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First published online 30 June 2004
doi: 10.1242/dev.01228

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PlexinA4 is necessary as a downstream target of Islet2 to mediate Slit signaling for promotion of sensory axon branching

Toshio Miyashita^1,*, Sang-Yeob Yeo^1,, Yoshikazu Hirate¹, Hiroshi Segawa¹, Hironori Wada¹, Melissa H. Little², Toshiya Yamada^2,, Naoki Takahashi⁴ and Hitoshi Okamoto^1,3,

¹ Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan
² Center for Molecular and Cellular Biology, The University of Queensland, Queensland 4072, Australia
³ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 3-4-5 Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan
⁴ Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan

View larger version (92K): [in a new window]   Fig. 7. Loss of function of PlexinA4 rescued the Slit2 overexpression phenotype in the primary sensory neurons. Ubiquitously overexpressed Slit2 induced excessive branching in the peripheral axons of Rohon-Beard neurons (black circles) (B) compared with branching in normal embryos (A). Simultaneous overexpression of Slit2 with dnPlexinA4 (C) and with injection of the AMO against PlexinA4 (D) rescued this phenotype. Overexpression of Slit2 before the central axons of trigeminal sensory ganglion neurons (arrows) enter the hindbrain prevents these axons from entering the hindbrain and induce abnormal defasciculation (F). The embryo in E is a normal control. In 40% of transgenic zebrafish that carry the transgenes for both Slit2 and dnPlexinA4 under control of the hsp70 promoter (G), failure of the central axons to enter the hindbrain by Slit2-overexpression was rescued. In addition, the same defects were also rescued in 44% of the AMO-injected embryos (H). (A-D) Lateral view; anterior is leftwards; dorsal is towards the top. (E-H) Dorsolateral oblique view; anterior is leftwards. Scale bars: in D, 20 µm for AD; in H, 20 µm for E-H. Repression of PlexinA4 function could not rescue the abnormal axonal pathfinding of Mauthner neurons induced by Slit2 overexpression. (I) The axons from Mauthner neurons of the normal embryo immunostained with 3A10 antibody cross the midline (broken line) and extend posteriorly near the midline. (J) In the Slit2-overexpressing embryo, the axons of Mauthner neurons take irregular trajectories. (K) Simultaneous overexpression of dnPlexinA4-GFP could not rescue this phenotype. ot.v, otic vesicle. Scale bar: 10 µm. Induction of excessive branching of the peripheral axons of Rohon-Beard neurons observed in the Slit2-overexpressing embryos (SG) was canceled in the embryos overexpressing both Slit2 and dnPlexinA4-GFP in a dose-dependent manner. (L,M) The number of branching points was reduced more severely in the embryos carrying the dnPlexinA4-GFP transgene homozygously (SG;dP/dP) than in the heterozygous embryos (SG;dP/-). (N,O) Injection of the AMO against PlexinA4 (SG;AMO) also rescued the excessive branching of the peripheral axons of Rohon-Beard neurons induced by Slit2 overexpression (SG), whereas injection of control-MO (SG;contMO) could not rescue this phenotype. wt, wild-type embryos.

View larger version (86K): [in a new window]   Fig. 1. D204 is a candidate downstream gene of Islet3 and Islet2. (A,A') D204 mRNA expression in 24 hpf normal (A) and LIMIsl-3-overexpressing (A') zebrafish embryos. The expression in tectum was reduced in LIMIsl-3-overexpressing embryo (arrows in A and A'). The embryos overexpressing LIMIsl-3 lack optic vesicles (ov) and MHB (brackets). (B,B',D,D') The peripheral axons of both the trigeminal sensory ganglion neurons (arrowheads in B and black circles in B') and Rohon-Beard neurons (black circles in D and D') were eliminated by overexpression of LIMIsl-2 (B',D'), while their central axons (arrows in B and B') remained intact as in normal embryos at 24 hpf. The axons were stained by anti-acetylated -tubulin antibody. Embryos in B and D are normal controls. (C,C',E,E') D204 expression in the trigeminal sensory ganglion neurons (arrows) and Rohon-Beard neurons (arrowheads) were reduced in LIMIsl-2-overexpressing 21 hpf embryos (C',E'), while the expression of D204 in the hindbrain remained intact (arrowheads in C'). Embryos in C and E are normal controls. (A,A',C,C') Dorsal views; anterior leftwards. (B,B',D,D',E,E') Lateral views; anterior leftwards; dorsal towards the top. Scale bar: 20 µm in A,C; 10 µm in B,D,E.

View larger version (86K): [in a new window]   Fig. 4. Inhibition of PlexinA4 translation by injection of AMO against plexinA4 mRNA caused reduction in the number of branches of the peripheral axons of Rohon-Beard neurons. (A-F) The peripheral axons of Rohon-Beard neurons (black circles) of 24 hpf embryos were visualized by anti-acetylated -tubulin antibody staining (A-F), and camera lucida drawings of them were made (A'-C'). The AMO-injected embryos showed reduction in the number of the peripheral axons of Rohon-Beard neurons, as shown in the lateral view (B,B') compared with normal (A,A') and control MO-injected embryos (C,C'). But in the dorsal view (E), a similar number of main trunks of the peripheral axons (arrowheads) extended out of the spinal cord as in the normal (D) and control MO-injected (F) embryos. (AC,A'-C') Lateral view; anterior is leftwards; dorsal is towards the top. (D-F) Dorsal view; anterior is leftwards. Scale bars: in C, 10 µm for A-C; in F, 10 µm for D-F. (G-I) We subdivided the trunk region of the embryos into six longitudinal areas along the dorsoventral axis (D1-D3, V3-V1), and counted the number of branching points of the peripheral axons of Rohon-Beard neurons stained by anti-acetylated -tubulin antibody (G). In AMO-injected embryos, the number of the branching points was prominently reduced (H), especially in the D2 region (I).

View larger version (26K): [in a new window]   Fig. 2. D204 encodes the zebrafish ortholog of PlexinA4 (Accession Number, AB103158). (A) The phylogenic tree indicates that D204 encodes the zebrafish ortholog of PlexinA4. (B) Schematic drawing of the domain structure of the Plexin family and identity of amino acids between zebrafish and mouse PlexinA4. MRS/PSI domain is Met related sequence/plexin, semaphorin, integrin domain. G-P/IPT motif is glycine-proline rich/immunoglobin-like fold shared by plexins and transcription factors motif. SP domain is plexin-specific sex plexin domain. m, mouse; x, Xenopus laevis; z, zebrafish.

View larger version (69K): [in a new window]   Fig. 3. plexinA4 mRNA expression in the embryonic zebrafish brain. (A) Lateral view of a 24 hpf zebrafish embryo. plexinA4-positive neurons and their axons were stained by in situ hybridization (purple signals) for plexinA4 mRNA and by immunohistochemistry (brown signals) for the acetylated -tubulin. plexinA4 is expressed in the nucleus of the anterior commissure (nAC), the nucleus of the posterior commissure (nPC), the nucleus of the postoptic commissure (nPOC), the epiphysis (ep), the nucleus of the medial lateral fascicle (nMLF), the tectum and the hindbrain. MB, midbrain; HB, hindbrain; anterior, leftwards; dorsal towards the top. (B) Dorsal view of a 22 hpf Tg(-actin:GFP) transgenic embryo which expresses GFP (brown signals) in Mauthner neurons (arrows) in the hindbrain. plexinA4 expression (purple signals) is detected in GFP-positive Mauthner neurons (arrows) in the rhombomere 4 (r4) and the segmentally distributed reticulospinal neurons in the other rhombomeres. (C) Dorsal view of an 18 hpf Tg(-actin:GFP) transgenic embryo that expresses GFP in Mauthner neurons (asterisks). White arrowheads indicate the axons from Mauthner neurons and a white arrow indicates 8th neurons. r3-r6, rhombomere 3 to rhombomere 6; ot.v, otic vesicles; anterior is towards the top. (D-I) Cellular localization of the transiently expressed PlexinA4-GFP and dnPlexinA4-GFP fusion proteins in the primary sensory neurons. To visualize the cellular localization of both PlexinA4-GFP and dnPlexinA4-GFP in the primary sensory neurons, we injected plasmids (D,G) that can induce expression of PlexinA4-GFP and dnPlexinA4-GFP fusion protein under control of the primary sensory neuron-specific enhancer (SS) of the zebrafish isl1 gene. In the trigeminal sensory ganglion neurons (E,H) and Rohon-Beard neurons (F,I), both PlexinA4-GFP (E,F) and dnPlexinA4-GFP (H,I) fusion protein were localized in both of the central (arrows in E,F,H,I) and peripheral (arrowheads in E,F,H) axons. The stable transgenic zebrafish line that carries pSS-hsp70:dnPlexinA4-GFP (G) was established. In this transgenic zebrafish [Tg(SS-hsp70:dnPlexinA4-GFP)], we can induce expression of dnPlexinA4-GFP fusion protein ubiquitously by heat-shock treatment (J). e, eye; ot.v, otic vesicles; SS, sensory neuron-specific enhancer of the zebrafish isl1 gene; hsp, zebrafish heat-shock protein 70 (hsp70) promoter; gfp, green fluorescence protein; SV40 polyA, SV40 late polyadenylation site. (E,F,H-J) Lateral view; anterior is leftwards; dorsal is towards the top. Scale bars: 20 µm in A; 10 µm in B and C.

View larger version (52K): [in a new window]   Fig. 5. Time-lapse observation of Rohon-Beard neurons that were separately labeled with Kaede in the embryos with a reduced level of PlexinA4 expression or with ubiquitous overexpression of Slit2. Stacked confocal images of a single Rohon-Beard neuron expressing Kaede transiently under control of the primary sensory neuron-specific enhancer (SS) of the zebrafish isl1 gene (A-F). In comparison with normal embryo (A,D,A',D'), the number of branching points of peripheral axons was reduced in the embryo injected with AMO against plexinA4 (B,E,B',E'). However, the number of branching points increased in the Slit2-overexpressing embryo (C,F,C',F'). (A-F) Confocal images; lateral view; anterior leftwards; dorsal towards the top. (A-C) 23 hpf embryos. (D-F) The same embryos as shown in A-C at 27 hpf. (A'-F') Schematic drawings of A-F in which cell bodies and branching points of the peripheral axons were indicated by black circles and red dots, respectively. Scale bar: 10 µm.

View larger version (62K): [in a new window]   Fig. 6. Transgenic overexpression of dnPlexinA4-GFP in the Rohon-Beard neurons caused reduction in the number of branching of the peripheral axons of Rohon-Beard neurons. Anti-acetylated -tubulin antibody staining of 26 hpf embryos of the transgenic zebrafish (B), which expresses dnPlexinA4-GFP fusion protein in the Rohon-Beard neurons (black circles), showed significant reduction in the number of branching points of the peripheral axons compared with that in the normal 26 hpf embryo (A,C), especially in the D2 region (D). Lateral view; anterior is leftwards; dorsal is towards the top. Scale bar: 10 µm.

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