Fignl1 colocalizes with the kinesin motor KIF1Bβ and is actively transported in zebrafish sMN axons. (A) Whole-mount in situ hybridization with kif1bβ antisense riboprobe at the 18-somite stage (18 s), 24 and 48 hpf. (B) Whole-mount in situ hybridization of 24-hpf embryos with fignl1 antisense probe. (A and B) Images are lateral views of the embryo, anterior to the left. Insets are higher magnifications of the spinal cord. Scale bars, 200 µm. (C) Z-stack of spinning-disk confocal images showing Fignl1 and KIF1Bβ colocalization in zebrafish axons of 56-hpf Tg(HuC:GAL4; 14UAS: Fignl1-TRFP; 14UAS: YFP-Hu_KIF1Bβ) transgenic larvae. Right-hand panels are higher magnifications of the boxed region of the corresponding left-hand panel. Scale bars, 10 µm. (D) Fluorescence intensity profile of Fignl1-TRFP and YFP-Hu_KIF1Bβ along the axon boxed in C. (C and D) White and black arrows indicate Fignl1 and KIF1Bβ colocalization. (E) Still images from time-lapse recordings of Fignl1-TRFP axonal transport in sMN axons of 56-hpf Tg(HuC:GAL4; 14UAS: Fignl1-TRFP) transgenic larvae. Right-hand panels are higher magnifications of the axon boxed in the left-hand panel. Yellow arrowheads track a Fignl1-TRFP-positive vesicle moving in the retrograde direction. Scale bars, 7.4 µm. (F) Left: Representative kymogram of Fignl1-TRFP motility in sMN axons. Right: schematic kymogram illustrating anterograde (black) and retrograde (gray) Fignl1-TRFP traces. Vertical scale bar, 85 s; horizontal scale bar, 5.6 µm. (G) Mean anterograde and retrograde velocities of Fignl1-TRFP-positive vesicles in sMN axons (n = 6) selected randomly from four transgenic fish. Mean velocities per axon were extracted from 50 kymogram traces. A.U., arbitrary units.