Statistics see Supplementary DataNATURE COMMUNICATIONS | (2019)10:3506 | 41467-019-11408-1 | www.nature.comnaturecommunicationsARTICLEaC2da3da4da C4da A08nNATURE COMMUNICATIONS | 41467-019-11408-27H06-LexALexAop-Brpshort-mCherry; 82E12-Gal4UAS-Drep2-GFPfas3-Cybrp-sh-mCherryDrep2-GFPMerge zUAS-TaoRNAiz UAS-TaoRNAi +UAS-hTaoK2WTb400 C4da presynapses 300 200 100nsz UAS-TaoRNAi +UAS-hTaoK2 A135PwoKTa-hASUzUAS-TaoRNAic250 A08n PB28 Autophagy postsynapses 200 150 100 50P t wd C4da-A08n synapses nsnsens Ectoptic A08n postsynapses P t wwUAS-hTa oKA1 35 P tKAoKAao-h TaTaTa-h TaoK-h ToKASAS-h-hASUUASASUUUUAS-TaoRNAiUAS-TaoRNAiU27H06-LexA LexAop-Brpshort-mCherry; 82E12-Gal4 UAS-Drep2-GFPFig. 5 Conserved Tao kinase activity regulates growth and connectivity. a Confocal pictures of larval VNC hemisegments (96 h AEL) in handle or with TaoRNAi expression in A08n neurons, without the need of or with co-expression of hTaoK2wt or hTaoK2A135P. Pictures show anti-Fas3 immunostaining labeling C2da, C3da, and C4da sensory axons (blue), with synaptic marker expression labeling C4da presynapses (magenta), and A08n postsynapses (green). Scale bar = five . XZ projections of each and every channel are shown below. Scale bar = 2 . b Quantitative analysis of synaptic profiles in handle or with TaoRNAi expression in A08n neurons, without having or with co-expression of hTaoK2wt or hTaoK2A135P for b C4da neuron presynapses, c A08n postsynapses, d C4da 08n synapses and e ectopic A08n postsynapses inside the C2daC3da domain. P 0.05, P 0.01, P 0.0001 SD, ANOVA with a number of comparisons and Dunnett’s post-hoc test (for exact P-values and statistics see Supplementary Information 1). Control: n = 18, UAS-TaoRNAi: n = 9, UAS-TaoRNAi + hTaoK2wt: n = 22, UAS-TaoRNAi + hTaoK2A135P: n =optogenetic actuator CsChrimson42 and monitored calcium signals in A08n neurons with or without having Tao perturbation applying the calcium sensor GCaMP6m (Fig. 6c ). Under control conditions, C3dacho neuron activation didn’t drive calcium responses in A08n (Fig. 6d, e). In contrast, activation of C3dacho neurons in larvae expressing TaoRNAi in A08n neuronsreproducibly resulted in A08n calcium responses, demonstrating that ectopic C3da and A08n synapses are functional. We also tested if loss of Tao affects functional connectivity of C4da and A08n neurons. Employing optogenetic activation of C4da neurons, we detected a considerable decrease in A08n neuron responses right after loss of Tao in comparison with controls (SupplementaryNATURE COMMUNICATIONS | (2019)10:3506 | 41467-019-11408-1 | www.nature.comnaturecommunicationsASUAS-TaoRNAi-hTaoKoKAPtNATURE COMMUNICATIONS | 41467-019-11408-ARTICLEC3da-A08n synapsesaC2da3da4daC3da presynapses82E12-Gal4AD,LexOP-syb-spGFP1-10, UAS-CD4-spGFP-11; six.14.3-Gal4DBD,NompC-LexAfas3-Cy24 hSyb-spGFP1-recGFPMerge96 hbSyb-GRASP SynapsesUAS-TaoRNAiUAS-TaoRNAiControlUAS-TaoRNAic80 60 40 20 0 24 h 48 h72 h96 h120 h82E12-Gal4 (A08n) 3clpro Inhibitors medchemexpress UAS-GCaMP6m nompC-LexA (C3dacho) LexAop-CsChrimson82E12-Gal4AD,LexOP-syb-spGFP1-10, UAS-CD4-spGFP-11 ; 6.14.3-Gal4DBD, NompC-LexAd20Fx F0 [ ]A08n responseTaoRNAieControlFmax F0 [ ]A08n response+0 0 tro ao U AS -T CSecondsFig. 6B ). These information show that loss of Tao in A08n neurons offers rise to functional ectopic connectivity with C3da sensory neurons although partially impairing C4da 08n neuron physiological output. Ectopic connectivity alters somatosensory network function. To dissect the effect of Tao-dependent connectivity changes, weanalyzed behavioral consequences of Tao loss of function in this method. We focu.
