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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 {alpha}-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({alpha}-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({alpha}-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.