FGF9/FGFR signaling inhibits the migration of RG/BG precursors/cells in cerebellar microexplants in vitro.

(A) Migration assays were performed with similarly sized (black dotted bars) CbA microexplants from E16.5 wild-type (CD-1) mice, containing Pax6⁺/Ccnd1⁺ GCPs in the outer EGL (yellow), Ccnd1⁺ RG/BG precursors and cells (green), Pax6⁺ postmitotic GCs and inner CbA cells (pink), and DAPI⁺ PCs (blue). ChPl, choroid plexus; EGL, external granular layer; IC, inferior colliculus; VZ, cerebellar ventricular zone. CbA microexplants were treated with control medium or medium containing 100 ng/ml FGF9 or 20 µM SU5402. The distance (d) migrated by each Pax6⁻/Ccnd1⁺ (green), Pax6⁺/Ccnd1⁺ (yellow) and Pax6⁺/Ccnd1⁻ (pink) cell from the border of the microexplant was measured after 36 h of incubation. (B–D) Representative confocal overviews of Ccnd1⁺ (green) and/or Pax6⁺ (red) cells (double-positive cells appear in yellow), counterstained with DAPI (blue) (overlays with single-positive cells appear in light green and pink, respectively), that migrated from the border of the CbA microexplant (white line) in control (B), FGF9- (C) or SU5402- (D) containing medium. Red arrowheads point at the front-most Ccnd1⁺/Pax6⁻ (green) cells. (E) Quantification of the average distance migrated by RG/BG precursors/cells (green bars) and GCPs (yellow bars) in control- (n = 8 explants), FGF9- (n = 8 explants) or SU5402- (n = 6 explants) treated microexplant cultures (Distance (µm): RG/BG precursors/cells, control, 86.2±16.8; +FGF9, 52.6±20.9; +SU5402, 89.3±33.2; GCPs, control, 111.7±15.3; +FGF9, 97.7±16.8; +SU5402, 100.2±15.6; one-way ANOVA). (F) Quantification of the proportion of Ccnd1⁺/Pax6⁻ RG/BG precursors/cells among the total number of migrating Ccnd1⁺ and/or Pax6⁺ cells in control-, FGF9- or SU5402-treated microexplant cultures (% Ccnd1⁺/Pax6⁻ RG/BG precursors/cells: control, 3.66, 95% confidence interval [2.95,4.48] (8 experiments for controls with 128–589 migrated cells, among them 2-29 RG/BG cells); +FGF9, 1.31, 95% confidence interval [0.84,1.92] (8 experiments for FGF9 with 79-501 migrated cells, among them 0–9 RG/BG cells); +SU5402, 2.89, 95% confidence interval [1.99,4.04] (6 experiments for SU5402 with 78–371 migrated cells, among them 0–15 RG/BG cells); P-values from contrasts of a logistic model). (G) Average proportions of Ccnd1⁺/Pax6⁻ RG/BG precursors/cells among the total number of migrating Ccnd1⁺ and/or Pax6⁺ cells in each 50-µm bin in control- (grey bars), FGF9- (red bars) or SU5402- (green bars) treated microexplant cultures were estimated with a logistic model. Values are given in Table S2 in File S1. Scale bar (D): 100 µm.