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Fig. 5. Repulsive activity of Sema3F against trochlear axons in ovo. (A) Normal trochlear nerve trajectory after decussation at stage 23, immunostained with anti-neurofilament antibody (magenta). The figure is the rostral view of a thick transverse section of the MHB region made by micro scissors. Thin arrows indicate trochlear axons exiting the brain contralaterally. (B-D) Trochlear nerve trajectory of Sema3F-transfected embryos. (B) Rostral view of a thick transverse section of the MHB region. The transfection domain was visualized with GFP (green). Thin arrows indicate trochlear axons exiting the brain ipsilaterally at the point of repulsion (short arrow). In the transfected side, trochlear axons have stopped at a distance from the transfected domain (arrowhead). (C) Flat-mount view of the contralateral half to the Sema3F-misexpressing side shown in (B). Repelled axons (thin arrows) elongate toward the ipsilateral target. (D) Flat-mount confocal image of trochlear axons denoted in C. (E-K) Flat-mount confocal images of trochlear nerve trajectory of normal (E,F) and Sema3F-transfected (G-K) embryos. Rotated images of E, G, I are shown in F, H, J and K, respectively. Trochlear axons repelled by ectopic Sema3F converge once in neuroepithelium (H,K; arrowhead) and then exit the brain ipsilaterally (I-K; thin arrow). Mid, midbrain; Hind, hindbrain. Scale bars: 200 µm in A-C,E-K; 50 µm in D.

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