Figure 1
dI induction, Smad activation and growth cone collapse by BMPs. (A) Lhx2/9 induction in [i] explants treated with BMP7 or BMP6. Scale = 50 μm. (B) pSmad1/5/8 response to BMP7 or BMP6 in dI neurons. Western blots of whole cell lysates. Results (mean ± SEM), normalized to total cellular Smad1/5/8, are expressed as percent control pSmad1/5/8 (at 0.01 ng/ml BMP7 = 68 ± 13% and BMP6 = 66 ± 4% (n = 2); at 50 ng/ml BMP7 = 780 ± 247% and BMP6 = 675 ± 199% (n = 5)). Mann-Whitney tests: the pSmad1/5/8 response at 50 ng/ml BMP7 and BMP6 was significantly different from control (**P < 0.01); the pSmad1/5/8 response at 0.01 ng/ml BMP7 and BMP6 was not different from control (P = 0.0952). (C) Dissociated dI neurons ±50 ng/ml BMP7 or BMP6, double-labeled with phospho-specific α-Smad1/5/8 (red) and DAPI (blue): 95.2 ± 2.0% (BMP7) and 97.9 ± 1.5% (BMP6) dI neurons were pSmad1/5/8+. Scale = 20 μm. (D) ERM)-labeled dissociated dI neurons ±BMP7 at 0.01 or 50 ng/ml. Arrowheads indicate typical widespread growth cones and asterisks indicate collapsed growth cones. Scale = 20 μm. (E) Average growth cone areas (mean ± SEM): control = 107.6 ± 0.44 μm2; BMP7 (0.01 ng/ml) = 57.8 ± 1.11 μm2; BMP7 (50 ng/ml) = 63.4 ± 0.85 μm2. Areas measured in 0.01 or 50 ng/ml BMP7 differ significantly from control (***P < 0.001, Student's t-test). Results are for 140 to 380 growth cones/condition/experiment; n = 2. (F) Comparison of dI growth cone responses to BMP7 and BMP6 (50 ng/ml), measured as in (E): control = 114.3 ± 10.7 μm2; BMP7 = 79.5 ± 7.4 μm2; BMP6 = 127.4 ± 11.9 μm2. Student's t-tests: area in BMP7 differs significantly from control (***P < 0.001); area in response to BMP6 was not different from control (P = 0.0607). Results are for 100 to 115 growth cones/condition/experiment; n = 2.