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Fig. 1. Development of trochlear motor axons in chick embryos. The trochlear nerve (magenta) in chick embryos. (B-D) Immunostaining of the trochlear nerve fibers at the initial phase of their development. Flat-mount specimens after staining with anti NF-M (magenta) and anti-ISL1 antibody (green) at stage 17 (B), stage 18 (C) and stage 19 (D). Rostral is towards the right. The cells stained by anti-ISL1 antibody in the isthmus region are trochlear neurons (this antibody stains the cell nuclei of motoneurons). Trochlear axons cross the medial longitudinal fasciculus (mlf; horizontally running fibers indicated by thin arrows in B) and elongate dorsally. (E-J) Whole-mount immunostaining with anti-neurofilament antibody (3A10; magenta) at stage 20 (E,F), stage 23 (G,H) and stage 26 (I,J). (E-G) Rostral view of the MHB at the stage before (E,F) and after (G) the dorsal decussation. Distal position of trochlear axons and the trochlear nucleus are indicated (arrows and arrowheads, respectively). (H-J) Lateral view after the decussation. (J) Staining with anti-myosin antibody (MF20; green) is merged with I. The trajectory of the trochlear nerve outside the brain is indicated (thin arrows in G-J). Trochlear axons exit the brain contralaterally to innervate the superior oblique muscle (SOM). (K) Sagittal section of the MHB region of stage 24 embryo. (L,M) Higher magnification of the dorsal isthmus at different focal planes (L, parasagittal; M, midline). The trochlear axons run in the brain before the decussation (a), decussate within the brain at the midline (b) and exit from the brain after the decussation (c) (solid arrows). Mid, midbrain; Hind, hindbrain; MHB, midbrain-hindbrain boundary; r1, rhombomere 1; III, oculomotor nucleus; IV, trochlear nucleus; fp, floor plate; mlf, medial longitudinal fasciculus; tec, tectum; V1, ophthalmic nerve; SOM, superior oblique muscle; nIII, oculomotor nerve; NF, neurofilament. Scale bars: 50 µm in B-D; 100 µm in E-G,K-M; 1 mm in H-J.





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