Figure 6

BMP7-evoked growth cone collapse and axon orientation is dependent on PI3K activity. (A) [d] explants cultured as in Figure 5C with control- or BMP7- (blue; α-flag) or Netrin-1- (blue; α-Netrin) expressing COS-1 cells, with or without 2.5 μM LY (+LY). Green shows TAG-1-labeled axons. Dashed lines mark COS-1 cell aggregate and [d] explant appended borders. The closed single arrowhead indicates dI axons repelled by BMP7. The double closed arrowheads indicate angle of axons with LY. Open arrowheads indicate dI axons attracted by Netrin-1. Scale = 50 μm. (B) Angle of reorientation in [d] explants co-cultured with or without BMP7 and with or without LY (mean ± SEM for each condition): pMT23 = 0.8 ± 1.7° (n = 9); pMT23 + LY = -4.3 ± 1.8° (n = 3); BMP7 = 32.5 ± 1.9° (n = 18); BMP7 + LY = 11.6 ± 1.7° (n = 29). Student's t-tests: the straight D-V projection of axons in control [d] explants was unchanged by LY (pMT23 - LY versus pMT23 + LY, P = 0.1412). BMP7-evoked reorientation was significantly inhibited by LY (BMP7 - LY versus BMP7 + LY, ***P < 0.0001). Attraction of dI axons by Netrin-1 was unchanged by LY (Netrin - LY = 51.5 ± 3.5° (n = 3); Netrin + LY = 49.0 ± 1.5° (n = 4); Netrin - LY versus Netrin + LY, P = 0.4621). (C) BMP7-evoked dI growth cone collapse measured with or without LY or WM. Percentage decrease of growth cone area relative to control cultures (mean ± SEM): BMP7 = 44 ± 4% (n = 3); BMP7 + LY = 14 ± 7% (n = 2); BMP7 + WM = 19 ± 2% (n = 3). BMP7-stimulated reduction in growth cone area was significantly different from cultures treated with BMP7 + LY or BMP7 + WM (BMP7 - LY versus BMP7 + LY, *P < 0.05; BMP7 - WM versus BMP7 + WM, **P < 0.005; (Student's t-test)). Results are for 200 to 400 growth cones/condition/experiment.