Fig. 1

Robust and directional regrowth of Mauthner axons following laser-mediated transection. a, b 6-day-old double transgenic zebrafish larvae with labeling of the Mauthner neuron: Tg(hspGFF62a) and Tg(UAS:gap43_1-20-citrine), driving expression of membrane-bound citrine in the Mauthner using the Gal4-UAS system; bright field in (a), citrine labeled Mauthner axonal membrane along the ventral spinal cord in (b). Yellow arrowheads mark the future axon transection site at the ninth body segment. c, d In 5-day-old larval zebrafish, the Mauthner axon, labeled by Tg(hspGFF62a) and Tg(UAS:gap43_1–20-RFP) is surrounded by GFAP-positive glial cells in TgBAC(GFAP:GFAP-GFP) (c) and myelinating oligodendrocytes in Tg(MBP:EGFP-CAAX) (d). e–k Laser-mediated transection of the Mauthner axon, intact axon before transection with the future transection site marked by a yellow arrowhead (e); retracted and sealed proximal and distal axon stump at 1 hpt (f), both at the ninth body segment. The axon stump distal to the transection site (here at the level of the anus) remains intact for several hours after transection (g) and then undergoes Wallerian degeneration, leaving behind the fragmented axon (h). Of eight axons, one fragmented earlier than 13.5 hpt and another later than 36 hpt. The mean time of fragmentation at the level of the anus for the remaining six axons was 28.1 ± 7.6 hpt. At 15 hpt, the distal stump was still intact in this example (white asterisk) and the proximal stump had started to regrow (i). Initially, the Mauthner axon regrew multidirectionally in seven of eight larvae, with some sprouts pointing caudally across the transection site; others pointed rostrally toward the cell body in the brain stem (white arrowheads; i). j, k At 48 hpt, the regrowing proximal axon had successfully crossed the transection site (yellow arrowhead). Misdirected axonal projections at the transection site were corrected (j) and the axon had regrown caudally. The entire length of the regrown axon is shown in (k). Scale bars: 200 µm in (a); 30 µm in (c), 30 µm in (e), and 30 µm in (k)