Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP⁺ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP⁺ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP⁺ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ^#p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ^##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ^###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.