ATG1 and ATG2 morphants exhibit different spinal motor neuron defects. (A) Immunolabelling of secondary motor neurons (sMN) in 72 hpf COMOspATG1 (n=29), COMOspATG2 (n=29), MOspATG1 (n=41) and MOspATG2 (n=32) Tg(Hb9:GFP) transgenic larvae using zn-5 antibody. Lateral views of the trunk, anterior towards the left. Dotted lines indicate the horizontal myoseptum (i.e. axon guidance choice point). White arrowheads indicate misguided rostral nerves of ATG1 morphants while arrows indicate the ectopic sorting of sMN somata from the spinal cord of ATG2 morphants. Empty arrowheads and asterisks indicate missing rostral and dorsal nerves, respectively. Scale bar: 50 µm. (B-E) Quantification of sMN defects in 72 hpf control and spastin morphant larvae (24 spinal hemisegments were analysed per larva). *P≤0.05, ***P≤ 0.001; Kruskal–Wallis ANOVA test with Dunn's post test. The number (n) of larvae analysed per condition is indicated under the corresponding histogram bar. Error bars are s.e.m. (F) Distinctive navigational behaviour of ATG1 and ATG2 morphant sMN axons at the choice point. Representative stills of time-lapse recordings of sMN axon outgrowth monitored in 32 spinal hemisegments of 40-72 hpf Tg(Hb9:GFP) transgenic larvae injected with MOspATG1, MOspATG2 or a control morpholino (MOscr). White arrowheads indicate the aberrant caudal turning of ATG1 morphant sMN rostral axons at the horizontal myoseptum (dotted line) while empty arrowheads indicate the erroneous ventral growth of ATG2 morphant sMN axons beyond this choice point. Arrows track the aberrant migration of sMN somata along motor tracts of ATG2 morphants. Scale bar: 50 µm.