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Supplementary Figure 1

Characterization of A08n neuron connectivity

(a) Co-labeling of C4da (ppk-CD4-tdTomato) and A08n neurons (6.14.3-Gal4, UAS-CD4-tdGFP). C4da axon terminals overlap with

ladder-like projections of A08n neurons. A08n somata and additional neurons labeled by 6.14.3-Gal4 are indicated by arrows. Scale

bar, 50 m. (b) Flip-out labeling of 6.14.3-Gal4 expressing cells (UAS-CD4-tdTomato) shows that ladder-like projections are specific

to A08n neurons. Scale bars, 50 m. (c) A08n neurons were co-labeled by 6.14.3-Gal4 and 82E12-LexA expressing CD4-tdGFP and

CD4-tdTomato, respectively. A08n somata are indicated by arrows. Scale bar: 50 m. (d) A08n split-Gal4 (82E12-Gal4AD, 6.14.3-

Gal4DBD, UAS-CD4-tdGFP) expression pattern and co-labeling with C4da axon terminals (ppk-CD4-tdTomato). A08n somata and

additionally weakly labeled A01b neurons are indicated by arrows. Scale bar, 50 m. (e) Model of A08n and C4da neuron innervation

A08n and synaptic overlap in VNC cross-sections. (f) A08n neurons expressing presynaptic Brp-short-mCherry and postsynaptic

D 7-GFP markers. Scale bar: 30 m. (g) Syb-GRASP control. Expression of spGFP1-10-Syb in C4da neurons alone shows no

reconstituted GFP signal. Scale bar, 25 m.

Nature Neuroscience: doi:10.1038/nn.4580


Characterization of DP-ilp7 neuron connectivity

(a) Co-labeling of C4da axon terminals (ppk-CD4-tdTomato) with ilp7 neurons (ilp7-Gal4, UAS-CD4-tdGFP) with arrows indicating

DP-ilp7 somata. Scale bar, 50 m. (b,b’) DP-ilp7 neuron morphology visualized by Flp-mediated CD4-tdTomato labeling together

with Fas3-labelled sensory axons (C2da-C4da). Scale bar, 25 m. (b’) Enlarged view of boxed area in (a) and resliced maximal

projections along the YZ and XZ axis (indicated by dotted lines) show overlap of DP-ilp7 neurites with anti-Fas3 labeled sensory

terminals. Scale bars, 5 m. (c) Schematic projection patterns of sensory da neurons and DP-ilp7 neurons is shown. Boxed region is

displays enlarged dorsal view (top panel) and cross-section view (bottom panel) indicating sites of sensory and DP-ilp7 neuron contact

in ventromedial and -lateral regions of the VNC neuropil. (d) ilp7 neurons expressing presynaptic Brp-short-mCherry and postsynaptic

D 7-GFP markers together with anti-Fas3 labeling. Scale bar, 25 m. (d’) Corresponding 3D-view of the boxed regions in (d) shows

specific overlap of DP-ilp7 postsynaptic sites with both ventro-medial and -lateral Fas3 positive sensory terminals. Scale bar, 5 m.

(e) Syb-GRASP control. Expression of spGFP1-10-Syb in sensory da neurons (2-17-Gal4) alone shows no reconstituted GFP signal.

Scale bar: 25 m. (f) Pan-da neuron expression of CD4-tdGFP (21-7-Gal4) shows sensory terminal overlap with DP-ilp7 neurons

(ilp7-LexA, LexOP-CD4-tdTomato). XY maximum projections and XZ projections of indicated regions (dotted lines) are shown. Scale

bars, 10 m.



Analysis of nociceptive circuit elements in larval locomotion and innocuous-touch response

(a) Larval locomotion analysis of freely moving larvae expressing Kir2.1 in C4da, A08n or ilp7 neurons and controls. Animals were

video-captured for 1 min and average locomotion speed was analyzed. A mild increase in locomotion speed was observed for C4da

neuron silencing with Kir2.1 compared to Gal4 control, but not for any other tested neuronal subsets (n as indicated, p<0.001, p>0.05,

Mann-Whitney U test). (b) Cumulative gentle touch scores for C2/3da, C4da, A08n and ilp7 driver lines expressing Kir2.1. C2/3da

neuron inactivation decreased gentle touch behavior (n as indicated, p<0.0001, Mann-Whitney U test), while silencing of other subsets

showed no defect in innocuous touch behavior (n as indicated, p>0.05, Mann-Whitney U test). (b’) Touch response scores according

to Kernan et al.79.



Functional analysis of A08n and ilp7 neuron connectivity and behavior

(a-b) Expression pattern of CsChrimson-GFP using (a) ppk-Gal4 or (b) 27H06-LexA in the (a,b) periphery and (a’,b’) CNS. High level

expression of CsChrimson with ppk-Gal4 resulted in partial dendrite and axon degeneration of C4da neurons. Scale bars, 50 m. (c-f)

A08n neurons are sufficient to elicit nociceptive responses. Flipase-induced mosaic expression of CsChrimson-GFP in only one or few

neurons. 3rd instar larvae were tested for light induced nociceptive behavior and subdivided into rollers/non-rollers. Larval brains of

each group were dissected and examined for their expression pattern. (c) Schematic larval brain with expression pattern of 82E12-

Gal4 showing the nomenclature used for labeled cell clusters. (d) Representative images of larval brains dissected after behavioral

analysis. CsChrimson-GFP expressing neurons of larvae displaying no reaction (“non-rollers”) or nocifensive rolling (“rollers”) after

activation were identified and assigned according to (c). Scale bars, 50 m. (e) Percentage of animals displaying non- vs. rolling with

particular cells expressing CsChrimson. Labeling of A08n neurons was significantly enriched in rolling vs. non-rolling larvae

(p<0.001, two-tailed unpaired Student’s t-test). All other neurons except s2 and l2 showed no enrichment in labeling in rolling larvae

(p>0.05, two-tailed unpaired Student’s t-test). (f) To exclude a role for s2 and l2 cells, the number of larvae showing light induced

rolling (“rollers”) labeling A08n plus the indicated cell types showed that s2 and l2 cells are not correlated with nocifensive responses,

but were co-expressing CsChrimson together with A08n. (g) Kir2.1 mediated inactivation of C4da neurons did not impair A08n

induced optogenetic rolling responses (n as indicated, p>0.05, 2 test). (h) Rolling induced by optogenetic activation of A08n neurons

was not affected by co-activation or silencing of ilp7 neurons (n as indicated, p>0.05, 2 test). (i) Syb-GRASP between ilp7

presynaptic sites and A08n neurons shows no reconstituted GFP signal. Scale bar, 25 m. (j) Syb-GRASP between A08n presynaptic

sites and ilp7 neurons shows reconstituted GFP signal along the medial axon projections of A08n neurons. Scale bar, 25 m. (j’)

Enlarged view from (j’). Scale bar, 10 m. (k) Syb-GRASP control. Expression of spGFP1-10-Syb in A08n neurons alone shows no

reconstituted GFP signal. Scale bar, 25 m. (l) Optogenetic activation of A08n or ilp7 neurons does not result in calcium responses of

DP-ilp7 or A08n neurons respectively (n as indicated, p>0.05, Mann-Whitney U test).



Behavioral analyses of DP-ilp7 and innocuous-touch neuron function

(a) CsChrimson-GFP expression pattern using DP-ilp7-LexA shows specific signal in DP-ilp7 neurons only. Scale bar: 50 m. (b)

Characterization of C2da neuron specific expression of R78A01-Gal4 using UAS-CD4-tdGFP together with ppk-CD4-tdTomato co-

labeling C4da neurons. C2da neuron specific axon terminals are labeled in abdominal segments. Thoracic segments show partial

labeling of C1da neuron projections. Arrows indicate medial terminal projections, arrowheads lateral, C2da neuron specific terminal

projections. (b’) R78A01-Gal4 expresses in a subset of C2da neurons located on the dorsal (ddaB) and ventral (ldaA) body wall.

However, the two ventral C2da neurons are not labeled. Scale bars, 30 m. (c) Mechanonociceptive behavior of 3rd instar larvae at 96h

AEL after the first (I) and second (II) mechanical stimulation with a 45 mN von Frey filament. Kir2.1 mediated silencing of C3da or

C2/3da neurons inhibits mechano-nociceptive responses (n as indicated, p<0.001, 2 test). (d) Tetanus toxin light chain (TnT)

mediated silencing of C2/3da or A08n neurons strongly inhibits mechano-nociceptive responses (n as indicated, p<0.001, 2 test). (e)

Tetanus toxin light chain (TnT) mediated silencing of C2/3da but not C2da neurons alone impairs innocuous touch behavior (n as

indicated, p<0.001, Mann-Whitney U test). (f) Animals lacking ilp7 peptide (ilp7ko) show significantly increased mechano-nociceptive

responses after the 1st stimulus (p<0.001, chi2 test) but not after the 2nd stimulus (n as indicated, p>0.05, 2 test).



Ilp7 neuron-derived sNPF is required for mechanonociception

(a) sNPF is expressed in DP-ilp7 but not in other ilp7 neurons. The sNPF expression pattern was visualized with a MIMIC enhancer

trap expressing GFP under control of the endogenous sNPF promoter (sNPFMI01807). Co-labeling of ilp7 neurons (ilp7-LexA,LexAop-

CD4-tdTomato) showed overlapping expression in DP-ilp7 (arrowheads) but not posterior ilp7 neurons (arrows). Scale bar, 50 m. (b)

Anti-sNPF and anti-Fas3 staining of 3rd instar larval brain preparations of w1118 control, sNPFc00448 or sNPFMi01807 homozygous

animals showed strongly reduced sNPF levels in both mutant alleles. Scale bar, 50 m. (c) sNPF-RNAi in ilp7 but not C4da neurons

impairs mechano-nociceptive responses (n as indicated, p<0.05, 2 test). (d) Heterozygous sNPF mutant animals (sNPFc00448) show

impaired mechano-nociceptive responses which are enhanced by ilp7 neuron specific sNPF knockdown by RNAi. Expression of

sNPF1/2 in ilp7 neurons partially rescues rolling behavior compared to sNPF-RNAi (n as indicated, p<0.05, 2 test). (e) Inhibition of

sNPF-R function in C2/3da or C4da neurons expressing a dominant negative variant did not impair innocuous touch responses (n as

indicated, p>0.05, Mann-Whitney U test). (f) Inhibition of sNPF-R function in basin neurons using RNAi mediated knockdown or a

dominant negative variant did not impair mechano-nociceptive responses (n as indicated, p>0.05, 2 test).



Modality-specific requirement of DP-ilp7 neuron activity and sNPF function in mechanonociception

(a) Latency of nociceptive rolling responses was measured in 3rd instar larvae at 96h AEL after local simulation with a probe heated to

46 °C. Kir2.1 mediated silencing of ilp7 neurons does not induce thermo-nociceptive defects (n as indicated, p>0.05, Kruskal-Wallis

ANOVA). (b) Inhibition of sNPF-R function in C4da neurons expressing a dominant negative variant did not significantly impair

thermo-nociceptive responses (n as indicated, p>0.05, Kruskal-Wallis ANOVA). (c) Schematic of testing activity and sNPF function

in C4da presynaptic and A08n neuron calcium responses. (c’) shows larval preparation used for calcium imaging with optical access

to the VNC and von Frey filament stimulation of an abdominal segment (a4) on the intact posterior body wall. (d) Mechano-

nociceptive calcium responses of A08n neurons (82E12-Gal4, UAS-GCaMP6m) after mechanical stimulation of abdominal segment

4-6 (45 mN) with and without ilp7 neuron inhibition by Kir2.1. (d’) Maximum responses ( Fmax/F0) were plotted and compared

(n=11/genotype, p<0.05, Mann-Whitney test). (e) Modality specific nociceptive circuit function requires distinct circuit and

neuromodulatory components. Harsh mechanical touch activates C2-C4da neurons, which results in activation of DP-ilp7 neurons

(indicated by color coded arrows). sNPF derived from DP-ilp7 neurons is required for paracrine feedback signaling via sNPF-R in

C2/3/4da neurons (light blue arrows). sNPF-R signaling in turn results in C4da neuron presynaptic facilitation and successive increase

in A08n response to trigger nociceptive escape behavior. Thermo-nociceptive behavior is likely encoded by additional circuit

components independent of the identified network elements. (f) Synaptic model of sNPF and sNPF-R action. Activity results in local

release of sNPF from DP-ilp7 neurons. Released sNPF can then activate sNPF-Rs expressed in sensory C2/C3/C4da neurons in a

paracrine fashion.


Supplementary Table 1: Full experimental genotypes Figure 1 panel genotype (a,a’) w,hs-Flp/w;tub>FRT>Gal80>FRT>/6.14.3-Gal4,UAS-CD4-GFP,ppk-CD4-

tdTomato (b-b’’) w/w; 82E12-Gal4AD/UAS-Brp-short-mCherry; 6.14.3-Gal4DBD/UAS-Da7-GFP (c,c’) w/w; 82E12-Gal4AD/27H06-LexA,LexAop-brp-short-mCherry; 6.14.3-

Gal4DBD/UAS-Da7-GFP (d,d’) w/w; 27H06-LexA/UAS-spGFP11-CD4,LexAop-spGFP1-10-Syb;82E12-Gal4/+ (f,f’) w/w; +/+; trpA1-QF,QUAS-Rab3-GFP/6.14.3-Gal4,UAS-CD4-tdGFP Figure 2 (a) w,hs-Flp/w; +/+ ;ilp7-Gal4/UAS->FRT> spGFP1-10-CD4-3xflag6xHis>FRT>

spGFP11-CD4-tdTomato (b-b’’) w/w; UAS-Brp-short-mCherry/+; ilp7-Gal4/UAS-Da7-GFP (c,c’) w/w; LexAop-spGFP11-CD4,UAS-spGFP1-10-Syb/+; pkk-Gal4/ilp7-LexA (d,d’) w/w; 21-7-Gal4/UAS-spGFP1-10-Syb,LexAop- spGFP11-CD4; ilp7-

LexA,LexAop- spGFP11-CD4-tdTomato/+ (f-f’’) w/w; 21-7-Gal4/UAS-Brp-short-mCherry; ilp7-LexA,LexAop- spGFP11-CD4-

tdTomato/+ Figure 3 (a-b) Ln/6.14.3-

Gal4: w/w;27H06-LexA/LexAop-CsChrimson-GFP; 6.14.3-Gal4/ UAS-GCaMP6m

82E12-Gal4:

w/w;27H06-LexA/LexAop-CsChrimson-GFP; 82E12-Gal4/ UAS-GCaMP6m

45mN: w/w;+/+;82E12-Gal4/UAS-GCaMP6m (c) w1118: w1118/w1118 TrpA11: TrpA11 Kir2.1: w/w;+/+;UAS-Kir2.1/+ C4da: w/w;+/+;ppk-Gal4/+ w/w;+/+;ppk-Gal4/UAS-Kir2.1 A08n: w/w;+/+;6.14.3-Gal4/+ w/w;+/+; 6.14.3-Gal4/UAS-Kir2.1 w/w;+/+;82E12-Gal4/+ w/w;+/+; 82E12-Gal4/UAS-Kir2.1 w/w; 82E12-Gal4AD/ +; 6.14.3-Gal4DBD/ + w/w; 82E12-Gal4AD/UAS-Brp-short-mCherry+; 6.14.3-Gal4DBD/ UAS-Kir2.1 (d) Ctl: w/w:+/+;UAS-CsChrimson-GFP/+ w/w:+/+;LexAop-CsChrimson-GFP/+ C4da: w/w;+/+;ppk-Gal4/ UAS-CsChrimson-GFP w/w;27H06-LexA/+; LexAop-CsChrimson-GFP/+ A08n: w/w;+/+; 82E12-Gal4/ UAS-CsChrimson-GFP w/w;+/+; 6.14.3-Gal4/ UAS-CsChrimson-GFP w/w; 82E12-Gal4AD/ +; 6.14.3-Gal4DBD/ UAS-CsChrimson-GFP w/w;+/+; ilp7-Gal4/ UAS-CsChrimson-GFP (e) C4da+: w/w;27H06-LexA/+;LexAop-CsChrimson-GFP,UAS-Kir2.1/+ A08nKir2.1: w/w;27H06-LexA/+;LexAop-CsChrimson-GFP,UAS-Kir2.1/82E12-Gal4 w/w;27H06-LexA/+;LexAop-CsChrimson-GFP,UAS-Kir2.1/6.14.3-Gal4 ilp7Kir2.1: w/w;27H06-LexA/+;LexAop-CsChrimson-GFP,UAS-Kir2.1/ilp7-Gal4 A08nKir2.1,

ilp7Kir2.1: w/w;27H06-LexA/+;LexAop-CsChrimson-GFP,UAS-Kir2.1/ ilp7-Gal4,82E12-Gal4


Figure 4 panel (a) ilp7: w/w;+/+;ilp7-Gal4/+ w/w;+/+; ilp7-Gal4/+ w/w;+/+;UAS-TnTE/ilp7-Gal4 w/w;+/+;UAS-Kir2.1/ilp7-Gal4 DP-ilp7: w/w;+/+; LexAop-Kir2.1/+ w/w;+/+; DP-ilp7-LexA /+ w/w;+/+; LexAop-Kir2.1/DP-ilp7-LexA (b) ctl: w/w;+/+;ilp7-Gal4/+(625nm exposure) ilp7 CsChrimLo

w: w/w;+/+; ilp7-Gal4/UAS-CsChrimson-GFP(low light exposure)

CsChrim62

5nm: w/w;+/+; ilp7-Gal4/UAS-CsChrimson-GFP(625nm exposure)

DP-ilp7 ctl: w/w;+/+; LexAop-CsChrimson-GFP/+(625nm exposure) w/w;+/+; DP-ilp7-Gal4/+(625nm exposure) CsChrim

Low: w/w;+/+; DP-ilp7-Gal4/LexAop-CsChrimson-GFP(low light exposure)

CsChrim 625nm:

w/w;+/+; DP-ilp7-Gal4/ LexAop-CsChrimson-GFP(625nm exposure)

(c,c’) C1/bd: w/w;LexAop-GCaMP6sUAS-CsChrimson-GFP/+;ilp7-LexA,LexAop-spGFP11-

CD4- tdTomato/2-21-Gal4 C2: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/+;ilp7-LexA,LexAop-spGFP11-

CD4- tdTomato/R78A01-Gal4 C2/4: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/ppk-Gal4;ilp7-LexA,LexAop-

spGFP11-CD4- tdTomato/R78A01-Gal4 C3: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/nompC-Gal4;ilp7-LexA,LexAop-

spGFP11-CD4- tdTomato/+ C4: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/+;ilp7-LexA,LexAop-spGFP11-

CD4- tdTomato/ ppk-Gal4 C2/3: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/+;ilp7-LexA,LexAop-spGFP11-

CD4- tdTomato/C1003.3-Gal4 C2-4: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/ppk-Gal4;ilp7-LexA,LexAop-

spGFP11-CD4- tdTomato/C1003.3-Gal4 C1-3: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/ppk-Gal80,21-7-Gal4;ilp7-

LexA,LexAop-spGFP11-CD4- tdTomato/+ C1-4: w/w;LexAop-GCaMP6s,UAS-CsChrimson-GFP/21-7-Gal4;ilp7-LexA,LexAop-

spGFP11-CD4- tdTomato/+ 45mN: w/w; w/w;LexAop-GCaMP6s /+;ilp7-LexA,LexAop-spGFP11-CD4- tdTomato/+ (d) TnT: w/w;+/+;UAS-TnTE/+ C2da: w/w;+/+;R78A01-Gal4/+ w/w;+/+;UAS-TnTE/R78A01-Gal4 C3da: w/w;tsh-Gal80/+;19-12-Gal4/+ w/w; tsh-Gal80/+;UAS-TnTE/19-12-Gal4 C4da: w/w;+/+;ppk-Gal4/+ w/w; +/+;UAS-TnTE/ppk -Gal4 (e) C2da: w/w; R78A01-LexA/+; LexAop-CsChrimson-GFP /+ w/w;ppk-Gal80/+; UAS-CsChrimson-GFP/R78A01-Gal4 C3da: w/w;tsh-Gal80/+;19-12-Gal4/ UAS-CsChrimson-GFP C2/3da: w/w; tsh-Gal80/+;C1003.3-Gal4/ UAS-CsChrimson-GFP (f) C2da+: w/w; R78A01-LexA/+; LexAop-CsChrimson-GFP,UAS-Kir2.1/+ ilp7Kir2.1: w/w; R78A01-LexA/+;LexAop-CsChrimson-GFP,UAS-Kir2.1/ilp7-Gal4


Figure 5 panel (a-b) w/w;+/+;ilp7-Gal4,UAS-CD4-tdGFP/ppk-GFPsp11-CD4-tdTomato (c) w1118: w1118/w1118 w/w; sNPFc00448/sNPFc00448

w/w; sNPFMI01807/sNPFMI01807 w/w; sNPFMI01807/sNPFc00448 (d) ctl: w/w;+/+;UAS-sNPF-RNAi w/w;+/+;UAS-sNPF1 w/w;+/UAS-sNPF2 w/w; sNPFMI01807/+;ilp7-Gal4/+ w/w; sNPFMI01807/+;ilp7-Gal4/ UAS-sNPF-RNAi w/w; sNPFMI01807/+;ilp7-Gal4/ UAS-sNPF1 w/w; sNPFMI01807/UAS-sNPF2;ilp7-Gal4/+ (e) w/w; sNPF-RMI08722/ sNPF-RMI08722

(f) ctl: w/w;+/+;UAS-sNPF-RRNAi#1 w/w;+/+;UAS-sNPF-RRNAi21 w/UAS-sNPF-RDN

C2/3da: w/w; tsh-Gal80/+;C1003.3-Gal4/+ w/w;tsh-Gal80/+;C1003.3-Gal4/UAS-sNPF-RRNAi#1 w/w; tsh-Gal80/+;C1003.3-Gal4/UAS-sNPF-RRNAi21 w/UAS-sNPF-RDN; tsh-Gal80/+;C1003.3-Gal4/+

C4da: w/w; +/+;ppk-Gal4/+ w/w;+/+; ppk-Gal4/UAS-sNPF-RRNAi#1 w/w;+/+; ppk-Gal4/UAS-sNPF-RRNAi21 w/UAS-sNPF-RDN;+/+; ppk-Gal4/+

Figure 6 (a) C4da: w/w; +/+;UAS-TnTE/+ w/w;+/+;ppk-Gal4/+ w/w; +/+;UAS-TnTE/ppk -Gal4 (b) A08n: w/w; +/+;UAS-TnTE/+ w/w; 82E12-Gal4AD/+; 6.14.3-Gal4DBD/+ w/w; 82E12-Gal4AD/+; 6.14.3-Gal4DBD/UAS-TnTE (c) C3da: w/w; +/+;UAS-TnTE/+ w/w;tsh-Gal80/+;C1003.3-Gal4/+ w/w;tsh-Gal80/+;C1003.3-Gal4/UAS-TnTE (d) ilp7: w/w; +/+;UAS-TnTE/+ w/w;+/+;ilp7-Gal4/+ w/w;+/+;ilp7-Gal4/UAS-TnTE

(e) sNPF: w1118: w1118/w1118 w/w; sNPFc00448/sNPFc00448

w/w; sNPFMI01807/sNPFMI01807 (f) sNPF-R: w/w;+/+; ppk-Gal4/UAS-sNPF-RRNAi#1

w/w; +/+;ppk-Gal4/+ w/w;+/+; ppk-Gal4/UAS-sNPF-RRNAi#1

Figure 7 (a,a’) w/w;27H06-LexA/LexAop-GCaMP6m; UAS-Kir2.1/+ w/w;27H06-LexA/LexAop-GCaMP6m; ilp7-Gal4/UAS-Kir2.1 (b,b’) w/w;27H06-LexA,LexAop-GCaMP6m/+; ilp7-Gal4/+


panel w/w;27H06-LexA,LexAop-GCaMP6s/sNPFc00448; ilp7-Gal4/+ w/w;27H06-LexA,LexAop-GCaMP6s/sNPFc00448; ilp7-Gal4/UAS-sNPF1 (c,c’) w/w;82E12-LexA/LexAop-GCaMP6s; UAS-Kir2.1/+ w/w;82E12-LexA/LexAop-GCaMP6s; UAS-Kir2.1/C1003.3-Gal4 w/w;82E12-LexA/LexAop-GCaMP6s; UAS-Kir2.1/ppk-Gal4 w/w;82E12-LexA/LexAop-GCaMP6s; UAS-Kir2.1/ilp7-Gal4 (d,d’) w/w;82E12-LexA,LexAop-GCaMP6s/+; C1003.3-Gal4/+ w/UAS-sNPF-RDN;82E12-LexA,LexAop-GCaMP6s/+; C1003.3-Gal4/+ w/w; 82E12-LexA,LexAop-GCaMP6s/+; C1003.3-Gal4/UAS-sNPF-RRNAi#1 w/w; 82E12-LexA,LexAop-GCaMP6s/+; C1003.3-Gal4/UAS-sNPF-RRNAi#2

(e,e’) w/w;82E12-LexA,LexAop-GCaMP6s/+; ppk-Gal4/+ w/UAS-sNPF-RDN;82E12-LexA,LexAop-GCaMP6s/+; ppk-Gal4-Gal4/+ w/w; 82E12-LexA,LexAop-GCaMP6s/+; ppk-Gal4-Gal4/UAS-sNPF-RRNAi#1 w/w; 82E12-LexA,LexAop-GCaMP6s/+; ppk-Gal4-Gal4/UAS-sNPF-RRNAi#2 Supplementary Figure 1

(a) w /w; +/+; 6.14.3-Gal4,UAS-CD4-GFP,ppk-CD4-tdTomato/+ (b) w,hs-Flp/w; +/+ ; 6.14.3-Gal4/UAS->FRT> spGFP1-10-CD4-

3xflag6xHis>FRT>spGFP11-CD4-tdTomato (c) w/w; 82E12-LexA,LexAop-spGFP11-CD4-tdTomato/+; 6.14.3-Gal4,UAS-CD4-

tdGFP (d) w/w; 82E12-Gal4AD/+; 6.14.3-Gal4DBD/UAS-CD4-GFP,ppk-CD4-tdTomato (f) w/w; 82E12-Gal4AD/27H06-LexA,LexAop-brp-short-mCherry; 6.14.3-

Gal4DBD/UAS-Da7-GFP (g) w/w; 27H06-LexA/UAS-spGFP11-CD4,LexAop-spGFP1-10-Syb;+/+ Supplementary Figure 2

(a) w/w; +/+; ilp7-Gal4,UAS-CD4-tdGFP/ppk-spGFP11-CD4-tdTomato (b) w,hs-Flp/w; +/+ ;ilp7-Gal4/UAS->FRT> spGFP1-10-CD4-

3xflag6xHis>FRT>spGFP11-CD4-tdTomato (d,d’) w/w; UAS-Brp-short-mCherry/+; ilp7-Gal4/UAS-Da7-GFP (e) w/w; 21-7-Gal4/UAS-spGFP1-10-Syb,LexAop-spGFP11-CD4; +/+ (f) w/w; 21-7-Gal4,UAS-CD4-tdGFP/+; ilp7-LexA,LexAop-spGFP11-CD4-

tdTomato/+ Supplementary Figure 3

(a) w1118: w1118/w1118 Kir2.1: w/w;+/+;UAS-Kir2.1/+ C4da: w/w;+/+;ppk-Gal4/+ w/w;+/+;ppk-Gal4/UAS-Kir2.1 A08n: w/w;+/+;82E12-Gal4/+ w/w;+/+; 82E12-Gal4/UAS-Kir2.1 w/w; 82E12-Gal4AD/ +; 6.14.3-Gal4DBD/ + w/w; 82E12-Gal4AD/ +; 6.14.3-Gal4DBD/ UAS-Kir2.1 ilp7: w/w;+/+;ilp7-Gal4/+ w/w;+/+; ilp7-Gal4/UAS-Kir2.1 (b) w1118: w1118/w1118 Kir2.1: w/w;+/+;UAS-Kir2.1/+ C2/3da: w/w;tsh-Gal80/+;C1003.3-Gal4/+ w/w; tsh-Gal80/+;UAS-Kir2.1/ C1003.3-Gal4 C4da: w/w;+/+;ppk-Gal4/+


panel w/w; +/+;UAS- Kir2.1/ppk -Gal4 A08n: : w/w;+/+;6.14.3-Gal4/+ w/w;+/+; 6.14.3-Gal4/UAS-Kir2.1 w/w;+/+;82E12-Gal4/+ w/w;+/+; 82E12-Gal4/UAS-Kir2.1 w/w; 82E12-Gal4AD/ +; 6.14.3-Gal4DBD/ + w/w; 82E12-Gal4AD/ +; 6.14.3-Gal4DBD/ UAS-Kir2.1 ilp7: w/w;+/+;ilp7-Gal4/+ w/w;+/+; ilp7-Gal4/UAS-Kir2.1 Supplementary Figure 4

(a,a’) w/w;+/+;ppk-Gal4/ UAS-CsChrimson-GFP (b,b‘) w/w;27H06-LexA/+;LexAop-CsChrimson-GFP/+ (d-f) w/hs-Flp; tub>FRT>Gal80>FRT>/+; 82E12-Gal4/ UAS-CsChrimson-GFP (g) A08n: w/w;+/UAS-CsChrimson-GFP;LexAop-Kir2.1/82E12-Gal4 C4daKir2.1: w/w; 27H06-LexA/UAS-CsChrimson-GFP;LexAop-Kir2.1/82E12-Gal4 (h) A08n: w/w; +/ UAS-CsChrimson-GFP;+/82E12-Gal4 A08n/

ilp7: w/w; +/UAS-CsChrimson-GFP;+/82E12-Gal4,ilp7-Gal4

A08n: w/w; 82E12-Gal4/ UAS-CsChrimson-GFP;LexAop-Kir2.1/+ A08n/

ilp7 Kir2.1: w/w; 82E12-Gal4/UAS-CsChrimson-GFP;LexAop-Kir2.1/ilp7-LexA

(i) w/w; LexAop-spGFP11-CD4,UAS-spGFP1-10-Syb/82E12-LexA; ilp7-Gal4/+ (j,j’) w/w; LexAop-spGFP11-CD4,UAS-spGFP1-10-Syb/+; 82E12-Gal4/ilp7-LexA (k) w/w; LexAop-spGFP11-CD4,UAS-spGFP1-10-Syb/+; 82E12-Gal4/+ (l) A08n>

ilp7: w/w; 82E12-Gal42nd/UAS-CsChrimson-GFP;ilp7-LexA/ LexAop-GCaMP6m

ilp7>A08n: w/w;82E12-LexA,LexAop-GCaMP6s / UAS-CsChrimson-GFP;ilp7-Gal4/+ Supplementary Figure 5

(a) w/w; +/+;DP-ilp7-LexA/LexAop-CsChrimson-GFP (b,b’) w/w; +/+; R78A01-Gal4/UAS-CD4-GFP,ppk-CD4-tdTomato (c) C3da: w/w;tsh-Gal80/+;19-12-Gal4/+ w/w; tsh-Gal80/+;UAS-Kir2.1/19-12-Gal4 C2/3da: w/w;tsh-Gal80/+;C1003.3-Gal4/+ w/w; tsh-Gal80/+;UAS-Kir2.1/ C1003.3-Gal4 (d) C2/3da: w/w;tsh-Gal80/+;C1003.3-Gal4/+ w/w; tsh-Gal80/+;UAS- TnTE /C1003.3-Gal4 A08n: w/w;+/+;82E12-Gal4/+ w/w; +/+;UAS-TnTE/82E12-Gal4 (e) TnT: w/w;+/+;UAS- TnTE/+ C2da: w/w;+/+;R78A01-Gal4/+ w/w;+/+;UAS-TnTE/R78A01-Gal4 C2/3da: w/w;tsh-Gal80/+;C1003.3-Gal4/+


w/w; tsh-Gal80/+;UAS-TnTE/ C1003.3-Gal4 (f) w1118: w1118/w1118 Ilp7ko: w,Ilp71 Supplementary Figure 6

(a) w/w; sNPFMI01807/+; ilp7-LexA,LexAop-spGFP11-CD4- tdTomato /+ (b) w1118: w1118/w1118 w/w; sNPFc00448/sNPFc00448

w/w; sNPFMI01807/sNPFMI01807 (c) ctl: w/w;+/+;UAS-sNPF-RNAi ilp7: w/w; +/+;ilp7-Gal4/+ w/w; +/+;ilp7-Gal4/ UAS-sNPF-RNAi C4da: w/w; +/+;ppk-Gal4/+ w/w; +/+;ppk-Gal4/ UAS-sNPF-RNAi (d) ctl: w/w;+/+;UAS-sNPF-RNAi w/w;+/+;UAS-sNPF1 w/w;+/UAS-sNPF2 w/w; sNPF c00448/+;ilp7-Gal4/+ w/w; sNPF c00448/+;ilp7-Gal4/ UAS-sNPF-RNAi w/w; sNPF c00448/+;ilp7-Gal4/ UAS-sNPF1 w/w; sNPF c00448/UAS-sNPF2;ilp7-Gal4/+ (e) ctl: w/UAS-sNPF-RDN

C2/3da: w/w; tsh-Gal80/+;C1003.3-Gal4/+ w/UAS-sNPF-RDN; tsh-Gal80/+;C1003.3-Gal4/+

C4da: w/w; +/+;ppk-Gal4/+ w/UAS-sNPF-RDN;+/+; ppk-Gal4/+

(f) ctl: w/w;+/+;UAS-sNPF-RRNAi#1

w/UAS-sNPF-RDN

basin: w/w; +/+;R72F11-Gal4/+ w/w;+/+; R72F11-Gal4/UAS-sNPF-RRNAi#1 w/UAS-sNPF-RDN; +/+; R72F11-Gal4/+


(a) ilp7: w/w; +/+;UAS-Kir2.1/+ w/w;+/+;ilp7-Gal4/+ w/w;+/+;ilp7-Gal4/UAS- Kir2.1

(b) C4da: w/UAS-sNPF-RDN

w/w; +/+;ppk-Gal4/+ w/UAS-sNPF-RDN;+/+; ppk-Gal4/+

(d,d’) w/w; 82E12-Gal4/UAS-GCaMP6m; ilp7-LexA/+ w/w; 82E12-Gal4/UAS-GCaMP6m; ilp7-LexA/LexAop-Kir2.1


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