Non-invasive in vivo imaging of mitochondrial transport in zebrafish M cells. (A) M cells of zebrafish larvae were labeled with fluorescent proteins by single-cell electroporation at 6 days post-fertilization (dpf). White arrows indicate the M soma (leftward) and the M axon terminal (rightward). White box 1, proximal axon area; White box 2, distal axon area. Asterisk, cloacal pores. (B) Schematic of constructs used to label M cells (DsRed2) and mitochondria (mito-EGFP) through single-cell electroporation in living zebrafish larvae. (C) The labeled M axon was imaged with a confocal microscope, showing multiple tracts of mitochondria (mito-EGFP, middle) in the axon (DsRed2, top); merged image, bottom. (D) Moving mitochondria were monitored in the axon (area in white box). Arrowheads, moving mitochondria (anterograde, green; retrograde, red). (E–G) Mitochondrial motility (E), transport direction (F; anterograde, blue; retrograde, pink), and speed (G) in axons at 7 dpf. Scale bars: (A) 500 μm, (C) 5 μm, (D) 2.5 μm. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Error bars represent SEM.

Nocodazole treatment disrupts mitochondrial trafficking. (A) The kymographs (left) depict moving mitochondria in vehicle- (top) and nocodazole-treated (below) zebrafish larvae at 6 dpf. Right kymographs are hand-drawn traces, and the diagonal lines represent moving mitochondria (anterograde, blue lines; retrograde, pink lines). (B–E) Mitochondrial motility (B), transport direction (C; anterograde, blue; retrograde, pink), and speed (D, in the area of proximal axons; E, in the a rea of distal axons) in vehicle- (DMSO; gray) and nocodazole- (red) treated zebrafish larvae at 6 dpf. ^∗∗p < 0.01, ^∗∗∗p < 0.001. Error bars represent SEM.

M axons were completely severed by laser and have regenerative capacity after injury. (A) M axons were axotomized by two-photon laser before (left) and after (right). Asterisk, injury site; white arrowheads, cloacal pores. (B) Injured axons regenerated to hundreds of microns 2 days after axotomy. Asterisk, injury site. (C) Using transgenic Tg (Tol-056) lines, the uncut axon (bottom) was longer than the severed axon (top) which stopped at the lesion site, implying that laser-induced injured axons were physically disconnected. White arrow, severed axon; white arrowhead, uncut axon. (D) One-photon laser was used to photobleach axons. Green fluorescence recovered after several minutes. When the photobleached axon was labeled with red dye at 24 hpb, its structure was still physically connected. Scale bars: (A–C) 100 μm, (D) 50 μm.

Dibutyryl cyclic adenosine monophosphate boosts axon regeneration and promotes mitochondrial motility. (A,B) The axon regeneration in vehicle- and db-cAMP-treated zebrafish at 2 dpa. (C) The kymographs display axonal transport of mitochondria before and after vehicle and db-cAMP treatments. Right kymographs are hand-drawn traces corresponding to the left kymographs (anterograde, blue lines; retrograde, pink lines). (D–F) Mitochondrial motility (D), transport direction (E), and speed (F) before and after vehicle and db-cAMP treatments. Scale bar: (A) 100 μm. ^∗p < 0.05, ^∗∗p < 0.01. Error bars represent SEM.