Mechanosensitive Neurons on the Internal Reproductive Tract Contribute to Egg-Laying- Induced Acetic Acid Attraction in Drosophila

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1 Cell Reports, Volume 9 Supplemental Information Mechanosensitive Neurons on the Internal Reproductive Tract Contribute to Egg-Laying- Induced Acetic Acid Attraction in Drosophila Bin Gou, Ying Liu, Ananya R. Guntur, Ulrich Stern, and Chung-Hui Yang

2 Fig S1. Recording setup and analysis of fly behavior, related to Figure 1. (A) The setup we constructed to record egg laying preferences and positional trajectories of egg laying females. A single apparatus is fitted with 4 cameras, each of which can record behavior of two females from two chambers. (B) A still picture showing 4 females in 4 egg laying chambers. The two vertical strips (one on the left and one on the right) denote the areas, or troughs, where we place agarose substrates. The large middle area is a plastic divider, on which flies will not lay eggs. The red dot in the middle of the chamber is where we place a drop of food (usually grape juice). Females consistently prefer to lay eggs on the AA substrate over the plain substrate (with an oviposition preference index of ~ 0.88, n= 38). One can see that there are many eggs (faint gray spots) on the AA substrate but not on the plain substrate. (C) Example of an one hour trajectory of a female (upper panel) and its corresponding speed (lower panel). In the upper panel, the x axis denotes time and the y axis denotes position. In the lower panel, the x axis denotes time and the y axis denotes speed. (D) When different speed thresholds (7, 5, 3.5, 2, and 0.35 mm/sec) were used to separate the trajectory into high vs. low locomotion states, different percentages of the egg laying events (100%, 98.6%, 94.7%, 87.4% and 16.7%) were covered in the low locomotion state. We used 7 mm/sec as the speed to separate the trajectories for the rest of the experiments we describe in this manuscript because it best separates the trajectories into egg laying and non egg laying states. Trajectories from 8 animals were used for this experiment. (E) Raster of aversive returns in trajectories of the same individual egg laying events in Fig. 1F. They occur very rarely as compared to attractive returns described in Fig. 1F. 2

3 (F) Neither yeast fed virgins that lay no egg nor yeast fed males show signs of AA attraction. (G) Egg laying females still show strong AA attraction whereas virgins that do not lay eggs show AA aversion when sucrose (150mM) was added to both AA and plain substrates. Note that these attraction indices are calculated from low locomotion state. *** p < Student s t test. Fig S2. Reproductive tract ppk1 Gal4 neurons send axons to the tip of the ventral nerve cord (VNC), related to Fig 3. (A) Axonal projection pattern of all ppk1 Gal4 neurons in the VNC. Aside from axonal processes originating in the reproductive tract, ppk1 Gal4 labels also axonal processes of sensory neurons located in appendages (e.g., wings) and body wall. These axons target both the thoracic (red arrow) and the abdominal ganglia. Scale bar is 100 μm for A, B, C, E, 25 μm for D and 50 μm for F, G. (B) Axonal projection of ppk1 Gal4 neurons in the VNC after ppk1.0 Gal80 is introduced to suppress ppk1 Gal4 activities in many of the appendage and body wall. Note that the axonal projection shown in this picture is mainly restricted to the posterior tip of the VNC. (C D) Dendritic and axonal projections of the 21 7 ppk1 (intersected) neurons. (C) The intersected neurons extend large dendrites (blue arrows) on the reproductive tract, similar to the ones labeled by ppk1 Gal4. (D) They also target their axons to the tip of the VNC in a pattern similar to panel (B). These flies contain the following transgenes: 21 7 Gal4, UAS > stop > Kir2.1 GFP, ppk1 lexa, lexaop2 flp. 3

4 (E) The piezo ppk1 intersected neurons also extend their large dendrites onto the tract (blue arrows). These flies contain the following transgenes: piezo Gal4, UAS > stop > Kir2.1 GFP, ppk1 lexa, lexaop2 flp. (F, G) Projection pattern of two reproductive tract ppk1 Gal4 neurons labeled by the FLP out method. (F) Dendrites (blue arrow) of these neurons arborize at the reproductive tract. (G) These neurons extend axonal processes onto the tip of the VNC (blue arrow). The general areas of the processes are shown in the schematic diagrams to the right of the pictures. (H I) Cell bodies of ppk1 neurons that project dendrites onto the common duct. (H) There are two clusters of ppk1 neurons that innervate the common duct (white arrows), each contain 2 3 somas. GFP and red stinger were used to show the processes and nucleus of ppk1 neurons, respectively. Scale bar is 50 μm. (J) Higher magnification picture of boxed area in (I). The cluster on the left (white arrow) contains common duct innervating ppk1 neurons. The cluster on the right contains neurons that do not innervate the common duct, and some of them are fru/ppk1 neurons. Scale bar is 12.5 μm. Fig S3. Anatomical relationship between ILP7 and ppk1 neurons in the VNC, related to Fig 3. (A) Co labeling of ILP7 and ppk1 neurons at the tip of the VNC. Left: ILP7 neurons labeled by ILP7 lexa driving lexaop2 GFP. Middle: ppk1 neurons labeled by ppk1 Gal4 driving UAS RFP. Right: merged picture of the two. Scale bar is 25 μm. (B C) ILP7 neurons that express P 2 X 2 show robust response to ATP even when their axons were severed. (B) Baseline GCaMP3 response. (C) Response after ATP perfusion. Arrows point to the cluster of ILP7 neurons that innervate reproductive tract. 4

5 (D) Quantification of experiment in (B C). As a control, ILP7 neurons that do not express P 2 X 2 do not show any response to ATP. *** p < Student s t test. Fig S4. Mushroom body and olfactory neurons are not essential for egg laying induced AA attraction, related to Fig. 4. (A) α FasII staining of adult brain showing that the mushroom body (MB) is largely absent in the hydroxyurea (HU) treated flies (left panel) but remains intact in the mock treated flies (right panel). Scale bar is 50 μm. (B D) Comparison of behaviors of mock treated vs. HU treated flies shows that they show similar egg laying preference (B), similar positional preference (C), and similar attraction index (D) for AA. Student s t test. p > (E, F) Olfactory mutants Or83b and Ir64 show egg laying preference and active attraction for AA similar to WT. Table S1. This table lists the average # of eggs laid on AA vs. plain sides for each of our experiment. Movie S1. The movie shows females exploring in our egg laying chamber that contains 1 AA substrate and 1 plain substrate. Sped up 30X. Related to Fig. 1. Movie S2. This movie shows that stimulating ILP7 axons can induce contraction of a reproductive tract severed from the CNS. P 2 X 2 and GCaMP3 were driven by ILP7 Gal4 in this fly. ATP was added at 3 minute. Related to Fig. 3. Movie S3. This movie shows that ppk1 neurons responded to tract contraction induced by activation of ILP7 neurons. P 2 X 2 was driven by ILP7 LexA and GCaMP3 was driven by ppk1 Gal4 in this fly. ATP was added at 3 minute. Related to Fig. 3. 5

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10 Table S1 Fed with yeast Egg number on AA side Egg number on plain side Total egg number n Figure related Wild type, mated yes 15.6± ± ± Fig. 1A, B Wild type,mated no 0.5± ± ± Fig. 1A, B Wild type,virgin yes 12± ± ± Fig. 1A, B Wild type, virgin no Fig. 1A, B ILP7 > Kir, mated yes Fig. 2E ILP7 > Kir, mated no Fig. 2E ILP7 Gal4/+, mated Yes 19.8± ± ± Fig. 2E UAS Kir/+, mated yes 19.2± ± ± Fig. 2E ppk1 > Kir, ppk1.0 Gal80, mated yes Fig. 4A UAS Kir, ppk1.0 Gal80/+, mated yes 19.2± ± ± Fig. 4A ppk1 Gal4/+, mated yes 21.6± ± ± Fig. 4A ppk1 gs > Kir, fed with RU486, mated yes 0.1± ± Fig. 4B ppk1 gs > Kir, without RU486, mated yes 20.9± ± ± Fig. 4B ppk1 gs Gal4/+, feed with RU486, mated yes 21.2± ± ± Fig. 4B UAS Kir/+, fed with RU486, mated yes 21.7± ± ± Fig. 4B 21 7 ppk1 > Kir, mated yes Fig. 4C ppk1 lexa, > stop > Kir, lexa FLP /+, mated yes 18.8± ± ± Fig. 4C 21 7 Gal4/+, mated yes 13.8± ± ± Fig. 4C UAS piezo RNAi/+, mated yes 13.0± ± ± Fig. 4C 21 7 > piezo RNAi, mated yes 0.0± ± ± Fig. 4 ppk1(double)>piezo RNAi, ppk1.0 Gal80, mated yes 8.3± ± ± Fig. 4C ppk1(double) Gal4/+, mated yes 17.5± ± ± Fig. 4C UAS piezo RNAi, ppk1.0 Gal80/+, mated yes 29.0± ± ± Fig. 4C ppk1 > NaChBac, ppk1.0 Gal80, virgin no Fig. 4D ppk1.0 Gal80/+, virgin no Fig. 4D ppk1 Gal4/+, virgin no Fig. 4D UAS NaChBac/+, virgin no Fig. 4D Wild type, young virgin yes Fig. S1F Wild type, mated, with sucrose yes 17.4± ± ± Fig. S1G Wild type, virgin, with sucrose yes Fig. S1G Wild type, fed with Hu, mated yes 15.6± ± ± Fig. S4B,C, D Wild type, without Hu, mated yes 16.2± ± ± Fig. S4B,C, D Or83b mutant, mated yes 16.5± ± ± Fig. S4E Ir64a mutant, mated yes 18.1± ± ± Fig. S4E Wild type, mated yes 11.4± ±1.1 13± Fig. S4E