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First published online January 16, 2004
doi: 10.1242/10.1242/dev.00970

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Navigation of trochlear motor axons along the midbrain-hindbrain boundary by neuropilin 2

¹ Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai 980-8575, Japan
² Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai 980-8575, Japan

View larger version (115K): [in a new window]   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.

View larger version (11K): [in a new window]   Fig. 2. Cloning of chick neuropilin 2 and Sema3F. Molecular phylogenetic tree of neuropilins (A) and class 3 semaphorins (B). Deduced amino acid sequences of isolated chick neuropilin 2 and Sema3F were clustered with human and mouse counterparts. The dendrogram was deduced by the ClustalW algorithm (Thompson et al., 1994). Bar corresponds to 10% difference between the sequences.

View larger version (98K): [in a new window]   Fig. 3. Expression of neuropilin 2 and trochlear nerve trajectory. (A-C) neuropilin 2 expression at stage 14 (A), stage 16 (B) and stage 18 (C). The position of the MHB is indicated by an arrowhead. (D,E) Staining of the same specimen for neuropilin 2 (D, dark blue) and neurofilament (E, magenta) at stage 20 during the dorsal decussation of the trochlear axons. The decussation point is indicated by an arrow. (F) neuropilin 2 expression (dark blue) on the transversal section. Neuroepithelium layer is indicated by double-headed arrows. (G) Merged image of neuropilin 2 expression in dorsal isthmus with neurofilament staining (G, magenta) in higher magnification. (H,I) Flat-mount specimen after double staining with anti-ISL1 (H,I, green) and anti NF-M antibody (I, magenta) during the dorsal projection of trochlear axons at stage 19. (J-L) Staining of flat-mount specimen for neuropilin 2 (J, dark blue) and neurofilament (K, magenta) at st19. (L) Negative image of J was merged with K. Trochlear nucleus along the rostrocaudal axis is indicated by double-headed arrows. neuropilin 2 is expressed in the neuroepithelium of the dorsal isthmus, in addition to the oculomotor and rostral trochlear nucleus. III, oculomotor nucleus; IV, trochlear nucleus; dI, dorsal isthmus; ne, neuroepithelium layer; Mid, midbrain; Hind, hindbrain. Scale bars: 200 µm for A-E,H-L; 50 µm for F,G.

View larger version (83K): [in a new window]   Fig. 4. Expression of Sema3F and trochlear nerve trajectory. (A-G) Expression of Sema3F (dark blue) in chick embryos at stage 10-22. Sema3F expression is detected at the caudal border of the midbrain along the MHB. (H-J) Expression of Sema3F (dark blue), Fgf8 (red), Otx2 (red), Gbx2 (dark blue) at stage 18. Sema3F expression is rostral to the molecular boundary of the Otx2 and Gbx2 expression. (K,L) Staining of same specimen for Sema3F (K, dark blue) and neurofilament (L, magenta) during the dorsal projection of trochlear axons at stage 19. (M) Image of K was merged with (L). Expression of Sema3F was prominent in caudal margin of the midbrain ventral to the lateral longitudinal fasciculus, and also in the rhombic lip. Position of Sema3F expression along the MHB is indicated with arrowheads (A-F,H,I). ov, otic vesicle; r3, rhombomere 3; r5, rhombomere 5; Mid, midbrain; Hind, hindbrain. Scale bars: 200 µm in A-D,K-M; 500 µm in E-J.

View larger version (83K): [in a new window]   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.

View larger version (80K): [in a new window]   Fig. 6. Misexpression of neuropilin 2 dramatically alters trochlear nerve trajectory. (A-C) Caudal view of neuropilin 2-transfected midbrain 2 days after electroporation. Neurofilament (A, magenta); GFP (B, green); merge (C). The broken line indicates the midline dissecting the control (untransfected) and experimental (transfected) sides. (D-E) Flat-mount view of MHB from the same embryo showing control (D) and experimental sides (E). (F) The GFP image (green) was merged with E. An arrow indicates the dorsal decussation point. (G,H) Confocal images of D,E at higher magnification. (I) Schematic drawing of the result of misexpression. Trochlear axons (magenta) project dorsally from the nucleus (IV; blue) but deflect rostrally at the point where the transfected domain is encountered (thin arrow), cross the MHB and invade the ipsilateral tectum. GFP-positive transfected domain is indicated (green). The trochlear axons from the contralateral nucleus are seen (purple). A broad arrow indicates the dorsal decussation point. A broken line marks the boundary between alar and basal plate. (J,K) Trochlear axon trajectory of normal (J) and experimental (K) embryos revealed by DiI labeling of trochlear nucleus. An arrow indicates the relative position of the dorsal decussation point. The images of E and F, H, K were reversed horizontally to compare with D, G, J, respectively. Mid, midbrain; Hind, hindbrain; tec, tectum; cont, control side; exp, experimental side; normal, normal embryo; teg, tegmentum; IV, trochlear nucleus. Scale bars: 200 µm in A-H,J,K.

View larger version (79K): [in a new window]   Fig. 7. Distribution of neuropilin 2 ligands in normal and in Sema3F- or neuropilin 2-transfected embryos. (A-F) Double staining with NP2dC-AP (A,B,E; dark blue) and anti-neurofilament antibody (C,F; magenta) on normal embryos at stage 17(A-C) and stage 19 (D-F). (D) Images in E and F merged. Stained brain specimens were flat-mounted and focused on the MHB (B-F). Trochlear nucleus along the rostrocaudal axis is indicated by double-headed arrows in C,F. Trochlear axons extend in the isthmic region devoid of NP2dC-AP staining. (G-J) Detection of GFP (G,I; green) and NP2dC-AP staining (H,J; dark blue) on Sema3F-(G,H) or neuropilin 2-(I,J) transfected embryos. NP2dC-AP can detect misexpressed Sema3F (G,H). NP2dC-AP staining was dismissed in the brain where neuropilin 2 was misexpressed (I,J). NF, neurofilament. Scale bars: 1 mm in A,G-J; 200 µm in B-F.

View larger version (33K): [in a new window]   Fig. 8. A model for navigation of trochlear motor axons by neuropilin 2. (A) Normal embryo. (B) neuropilin 2-transfected embryo. Expression domains of Sema3F (dark blue), neuropilin 2 (green) and the trochlear nerve trajectory (magenta) are indicated. Distribution of neuropilin 2 ligands is denoted (light blue). See Discussion for explanation. III, oculomotor nucleus; IV, trochlear nucleus; MHB, midbrain-hindbrain boundary.