Figure 3
Type I BMP receptor kinase activity required for BMP7-evoked Lhx2/9 induction, but not axon orientation. (A) [i] explants treated with BMP7 or BMP6 (20 ng/ml) with or without DM (5 μM): α-pSmad1/5/8 (upper) and α-Lhx2/9 (lower) panels. Scale = 20 μm. (B-E) [d] explants, with appended control or BMP7-expressing COS-1 cells, with or without 10 μM DM. Explants co-labeled with α-Lhx2/9 (red) (B), and α-TAG-1 (green) and α-flag (blue) (C). Scale = 50 μm. (B) Endogenous Lhx2/9, induced prior to explantation, present only in the dorsal-most region of explants, cultured alone (not shown) or with pMT23-expressing COS-1 cells (a). DM did not alter endogenous Lhx2/9 (b). Dashed lines indicate ectopic ventral Lhx2/9 expression in [d] explants + BMP7-expressing COS-1 cells (c). DM treatment greatly reduced BMP7-induced ectopic Lhx2/9 (d, arrows). (C) Integrated density of Lhx2/9 expression (mean × 105 ± SEM for each condition): pMT23 - DM = 5.41 ± 0.77 (n = 3); pMT23 + DM = 5.33 ± 0.94 (n = 2); BMP7 - DM = 16.46 ± 2.51 (n = 5); BMP7 + DM = 9.94 ± 1.90 (n = 6). (D) Dashed lines mark appended borders of COS-1 cell aggregates and [d] explants. Arrowheads indicate dI axons repelled by BMP7. (E) Angles of reorientation in [d] explants with pMT23- or BMP7-expressing COS-1 cells with or without DM (pMT23 = 5.1 ± 1.3° (n = 3); pMT23 + DM = 2.3 ± 0.3° (n = 2); BMP7 = 30 ± 3.1° (n = 5); BMP7 + DM = 28.4 ± 3.3° (n = 5). Results are mean ± SEM for each condition. Student's t-tests: DM had no effect on the D-V projection of dI axons either in control (pMT23 versus pMT23 + DM; P = 0.1936) or BMP7-orienting conditions (BMP7 versus BMP7 + DM; P = 0.7345).