Figure 4. EphA3 ectodomain increases the velocity of nasal RGCs axon growth and RGC axons choose growing on EphA3-expressing cells.

(A) Quantification of axonal growth rate (during the first 100 minutes since axon appearance) of nasal explants exposed to soluble clustered EphA3-Fc or Fc (Student's t test, 3 experiments, n: 12 axons for explant, 3 explants for condition). Results are shown as mean +/− SE. (B) Quantification of distance covered by nasal axons exposed to soluble clustered EphA3-Fc and Fc during the first 100 minutes after their appearance. Nasal axons grow faster with EphA3-Fc (p: 3.62^*10⁻⁸, Student's t test). (C–E) Representative microphotographs of a time-lapse experiment. They show the behavior of axonal growth cones of nasal and temporal explants making contact with EphA3ΔC-EGFP-transfected-HEK293 cells or EGFP-F-transfected-HEK293 cells (control) (see arrows). (C) Nasal growth cones indistinctly attach or retract from EGFP-F-transfected-293 cells (Total time: 147 minutes). Similar results were obtained with temporal growth cones. (D) Nasal axons tend to grow along EphA3ΔC-EGFP-transfected-293 cells (Total time: 183 minutes) whereas (E) temporal axons tend to adhere to EphA3ΔC-EGFP-transfected-293 cells (Total time: 471 minutes). Scale bars  = 20 µm (see Videos S1, S2, S3). (F) Proportions of positive events (elongation or adhesion) produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells versus EGFP-F transfected-HEK293 cells. Positive events significantly increase with EphA3 expressing-cells (Fisher's exact test; n: 79 nasal growth cones, 39 temporal growth cones; 3 nasal and 3 temporal explants for condition). (G) Discrimination of positive events between elongation and adhesion produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells. Nasal axons present a significantly higher proportion of elongation than temporal ones (Fisher's exact test; n: 58 growth cones; 3 nasal and 3 temporal explants). Nasal (N), temporal (T).