Spastin regulates endosomal tubulation in spinal motor axonal growth cones. (a) Gross phenotype of 52-hpf control, spastin morphant (MO^spast), and IST1 morphant (MO^IST1) zebrafish larvae. Bar, 500 µm. (b) Whole-mount immunohistochemistry of 26-hpf zebrafish control, MO^spast, and MO^IST1 embryos labeled with the motor neuron marker synaptotagmin 2 (znp-1). Images are lateral views of the trunk; anterior is to the left. Axonal stumps (asterisks) and abnormal branching (arrows) of spinal motor neuron axons were observed in all spastin and IST1 morphants. Bar, 25 µm. (c–i) Spinal neurons were cultured from control (c), spastin morphant (d), and IST1 morphant (e) 24-hpf Tg(Mnx1:mGFP) embryos, in which GFP is selectively expressed in motor neurons. Cultured neurons were labeled with SNX1 and GFP antibodies, and the mean number of SNX1-positive tubules and complex tubular structures per 100-µm² region of each motor axon growth cone (large arrows in GFP-labeled images and inset boxes in SNX1-labeled images; small arrows indicate the cell body) was calculated by counting ∼30 cells per condition. Bars, 5 µm. (f and g) The mean values obtained in three (f) or five (g) independent experiments were plotted. (h and i) In these experiments, spastin and IST1 depletion was verified by immunoblotting. Anti–histone H2b immunoblotting is shown to verify equal protein loading. Error bars show SEMs.