dose-dependence of the effect of exogenously added BMP4 on the number of hair cells in otocysts after seven days in culture. BMP4 at 3 ng/ml and at 5 ng/ml significantly increases the number of hair cells when compared with control conditions (asterisks indicate p < 0.05, unpaired Student's t-test, n = 4–5). Noggin at various concentrations and soluble BMPR 1a and 1b significantly reduced the number of hair cells detected in otocysts after seven days in culture when compared to the untreated control (asterisks indicate p < 0.003, unpaired Student's t-test, n = 6–7); the effect of 0.5 μg/ml noggin can be fully rescued by addition of 5 ng/ml BMP4. Error bars represent standard deviations. (F,G) Higher magnification to show the morphology of hair cells observed in (A) and in (B).