Extended Data Fig. 1: Activity-dependent remodelling of motor neuron dendrites during a motor circuit critical period.

a–e, Tonic activation of motor neurons during embryogenesis induces dendrite retraction. a, Schematic of the activation paradigm used in this study. For activation of aCC–RP2 motor neurons, RN2-gal4/lexA drove expression of UAS-CsChrimson::mCherry or lexAop/UAS-CsChrimson::mVenus. Crosses were established on day 0 and fed exclusively on yeast paste supplemented with 0.5 mM ATR (required for maximum Chrimson activity) and changed daily for a minimum of 3 days. Timed embryo collections were performed on day 3 for a duration of 1.5 h. Sustained light activation (10,550 lx) was followed by immediate dissection. Optogenetic silencing experiments using UAS-GtACR2::eYFP followed the same scheme. b–e, Activation of aCC–RP2 motor neurons by Chrimson channelrhodopsin induces dendrite retraction. b–d, Representative 3D projections of brains expressing Chrimson::mCherry in aCC–RP2 motor neurons at 0 h ALH following activation during embryonic stage 17 (st17). After activation, brains were categorized qualitatively as control (b), mildly reduced (c) or strongly reduced (d) based on the extent of aCC–RP2 dendritic elaboration (dashed white boxes). Scale bar, 5 μm. e, Distribution of each phenotypic class in control, dark-reared animals versus animals whose aCC–RP2 motor neurons were Chrimson-activated for 15 min, 1 h or 4 h. Dark-reared controls were used throughout as aCC–RP2 motor neurons show sensitivity to Chrimson in the absence of ATR after 15 min and 4 h of Chrimson activation. N (in histogram) represents number of larvae. f–i, Complementary assays to define the motor circuit critical period. f, g, Silencing of aCC–RP2 motor neurons for 1 h by GtACR2 (400 ms pulses of 488 nm light per second) (f) or expression of the temperature sensitive shibire^ts to block synaptic transmission (active at 30 °C) (g), resulted in significant dendrite extension at 0 h ALH, but had no effect at 8 h ALH. N represents number of larvae. 0 h GtACR2: control (N = 11), 1 h silencing (N = 12). 8 h GtACR2: N = 10 per condition. GtACR2 statistics within group (one-way ANOVA): 0 h (P < 0.0001), 8 h (P < 0.76). GtACR2 statistics across groups (two-way ANOVA): P < 0.003. 0 h shibire^ts: control (N = 7), 1 h silencing (N = 6). 8 h shibire^ts: control (N = 6), 1 h silencing (N = 7). shibire^ts statistics within group (one-way ANOVA): 0 h (P < 0.0002), 8 h (P < 0.86). shibire^ts statistics across groups (two-way ANOVA): P < 0.003. GtACR2 genetics: RN2-gal4, UAS-GtACR2::eYFP. shibire^ts genetics: RN2-gal4, UAS-shibire^ts, UAS-myr::GFP. h, i, Activation of aCC–RP2 motor neurons for 1 h by Chrimson (600 ms pulses of 561 nm light per second) (h) or expression of the thermogenetic activator TrpA1 (inactive at 22 °C, fires at ~30 Hz at 27 °C) (i), resulted in significant dendrite retraction at 0 h ALH, but had no effect at 8 h ALH. N represents number of larvae. 0 h Chrimson: control (N = 12), 1 h activation (N = 14). 8 h Chrimson: control (N = 12), 1 h activation (N = 10). Chrimson statistics within group (one-way ANOVA): 0 h (P < 0.0001), 8 h (P < 0.6). Chrimson statistics across groups (two-way ANOVA): P < 0.001. 0 h TrpA1: control (N = 6), 1 h activation (N = 11). 8 h TrpA1: control (N = 5), 1 h activation (N = 6). TrpA1 statistics within group (one-way ANOVA): 0 h (P < 0.0001), 8 h (P < 0.25). TrpA1 statistics across groups (two-way ANOVA): P < 0.0001. Chrimson genetics: RN2-gal4, UAS-Chrimson::mCherry. TrpA1 genetics: RN2-gal4, UAS-TrpA1, UAS-myr::GFP. Data are mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Diamonds denote significance following two-way ANOVA when one-way and two-way ANOVA are displayed together. N values reflect biological replicates from 2 independent experiments.