control of feeding by piezo-mediated gut mechanosensation ......2020/09/11 · gut mechanosensation...

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Control of feeding by Piezo-mediated gut mechanosensation in Drosophila 1

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Soohong Min1, Yangkyun Oh2, Pushpa Verma3, David Van Vactor3, Greg S.B. Suh2,4, and 3

Stephen D. Liberles1# 4

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1Howard Hughes Medical Institute, Harvard Medical School, Department of Cell Biology, 6

Boston, Massachusetts 02115, 2Skirball Institute, NYU School of Medicine, New York, NY 7

10016, 3Harvard Medical School, Department of Cell Biology, Boston, Massachusetts 02115, 8

4KAIST, Department of Biological Sciences, Daejeon, South Korea, 9

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#Corresponding Author and Lead Contact: e-mail: [email protected] 11

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.CC-BY-NC-ND 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprintthis version posted September 11, 2020. ; https://doi.org/10.1101/2020.09.11.293712doi: bioRxiv preprint

https://doi.org/10.1101/2020.09.11.293712

http://creativecommons.org/licenses/by-nc-nd/4.0/

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SUMMARY 1

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Across animal species, meals are terminated after ingestion of large food volumes, yet 3

underlying mechanosensory receptors have so far remained elusive. Here, we identify an 4

essential role for Drosophila Piezo in volume-based control of meal size. We discover a 5

rare population of fly neurons that express Piezo, innervate the anterior gut and crop (a 6

food reservoir organ), and respond to tissue distension in a Piezo-dependent manner. 7

Activating Piezo neurons decreases appetite, while Piezo knockout and Piezo neuron 8

silencing cause gut bloating and increase both food consumption and body weight. 9

These studies reveal that disrupting gut distension receptors changes feeding patterns, 10

and identify a key role for Drosophila Piezo in internal organ mechanosensation. 11

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INTRODUCTION 1

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Mechanosensory neurons detect a variety of environmental forces that we can touch or hear, as 3

well as internal forces from organs and tissues that control physiological homeostasis (Abraira 4

and Ginty, 2013; Ranade et al., 2015; Umans and Liberles, 2018). In many species, specialized 5

mechanosensory neurons innervate the gastrointestinal tract and are activated by tissue 6

distension associated with consuming a large meal (Williams et al., 2016; Zagorodnyuk et al., 7

2001). Gut mechanosensation may provide an evolutionarily conserved signal for meal 8

termination, as gut distension inhibits feeding in many species and evokes the sensation of 9

fullness in humans (Phillips and Powley, 1996; Rolls et al., 1998). However, how gut distension 10

receptors contribute to long-term control of digestive physiology and behavior is unclear, as 11

tools for selective pathway manipulation are lacking. Identifying neuronal mechanisms involved 12

in detecting the volume of ingested food would provide basic insights into this fundamental 13

mechanosensory process, and in humans, perhaps clinical targets for feeding and metabolic 14

disorders. 15

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Here, we investigated roles and mechanisms of food volume sensation in the fruit fly Drosophila 17

melanogaster. Volumetric control of feeding was classically studied in a larger related insect, the 18

blowfly, with relevant mechanosensory hotspots identified in the foregut and crop, an analog of 19

the stomach (Dethier and Gelperin, 1967; Gelperin, 1967). In Drosophila, chemosensory 20

neurons detect nutrients in the periphery and brain to control appetite, with some neurons 21

positively reinforcing feeding during starvation conditions (Bjordal et al., 2014; Dus et al., 2015; 22

Miyamoto et al., 2012). In contrast, the importance of gut mechanosensation in Drosophila 23

feeding control and digestive physiology has not been similarly investigated; mechanosensory 24

neurons of the gustatory system sense food texture and modulate ingestion (Sanchez-Alcaniz et 25

al., 2017; Zhang et al., 2016), and other mechanosensory neurons in the posterior gut control 26



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defecation and food intake (Olds and Xu, 2014; Zhang et al., 2014). In contrast, food storage 1

during a meal occurs primarily in the anterior gut (Lemaitre and Miguel-Aliaga, 2013; Stoffolano 2

and Haselton, 2013). Enteric neurons of the hypocerebral ganglion innervate the fly crop, 3

foregut, and anterior midgut, and lesioning of the recurrent nerve (which contains neurons of the 4

hypocerebral ganglion) in Drosophila and blowfly increases feeding duration (Dethier and 5

Gelperin, 1967; Gelperin, 1967; Pool et al., 2014). Together, these prior studies raise the 6

possibility that a subpopulation of enteric neurons in Drosophila could be specialized to sense 7

meal-associated gut distension. 8

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RESULTS AND DISCUSSION 10

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Piezo-expressing enteric neurons innervate the gastrointestinal tract 12

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To explore whether food volume sensation occurs in Drosophila and to investigate underlying 14

mechanisms, we first asked whether neurons expressing various mechanosensory ion channels 15

innervated the anterior gut. Several mechanosensitive ion channels have been reported in 16

Drosophila, including TRP channels (Nompc, Nanchung, and Inactive), the degenerin/epithelial 17

sodium channel Pickpocket (Ppk), transmembrane channel-like (Tmc) protein, and Piezo (Coste 18

et al., 2012; Montell, 2005; Zhang et al., 2016; Zhong et al., 2010). We obtained Gal4 driver 19

lines that mark neurons containing mechanoreceptor proteins or related family members, 20

induced expression of membrane-tethered (CD8-GFP) or dendritically targeted (DenMark) 21

fluorescent reporters, and visualized neuronal innervation of the anterior gut. We observed a 22

small group of Piezo-expressing enteric neurons located in the hypocerebral ganglion (~5-6 23

neurons per fly), and a dense network of Piezo fibers throughout the crop and anterior midgut 24

(Figure 1A, 1B). Hypocerebral ganglion neurons were similarly labeled and anterior gut 25

innervation similarly observed in three independent Piezo-Gal4 driver lines (Figure S1A), but not 26



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in other Gal4 lines analyzed. We noted Nanchung expression in some epithelial cells of the crop 1

duct, but not in crop-innervating neurons. The hypocerebral ganglion and adjacent corpora 2

cardiaca together contain ~35 neurons per fly based on Elav immunohistochemistry, and Piezo 3

neurons therein were distinct from other neurons that expressed the fructose receptor Gr43a 4

(~5 neurons per fly) or the glucagon analog adipokinetic hormone (Akh, ~20 neurons per fly) 5

(Figure 1C, 1D, S1B). Piezo neurites formed a muscle-associated lattice in the gut and 6

ascending axons contributed to the recurrent nerve (Figure 1E, 1F). Drosophila Piezo is 7

expressed in many cell types (Kim et al., 2012), but its expression in gut-innervating sensory 8

neurons was not reported previously. Drosophila Piezo was previously shown to confer 9

mechanically activated currents when expressed in human cells, and to mediate mechanical 10

nociception (Coste et al., 2012; Kim et al., 2012). Furthermore, vertebrate Piezo homologs play 11

diverse mechanosensory roles, including in internal sensation of airway volume and blood 12

pressure (Min et al., 2019; Nonomura et al., 2017; Zeng et al., 2018). We hypothesized that 13

Drosophila enteric neurons which express Piezo and innervate the anterior gut might mediate 14

volumetric control of appetite. 15

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Piezo neurons control feeding behavior 17

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To explore this model, we activated and silenced Piezo neurons using genetic approaches and 19

monitored feeding behavior. We expressed temperature-sensitive Shibire (Shits) that blocks 20

synaptic transmission at non-permissive temperatures (>32°C) in Piezo neurons using three 21

independent Piezo-Gal4 drivers (Piezo>Shits). Piezo>Shits flies were reared at a permissive 22

temperature (18°C) and later tested for physiological and behavioral changes at 32°C. To 23

measure feeding behavior, flies were fasted for 24 hours, and then given brief access to food 24

containing a dye for visualization and quantification of ingestion (Figure 2A). Piezo>Shits flies 25

from all three genotypes fed ravenously, and histological examination of the gastrointestinal 26



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tract showed gut bloating with increased crop size (Figure 2B). For comparison, genetic 1

silencing of other gut-innervating neurons labeled in GMR51F12-Gal4 flies did not impact 2

appetite or cause crop distension. These findings indicate that disrupting Piezo neurons 3

compromises gut volume homeostasis and associated control of feeding. 4

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To test the effects of activating Piezo neurons on food consumption, we drove expression of the 6

temperature-regulated ion channel Trpa1 in Piezo neurons using Piezo-Gal4 lines 7

(Piezo>Trpa1). Thermogenetic activation of Trpa1 in Piezo cells, achieved by transferring 8

Piezo>Trpa1 flies from 18°C to 30°C, suppressed food intake after a 24 hour fast, and also 9

blocked meal-associated increases in crop volume, with similar results observed using three 10

different Piezo-Gal4 drivers (Figure 2C). Since many cell types express Piezo (Kim et al., 2012), 11

we next used approaches for intersectional genetics involving Gal80, a dominant suppressor of 12

Gal4-mediated gene induction to restrict Trpa1 expression to fewer cells. First, we drove Gal80 13

expression broadly in neurons using Piezo>Trpa1; Elav-Gal80 flies, and observed restoration of 14

normal feeding behavior, indicating the relevant Piezo expression site to be neurons (Figure 2D, 15

2E). Among neurons, Piezo-Gal4 drove expression in various peripheral sensory neurons, the 16

ventral nerve cord, brain, and hypocerebral neurons. Differential expression control could be 17

partially achieved using a Cha-Gal80 driver which silences Gal4-mediated expression in the 18

ventral nerve cord and many central neurons, but not in gut-innervating hypocerebral neurons or 19

a few cells of the proboscis, intestine, and brain (Figure 2D, S2A). Thermogenetic experiments 20

in Piezo>Trpa1; Cha-Gal80 flies also caused robust suppression of feeding behavior (Figure 21

2E). Intestinal cells are unlikely to contribute to feeding phenotypes in Piezo>Trpa1; Cha-Gal80 22

flies based on experiments involving Piezo>Trpa1; Elav-Gal80 flies; to provide additional 23

evidence, thermogenetic activation of intestinal Piezo cells using Escargot-Gal4; UAS-Trpa1 24

flies also had no effect on feeding (Figure S2B, S2C). In separate studies (Greg Suh, 25

unpublished data), a role for central Piezo neurons in feeding regulation has also been 26



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observed, consistent with the significant differences we observe in feeding following 1

thermogenetic activation experiments involving Piezo-Gal4; UAS-Trpa1 and Piezo-Gal4; UAS-2

Trpa1; Cha-Gal80 flies (Figure 2E). Additional studies are needed to isolate the contributions of 3

hypocerebral ganglion Piezo neurons to feeding control. 4

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Piezo enteric neurons respond to crop-distending stimuli 6

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Next, we investigated the response properties of Piezo-expressing enteric neurons. We 8

analyzed neuronal activity using a transcriptional reporter system involving CaLexA through 9

which sustained neural activity drives expression of GFP. CaLexA reporter was expressed in 10

Piezo neurons using Gal4 drivers, along with an orthogonal activity-independent CD8-RFP 11

reporter for normalization. For validation and determination of response kinetics, Trpa1-induced 12

activation of Piezo neurons increased CaLexA reporter levels gradually, with maximal induction 13

by 24 hours (Figure S3A). First, we asked whether hypocerebral Piezo neurons, and for 14

comparison hypocerebral Gr43a neurons which function as peripheral sugar sensors, changed 15

activity with feeding state (Figure 3A, 3B). For both neuron types, we observed that CaLexA-16

driven GFP expression was low after a fast or in flies fed ad libitum, but was strikingly elevated 17

when flies engorged themselves on a sucrose diet (Figure 3A, 3B, S3B). Sucrose consumption 18

could potentially stimulate both gut chemosensors and mechanosensors, as an increase in crop 19

volume was observed compared with flies fed ad libitum (Figure S3C). We next asked whether 20

activity changes in enteric neurons depended on the content of ingested material. We compared 21

CaLexA-mediated GFP expression levels in flies fed for 24 hours with 1) sucrose, 2) sucralose, 22

a sweetener that lacks caloric value and stimulates peripheral gustatory receptors but not 23

internal Gr43a neurons, 3) water alone after a period of water deprivation, or 4) water alone ad 24

libitum. Flies extensively consumed sucrose, sucralose, and water when water-deprived, 25

resulting in acute increases in crop volume that were not observed in flies given only water ad 26



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libitum (Figure 3C). Enteric Gr43a neurons displayed elevated levels of CaLexA-mediated GFP 1

expression after engorgement on sucrose, which is converted into fructose and glucose, but not 2

sucralose or water, consistent with a role for these neurons in sensing nutritional carbohydrates 3

(Miyamoto and Amrein, 2014). In contrast, enteric Piezo neurons were activated more generally 4

by sucrose, sucralose, and deprivation-induced water ingestion, but not in controls given only 5

water ad libitum, with responses correlated to the extent of gut distension. The observation that 6

Piezo neurons were similarly activated by water- and sucrose-induced gut distension indicated a 7

sensory mechanism that does not require chemosensation of particular nutrients. Together, 8

these findings suggest a model of two segregated sensory pathways through the hypocerebral 9

ganglion, with Gr43a neurons responding to sugars and Piezo neurons responding to anterior 10

gut mechanosensation. 11

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Piezo knockout alters enteric neuron responses and fly feeding behavior 13

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Next, we asked whether the Piezo receptor mediates neuronal responses of hypocerebral 15

neurons. We obtained Piezo knockout flies, and crossed them with flies harboring alleles 16

enabling the CaLexA reporter system in Piezo neurons (using Piezo-Gal459266 flies with the 17

Piezo-Gal4 transgene remote from the endogenous Piezo locus). Remarkably, hypocerebral 18

ganglion neurons marked in Piezo-Gal4 flies but lacking Piezo expression did not respond to 19

engorgement by sucrose, sucralose or water, even though the crops of Piezo knockout flies 20

were distended (Figure 3A, 3B). (As shown below, the extent of distension is actually more 21

pronounced in Piezo knockout flies, yet CaLexA-mediated responses were not observed). A 22

lack of neuronal responses in Piezo knockout flies is not due to gross deficits in the ability to 23

produce reporter, as Trpa1-mediated activation of Piezo neurons in Piezo knockout flies was 24

sufficient to induce a CaLexA-mediated response (Figure S3D). Furthermore, Piezo neurons 25

displayed similar anatomical innervation of the anterior gut, suggesting that the deficit was not 26



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due to developmental miswiring (Figure S3E). Instead, enteric neurons of Piezo knockout flies 1

seemingly fail to respond to crop-distending stimuli due to a mechanosensory defect. 2

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Next, we asked whether Piezo knockout flies display changes in behavior or physiology. We 4

measured feeding behavior in Piezo knockout flies, and for comparison, isogenic w1118 flies. For 5

synchronization, flies were fasted for 18 hours and then given ad libitum access to dye-labeled 6

food for 30 minutes. Remarkably, Piezo knockout flies increased food intake and had visually 7

observable crop distension (Figure 4A, 4B, 4C). Moreover, Piezo knockout flies fed ad libitum 8

on normal fly food for 5-7 days showed an increase in body weight compared to control flies 9

(Figure 4D). Automated analysis of feeding patterns was performed involving an EXPRESSO 10

platform, and Piezo knockout flies displayed an increase in food intake and feeding bout 11

duration but a similar frequency of feeding bout initiation (Figure 4E). Abnormal gut distension 12

and feeding behavior were rescued by exogenous expression of Piezo-GFP in Piezo knockout 13

neurons driven by Piezo-Gal4 (Figure 4F, S4A). Unlike Drop-dead knockout flies which have an 14

enlarged crop due to defective food passage into the intestine (Peller et al., 2009), Piezo 15

knockout flies have normal food transit, a normal lifespan, and increased defecation rates, 16

presumably due to increased feeding (Figure S4B, S4C, S4D, S4E). Other than food-induced 17

distension, the anatomy of the crop appeared normal in Piezo knockout flies, as visualized by 18

histology of crop muscle, analysis of cell density, and volume measurements during starvation 19

(Figure S4F, S4G, S4H). As mentioned above, knockout of Piezo does not impact the extent of 20

gut innervation (Figure S3E); furthermore, thermogenetic Trpa1-mediated activation of Piezo 21

neurons in Piezo knockout flies suppressed feeding behavior (Figure S4I), indicating that neural 22

circuits downstream of enteric Piezo neurons were intact and remained capable of eliciting a 23

behavioral response after Piezo knockout. Piezo also functions to guide the differentiation of gut 24

enteroendocrine cells from mechanosensitive intestinal stem cells (ISCs) (He et al., 2018); 25

however selectively restoring Piezo expression in ISCs using Escargot-Gal4 (Esg-Gal4) that 26



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broadly marks Piezo-expressing ISCs did not rescue crop volume and feeding phenotypes 1

(Figure S4J). We also note that while the crops of Piezo knockout flies are distended, the flies 2

generally do not burst and do stop eating (although bursting does rarely occur), suggesting 3

eventual engagement of a secondary satiety pathway, perhaps through nutrient sensors or 4

posterior gut mechanoreceptors. Taken together, our data indicate a role for Piezo in sensing 5

anterior gut distension, and that disrupting the function of Piezo neurons, or Piezo itself, causes 6

substantial changes to gut physiology and feeding behavior. 7

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Food-induced gut distension is thought to be an evolutionarily conserved signal for meal 9

termination, yet underlying mechanisms and sensory receptors have long remained mysterious. 10

Furthermore, whether food volume sensors are required for normal feeding control has 11

remained unknown, as tools for selective loss-of-function were not available without knowing 12

underlying sensory mechanisms. Here, we reveal a role for Drosophila Piezo in neurons that 13

innervate the anterior gut and sense the size of a meal. Disrupting this pathway increases food 14

consumption and body weight, and causes swelling of the gastrointestinal tract. These studies 15

demonstrate that anterior gut mechanosensation contributes to the complex calculus that 16

underlies the decision to eat, and provide a foundation for the comparative physiology and 17

evolution of feeding control. Moreover, understanding related pathways in humans may enable 18

new therapies for treating obesity and other food consumption disorders. 19

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ACKNOWLEDGMENTS 21

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We thank Norbert Perrimon, Bryan Song, Dragana Rogulja for reagents and advice, Jinfei Ni for 23

blinded analysis of behavior, Norbert Perrimon, Craig Montell, Julie Simpson, Hubert Amrein, 24

and Bloomington Drosophila Stock Center for flies, and Hansine Heggeness and Exelixis facility 25



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at Harvard Medical School for fly food and stock maintenance. SM is supported by an American 1

Heart Association postdoctoral fellowship (20POST35210914); PV and DVV are supported by 2

NINDS award NS090994; GSBS is supported by NIH R01 grants (RO1DK116294, 3

RO1DK106636) and Samsung Science and Technology Foundation (SSTF-BA-1802-11). SDL 4

is an investigator of the Howard Hughes Medical Institute. 5

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AUTHOR CONTRIBUTIONS 7

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SM, SDL, and GSBS designed experiments and analyzed data, SM and SDL wrote the 9

manuscript, SM performed all experiments except YO performed EXPRESSO analyses and Akh 10

co-staining, and PV and DVV assisted with fly husbandry. 11

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DECLARATION OF INTERESTS 13

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The authors declare no competing interests. 15

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FIGURE LEGENDS 1

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Figure 1. Piezo neurons innervate the gastrointestinal tract. 3

(A) Wholemount image of the digestive tract from a Piezo-Gal4 (59266); UAS-DenMark fly 4

visualized with immunofluorescence for DenMark (red, anti-RFP) and Phalloidin (blue) to label 5

visceral muscle, HCG: hypocerebral ganglion, scale bar: 100 µm. (B) Immunofluorescence for 6

RFP (red) and Elav (blue) in the HCG from a Piezo-Gal4; UAS-CD8RFP fly, scale bar: 10 µm. 7

(C) Immunofluorescence for GFP (green) and Akh (magenta) in the corpora cardiaca (CC) and 8

HCG from a Piezo-Gal4; UAS-CD8GFP fly, scale bar: 10 µm. (D) Native GFP and RFP 9

fluorescence from the HCG of a Piezo-Gal4; UAS-CD8RFP; Gr43a-LexA; LexAop-CD8GFP fly, 10

scale bar: 10 µm. (E) Image of the recurrent nerve (arrows) labeled by native RFP fluorescence 11

in a Piezo-Gal4; UAS-CD8-RFP fly and Phalloidin immunofluorescence (blue), scale bar: 10 µm. 12

(F) The anterior midgut (left) and crop (right) of a Piezo-Gal4; UAS-DenMark fly visualized by 13

immunofluorescence for DenMark (green) and Phalloidin (magenta), scale bar: 50 µm. 14

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Figure 2. Piezo neurons control feeding behavior. 16

(A) Depiction of the colorimetric feeding assay. (B) Fasted flies with Shibire alleles indicated 17

were given brief access (30 min) to dye-labeled food at 32°C, and feeding indices and crop 18

sizes were calculated, n (feeding index): 10-16 trials involving 120-192 flies, n (crop size): 9-13 19

flies, mean ± SEM, ***p<0.0005, **p<0.005, *p<0.05, ns: not significant by ANOVA Dunnett’s 20

multiple comparison test. (C) Fasted flies with Trpa1 alleles indicated were given brief access 21

(30 min) to dye-labeled food at 30°C, and feeding indices and crop sizes were calculated, n 22

(feeding index): 10-20 trials involving 120-240 flies, n (crop size): 6-12 flies, mean ± SEM, 23

***p<0.0005, **p<0.005, ns: not significant by ANOVA Dunnett’s multiple comparison test. (D) 24

Native RFP fluorescence in brain (top), ventral nerve cord (VNC, middle) and hypocerebral 25



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ganglion (HCG, bottom) of Piezo-Gal459266; UAS-CD8RFP flies with Gal80 alleles indicated, 1

scale bar: 100 µm (brain, VNC), 20 µm (HCG). (E) Fasted flies with Trpa1 alleles indicated were 2

given brief access (30 min) to dye-labeled food at 30°C, and feeding indices were calculated, n: 3

14-20 trials involving 168-240 flies, mean ± SEM, ***p<0.0005, **p<0.005, ns: not significant by 4

ANOVA Dunnett’s multiple comparison test. 5

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Figure 3. Piezo mediates enteric neuron responses to crop-distending stimuli. 7

(A) Flies of genotypes indicated were provided solutions of 1) sucrose, 2) sucralose, 3) water 8

alone after a period of water deprivation (water), or 4) water alone ad libitum for 24 hours 9

(control). Representative images of native CaLexA-induced GFP reporter (green) and CD8RFP 10

(red) fluorescence visualized in enteric Gr43a neurons (left), Piezo neurons (middle) or Piezo 11

neurons lacking Piezo (right), scale bar: 10 µm. (B) Quantification of CaLexA-induced GFP 12

fluorescence in individual RFP-expressing neurons from flies in A, n: 33-67 neurons from 5-15 13

flies, mean ± SEM, ***p<0.0001, ns: not significant by ANOVA Dunnett’s multiple comparison 14

test. (C) Visualization of the crop from flies given stimuli indicated after 24 hours (sucrose, 15

sucralose, control) or 15 minutes (water), scale bar: 100 µm. 16

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Figure 4: Piezo knockout alters fly feeding behavior. 18

(A) Fasted wild type and Piezo knockout female flies were given brief access to dye-colored 19

food and imaged, scale bar: 0.5 mm. (B) Representative images of the crop (arrow) in wild type 20

and Piezo knockout flies, scale bar: 100 µm, (C) Calculated feeding indices (left) and crop sizes 21

(right) from flies in A, n (feeding index: 17 trials involving 204 flies, n (crop size): 14 flies, mean ± 22

SEM, ***p<0.0001 by unpaired t-test. (D) Body weights of wild type and Piezo knockout flies fed 23

regular food ad libitum, n: 31-34 trials involving 93-102 flies, mean ± SEM. ***p<0.0001 by 24

unpaired t-test. (E) Feeding parameters of fasted wild type and Piezo knockout male flies was 25



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analyzed using the EXPRESSO assay for 30 minutes after food introduction to determine 1

overall food consumption, feeding duration per bout, and the number of bouts, n: 21-22 flies, 2

mean ± SEM, ***p<0.0005, ns: not significant by unpaired t-test. (F) Calculated feeding indices 3

(left) and crop sizes (right) from Piezo rescue and control flies indicated, n (feeding index): 20-4

30 trials involving 240-260 flies, n (crop size): 13-18 flies, mean ± SEM, ***p<0.0005, **p<0.005 5

by ANOVA Dunnett’s multiple comparison test, ns: not significant by unpaired t test. 6

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Figure S1. Innervation of the gastrointestinal tract by Piezo neurons. 8

(A) Wholemount image of the digestive tract from two additional Piezo-Gal4; UAS-CD8RFP fly 9

lines visualized with native RFP fluorescence (red) and Phalloidin immunofluorescence (blue) to 10

label visceral muscle, HCG: hypocerebral ganglion, scale bar: 50 µm. (B) The average number 11

of neurons in the hypocerebral ganglion and corpora cardiaca labeled per fly by 12

immunofluorescence of Elav (total), Akh (AKH) and RFP (Piezo) in Piezo-Gal4; UAS-CD8RFP 13

flies, and native GFP fluorescence (Gr43a) in Gr43a-LexA; LexAop-CD8GFP flies, n: 7-10 flies, 14

mean ± SEM. 15

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Figure S2. Visualizing and manipulating subtypes of Piezo neurons. 17

(A) Native RFP fluorescence in tissues indicated from Piezo-Gal4 (59266); UAS-CD8RFP flies 18

with or without Cha-Gal80, scale bars: 100 µm. (B) Wholemount images of native RFP 19

fluorescence in the brain, proventriculus (orange outline: HCG) and midgut of Esg-Gal4; UAS-20

CD8RFP flies (left), and wholemount images of native GFP fluorescence in the midgut of Esg-21

Gal4, UAS-CaLexA, UAS-Trpa1 flies at 18°C and 30°C (right). Scale bars: 100 µm. (C) Fasted 22

flies with alleles indicated were given brief access (30 min) to dye-labeled food at 30°C, and 23

feeding indices were calculated, n: 8-14 trials involving 96-168 flies mean ± SEM, ***p<0.0005, 24

ns: not significant by ANOVA Dunnett’s multiple comparison test. 25



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1

Figure S3. Responses and innervation patterns of Piezo neurons in wild type and Piezo 2

knockout flies. 3

(A) Piezo-Gal4; UAS-CaLexA; UAS-Trpa1 flies were placed at 30°C for indicated time periods 4

with ad libitum food. Representative pseudocolor images (left) and quantification (right) of native 5

CaLexA-induced GFP reporter fluorescence in the hypocerebral ganglion. GFP fluorescence 6

was visually transformed to a color map indicating fluorescence intensity, n: 10-18 flies, mean ± 7

SEM, scale bar: 10 µm. (B) Piezo-Gal4; UAS-CaLexA; UAS-CD8RFP flies were provided for 24 8

hours with sucrose, water alone (starved) or regular food ad libitum (Ad libitum). Representative 9

images (left) of native CaLexA-induced GFP reporter (green) and CD8RFP (red) fluorescence, 10

and quantification (right) of CaLexA-induced GFP fluorescence in individual RFP-expressing 11

neurons, n: 44-82 neurons from 8-15 flies, mean ± SEM, ***p<0.0001, ns: not significant by 12

ANOVA Dunnett’s multiple comparison test, scale bar: 10 µm. (C) Crop sizes from wild type flies 13

fed as indicated, n: 17-28 flies, mean ± SEM, ***p<0.0001 by ANOVA Dunnett’s multiple 14

comparison test. (D) Piezo-Gal4; UAS-CaLexA; UAS-Trpa1 (Piezo neurons) and Piezo-Gal4; 15

UAS-CaLexA; UAS-Trpa1; Piezo knockout (Piezo-null neurons) flies were placed at 30°C with 16

ad libitum food for 24 hours. Representative pseudocolor images (left) and quantification (right) 17

of native CaLexA-induced GFP reporter fluorescence in the hypocerebral ganglion. GFP 18

fluorescence was visually transformed to a color map indicating fluorescence intensity, n: 22-23 19

flies, mean ± SEM, ns: not significant by unpaired t-test, scale bar: 10 µm. (E) Wholemount 20

images of the digestive tract from Piezo-Gal4; UAS-CD8RFP and Piezo-Gal4; UAS-CD8RFP; 21

Piezo knockout flies visualized with immunofluorescence for RFP (green, anti-RFP) and 22

Phalloidin (magenta) to label visceral muscle, scale bar: 50 µm. 23

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Figure S4. Physiological characterization of Piezo knockout flies. 25



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(A) Immunofluorescence for GFP (green) and Elav (magenta) in the hypocerebral ganglion 1

(HCG) of Piezo rescue flies [Piezo knockout; Piezo-Gal4 (59266); UAS-Piezo-GFP], scale bar: 2

20 µm. (B) Visualizing (left) and quantifying (right) intestinal transit of dye-colored food in wild 3

type (WT), Drop-dead knockout (Drd KO) and Piezo knockout (Piezo KO) flies, n: 10-11 flies, 4

mean ± SEM, scale bar: 200 µm. Measurements of (C) crop size, (D) survival, and (E) fecal spot 5

deposition in wild type, Piezo knockout, and Drop-dead knockout flies, n for C: 13-17 flies, n for 6

D: 7-8 trials involving 84-96 flies, n for E: 9-10 trials involving 90-100 flies, mean ± SEM, 7

***p<0.0001; ns: not significant by ANOVA Dunnett’s multiple comparison test; different 8

alphabets indicate statistical difference. (F) Visualization (left) and quantification (right) of 9

muscle fiber density by immunofluorescence for Phalloidin, n: 7-10 flies, mean ± SEM, scale 10

bar: 100 µm. (G) Quantification of crop cell density based on nuclear staining (TO-PRO-3), n: 7-11

10 flies, mean ± SEM. (H) Measurement of crop size in fasted flies, n: 9-15 flies, mean ± SEM. 12

(I) Fasted flies of genotypes indicated were given brief access (30 min) to dye-labeled food at 13

30°C, and feeding indices and crop sizes were calculated, n (feeding index): 14-20 trials 14

involving 168-240 flies, n (crop size): 12 flies, mean ± SEM, ***p<0.0005, ns: not significant by 15

ANOVA Dunnett’s multiple comparison test or unpaired t-test. (J) Fasted flies of genotypes 16

indicated were given brief access (30 min) to dye-labeled food, and feeding indices and crop 17

sizes were calculated, n (feeding index): 9-10 trials involving 108-120 flies, n (crop size): 10-17 18

flies, mean ± SEM, ***p<0.0005, ns: not significant by ANOVA Dunnett’s multiple comparison 19

test. 20

21

MATERIALS AND METHODS 22

23

Flies 24



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Fly stocks were maintained on a regular cornmeal agar diet (Harvard Exelixis facility) at 25°C, 1

with mating and collection performed under CO2 anesthesia. For Piezo knock-out studies, Piezo 2

knockout flies were isogenized by outcrossing five times into a wild type w1118 isogenic 3

background. We obtained Piezo knockout, knock-in (KI) Piezo-Gal4 and UAS-Piezo-GFP flies 4

(Norbert Perrimon), Tmc-Gal4 (Craig Montell), knock-in Gr43a-LexA and knock-in Gr43a-Gal4 5

(Hubert Amrein), and from Bloomington Drosophila Stock Center Piezo-Gal4 (BDSC# 59266), 6

Recombinase-Mediated Cassette Exchange (RMCE) gene-trap Piezo-Gal4 (BDSC# 76658), 7

UAS-CD8GFP (BDSC# 5137), UAS-CD8RFP (BDSC# 32218), UAS-Trpa1 (BDSC# 26263), 8

Cha-Gal80 (BDSC# 60321), UAS-CaLexA (BDSC# 66542), Nanchung-Gal4 (BDSC# 24903), 9

Inactive-Gal4 (BDSC# 36360), Painless-Gal4 (BDSC# 27894), Trp-Gal4 (BDSC# 36359), 10

Trpa1-Gal4 (BDSC# 36362), Nompc-Gal4 (BDSC# 36361), Ppk-Gal4 (BDSC# 32078), UAS-11

DenMark (BDSC# 33061 and 33062), Drop-dead KO (BDSC# 24901), w1118 (BDSC# 3605), 12

Escargot-Gal4 (BDSC# 26816), GMR51F12-Gal4 (BDSC# 58685). Cha-Gal80, Elav-Gal80, 13

and UAS-Shibirets were provided by Dragana Rogulja as published (Kitamoto, 2001; Sakai et al., 14

2009; Yang et al., 2009). 15

16

Feeding analysis 17

Acute feeding assays were performed as previously described with modifications (Albin et al., 18

2015; Min et al., 2016). Twelve adult female flies were collected upon eclosion and housed in a 19

vial with for five to seven days. Prior to testing, baseline hunger was synchronized by starving 20

flies for 15-18 hrs in a vial containing only on a dampened kimwipe section. Regular fly food was 21

dyed with green food coloring (McCormick, 70 µl dye per vial) and dried (24 hours). For testing, 22

starved flies were transferred to vials containing dyed food for 30 minutes. Trials were ended by 23

cooling the vials on ice, a feeding index was scored as described below (see quantification). For 24

thermogenetic experiments, flies expressing Trpa1 or Shibire were maintained and starved at 25

18°C prior to testing. Ten minutes prior to testing, starved flies and dye-labeled food were pre-26



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warmed to 30°C or 32°C for experiments with either Trpa1 or Shibire, and then tested as above. 1

Feeding behavior was scored by visual inspection of ingested dye with scores given from 0 to 5 2

based on dye intensity, as reported previously. A feeding index was expressed by averaging the 3

feeding scores for all flies per vial (~12 flies). For automated analysis of feeding patterns, fasted 4

male flies (3-5 days old) were individually introduced into chambers connected to an Expresso 5

machine (http://public.iorodeo.com/docs/expresso/hardware_design_files.html) and feeding 6

bouts were analyzed using Expression acquisition software 7

(http://public.iorodeo.com/docs/expresso/device_software.html). 8

9

Chronic studies of body weight, intestinal transit, and fecal rate, and life span 10

Chronic studies were performed on 5-7 day old male and female flies fed ad libitum with regular 11

fly food. Flies were anesthetized (ice, 10 minutes) and weighed in groups of three in a 1.5 ml 12

Eppendorf tube, with body weight expressed as the average weight per group of three. Lifespan 13

was analyzed for a group of 12 flies by counting the number of surviving flies each day. Fecal 14

rates were measured after feeding flies dye-colored food (dye-colored food is described above) 15

for 1 hour, with visual inspection of abdominal dye to ensure ingestion. Flies were transferred to 16

an empty vial containing a 1 cm X 1 cm filter paper floor for 30 minutes, and dye-labeled fecal 17

spots on the filter paper were counted. For analysis of fecal deposition, individual data points 18

reflect the mean behavior of ten flies. Intestinal transit was measured in flies given brief access 19

to dye-colored food, with dye location in the intestine determined visually. A transit index was 20

calculated based on the leading dye edge position, with scores of 1, 2, and 3 referring to dye 21

edge in the crop/anterior midgut, middle midgut, and hindgut/anus respectively. 22

23

Immunohistochemistry 24

Wholemount preparations of the gastrointestinal tract were fixed (4% paraformaldehyde, PBS, 25

20 min, RT), washed (2 x 5 min, PBS with 0.5% Triton X-100), permeabilized (10 min, PBS with 26



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0.5% Triton X-100), blocked [1 hr, RT, blocking solution: 5% normal goat serum (Jackson 1

ImmunoResearch, 005-000-121), PBS with 0.1% Triton X-100], incubated with primary antibody 2

(1:200, blocking solution, 4°C, overnight), washed (3 x 10 min, RT, PBS with 0.1% Triton X-3

100), incubated with secondary antibody (1:200, PBS with 0.1% Triton X-100, 2 hr, RT), washed 4

(3 x 10 min, RT, PBS with 0.1% Triton X-100 then 2 x 5 min, RT, PBS, mounted on a slide glass 5

with Fluoromount-G mounting medium (Southern Biotech, 0100-01), covered with a thin 6

coverslip, sealed with nail polish, and analyzed by confocal microscopy (Leica SP5). Primary 7

antibodies were anti-GFP (Aves labs, Chicken, GFP-1020), anti-RFP (Rockland, Rabbit, 600-8

401-379), anti-Elav (Developmental Studies Hydridoma Bank, Mouse, Elav-9F8A9), and anti-9

Akh (Kerafast, Rabbit, EGA261). Secondary antibodies were anti-Chicken-Alexa fluor-488 10

(Jackson ImmunoResearch, 703-545-155 ), anti-Rabbit-Alexa fluor-488 (Jackson 11

ImmunoResearch, 711-545-152), anti-Rabbit-Cy3 (Jackson ImmunoResearch, 711-165-152), 12

anti-Rabbit-Alexa fluor-647 (Jackson ImmunoResearch, 711-605-152), anti-Mouse-Alexa fluor-13

647 (Jackson ImmunoResearch, 715-605-150), anti-Mouse-Alexa fuor-488 (Jackson 14

ImmunoResearch, 715-545-150). For staining of visceral muscle and nuclei, anti-Phallodin-FITC 15

(Sigma, P5282-1MG), anti-Phalloidin-TRITC (Sigma, P1951-1MG) and TO-PRO-3 16

(ThermoFisher, T3605) were added together with the secondary antibody. 17

18

Quantification of crop size and composition 19

After the feeding assay, flies were fixed (4% paraformaldehyde, PBS, RT, 1 hr) and decapitated. 20

The anterior gastrointestinal tract was surgically removed after gentle displacement of 21

appendages and thoracic muscles. Dissected tissue was washed (3x PBS, RT, 5 min) and 22

mounted for bright field microscopy using the “Analyze-Measure” tool in Fiji to calculate crop 23

area. Crop muscle and cell density were quantified as detailed below. For quantification of crop 24

muscle density, the intensity of the Phalloidin-labeled muscle fibers in ROI was divided by the 25

total ROI area. For cell density, the number of nuclei labeled with TO-PRO-3 and counted using 26



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“Analyse-3D objects counter” function in Fiji (https://imagej.net/Fiji) was divided by total ROI 1

area. 2

3

Analyzing neuronal responses with CaLexA 4

CaLexA responses were measured in Piezo-Gal4 or Gr43a-Gal4 flies containing UAS-CaLexA 5

(LexA-VP16-NFAT, LexAop-rCD2-GFP and LexAop-CD8-GFP-2A-CD8GFP), and UAS-6

CD8RFP. Responses of Piezo knockout neurons were measured by introducing Piezo knockout 7

alleles into Piezo-Gal4; UAS-CaLexA; UAS-CD8RFP flies. For sucrose and sucralose 8

responses, flies we fed ad libitum with regular food, transferred to vials containing a kimwipe 9

soaked with 10% sucrose solution or 1% sucralose solution containing green food coloring for 10

24 hours, and analyzed for crop distension and CaLexA expression. For water responses, flies 11

were deprived of food and water for 6 hours, and transferred to vials containing a water-soaked 12

kimwipe. Some flies were harvested after 15 minutes for analysis of crop distension and others 13

were harvested after 18 hours for analysis of CaLexA expression. Control flies were placed in a 14

vial containing a water-soaked kimwipe but no food for 24 hours and harvested for analysis. For 15

TRPA1-mediated neuron stimulation, WT and Piezo KO flies bearing a Piezo-Gal4, UAS-16

CaLexA (LexA-VP16-NFAT, LexAop-rCD2-GFP and LexAop-CD8-GFP-2A-CD8GFP), and 17

UAS-Trpa1 were placed in a 30°C incubator for 24 hours prior to analysis. For analysis of 18

CaLexA expression, flies were anesthetized (ice, 10 minutes), and the anterior gastrointestinal 19

tract was surgically removed. Dissected tissue was fixed (4% paraformaldehyde, PBS, 20 min, 20

RT), washed (3 x 5 min, PBS), and slide mounted with Fluoromount-G mounting medium and a 21

coverslip. Native GFP (derived from CaLexA activation) and RFP (constitutive from a Gal4-22

dependent reporter) fluorescence were analyzed by confocal microscopy (Leica SP5). 23

24

For quantification of CaLexA-dependent reporter in Figure 3B and S3B, intensity of GFP and 25

RFP fluorescence was calculated per neuron and a CaLexA index expressed as GFP 26



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fluorescence divided by RFP fluorescence. For quantification of CaLexA-dependent reporter in 1

Figure S3A and S3D, which involved flies lacking an RFP allele for neuron identification and 2

normalization, GFP intensity was measured in the whole hypocerebral ganglion and a 3

background subtraction was performed involving a comparably sized region of the 4

proventriculus lacking Gal4-positive cell bodies. For S3A and S3D, background-subtracted GFP 5

fluorescence was divided by RFP fluorescence from a control Piezo-Gal4; UAS-CD8RFP fly to 6

generate a CaLexA index. 7

8

Statistical analysis 9

Sample sizes from left to right (except 3B is top to bottom): Figure 2B (16, 11, 13, 10, 10 and 13, 10

9, 11, 9, 9), Figure 2C (19, 20, 14, 10, 13 and 12, 6, 11, 10, 12), Figure 2E (15, 20, 14, 15), 11

Figure 3B (59, 64, 43, 67 for Gr43a neuron responses; 61, 61, 59, 66 for Piezo neuron 12

responses; 60, 60, 33, 37 for Piezo-null Piezo neuron responses), Figure 4C (17, 17 and 14, 14), 13

Figure 4D (32, 34, 33, 31), Figure 4E (21, 22 for each graph), Figure 4F (29, 22, 30, 20, 22 and 14

13, 13, 13, 18, 16), Figure S1B (10, 10, 10, 7), Figure S2C (8, 14, 9, 8), Figure S3A (54, 82, 44), 15

Figure S3B (10, 10, 18, 11, 12), Figure S3C (17, 19, 28), Figure S3D (22, 23), Figure S4B (11, 16

11, 10, 11, 11, 10), Figure S4C (13, 16, 17), Figure S4D (8, 7, 7 for WT, Drd KO and Piezo KO), 17

Figure S4E (9, 10, 10), Figure S4F (7, 10), Figure S4G (7, 10), Figure S4H (15, 9), Figure S4I 18

(14, 15, 15, 15, 20 and 12, 12, 12, 12, 12), Figure S4J (10, 9, 9, 9, 9, 9 and 17, 15, 14, 16, 16, 19

10). Data in graphs are represented as means ± SEM. Statistical significance was analyzed by 20

ANOVA Dunnett’s multiple comparison test or unpaired t-test using Prism 8 software 21

(Graphpad), as indicated in figure legends. 22

23

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5

6



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Figure 1

Crop

Piezo neuronsVisceral muscle

Head

Anus

Anterior midgutF

Piezo neurons Visceral muscle

Anterior midgut

Proventriculus

Crop nerve

Crop

Mid/hindgut

HCG

B

Piezo neuronsVisceral muscle

HCGRecurrent nerve

E

A

Piezo neuronsGr43a neurons

Proventriculus

DC

Piezo neuronsAkh neurons

HCG

CC

Elav RFP Merge

Piezo-Gal4; UAS-CD8RFP HCG



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Figure 2

D

A

Dyed food in abdomenFasted flies Given dyed food

***

*

ns

Piezo>CD8RFP; +

Bra

in

Piezo>CD8RFP;Cha-Gal80

ElavRFP

ElavRFP

Piezo>CD8RFP;Elav-Gal80

VN

C

ElavRFP

**

ns

*****

B

HC

G

****

***

ns

***

ns**

E

*** ******

ns

C

**



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Figure 3

A

Gr43a neurons Piezo-null Piezo neuronsPiezo neuronsB

***

ns

***

ns

CSucraloseSucrose Water

Gr43a>CaLexA,CD8RFPSucralose Water ControlSucrose

Piezo>CaLexA,CD8RFPSucralose Water ControlSucrose

Piezo>CaLexA,CD8RFP;Piezo KO

Su

cral

os

e

Gr43a neuronsGr43a>CaLexA,CD8RFP

Wat

er

CaLexA RFP Merge

CaLexA RFP Merge

Su

cro

se

CaLexA RFP Merge

Co

ntr

ol

CaLexA RFP Merge

Piezo neuronsPiezo>CaLexA,CD8RFP

CaLexA RFP Merge

CaLexA RFP Merge

CaLexA RFP Merge

CaLexA RFP Merge

Piezo-null Piezo neurons Piezo>CaLexA,CD8RFP; Piezo KO

CaLexA RFP Merge

CaLexA RFP Merge

CaLexA RFP Merge

CaLexA RFP Merge

Control



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Figure 4

Piezo KOWTA B

E

WT Piezo KO

Male Female

***

***

D

ns ns

*** ***

F

nsns

** **

*** ns***

*** ***

C



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A BAnterior

midgut nerve

Piezo(KI)>CD8RFPVisceral muscle

Crop nerve

Piezo(76658)>CD8RFPVisceral muscle

Crop nerve

Anterior midgut nerve

Figure S1



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RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue

A Piezo>CD8RFP;+


Piezo>CD8RFP;+

Pro

bo

scis

An

ten

na

Win

gL

egT

arsa

l tip

Bo

dy

wal

lH

CG

Gu

tV

NC

Bra

in

B

RFPTissue

RFPTissue

RFPTissue

RFPTissue

RFPTissue


RFPTissue

C30°C18°C

CaLexA CaLexA

Figure S2

Esg>CaLexA,Trpa1HCGBrain

RFP RFP

Esg>CD8RFPGut

RFP

***

nsns



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D Piezo neuronsPiezo>CaLexA,Trpa1;

+

Piezo-null neuronsPiezo>CaLexA,Trpa1;

Piezo KO nsE

Figure S3

Piezo>CD8RFP;+

Piezo>CD8RFP;Piezo KO



Piezo KOWT

B Sucrose Starved Ad libitum

High

Low

A 1hr 7hrs 24hrs

High

Low

***

***

C

***

***



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Feeding time

WT Piezo KO

10 min 1 hr10 min 1 hr

ED

(Days)

WT Piezo KO

nsns

G HF

C

A

ElavPiezo-GFP

HCG

B

*** ns

a

c

b

ns

ns

***

ns***

Figure S4

ns

ns

*** ***

I

ns

*** ***

ns

ns

ns

J*** ns***

ns



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