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.