. Author manuscript; available in PMC: 2015 Oct 23.

Published in final edited form as: Cell Rep. 2014 Oct 16;9(2):522–530. doi: 10.1016/j.celrep.2014.09.033

Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying-induced acetic acid attraction in Drosophila

Bin Gou ^1,², Ying Liu ^1,², Ananya R Guntur ¹, Ulrich Stern ^3,⁴, Chung-Hui Yang ^1,^*

PMCID: PMC4223655 NIHMSID: NIHMS631474 PMID: 25373900

Abstract

Selecting a suitable site to deposit their eggs is an important reproductive need of Drosophila females. While their choosiness towards egg-laying sites is well documented, the specific neural mechanism that activates females’ search for attractive egg-laying sites is not known. Here we show that distention/contraction of females’ internal reproductive tract triggered by egg-delivery through the tract plays a critical role in activating such search. We found that females start to exhibit acetic acid attraction prior to depositing each egg but no attraction when they are not laying eggs. Artificially distending the reproductive tract triggers acetic acid attraction in non-egg-laying females whereas silencing the mechanosensitive neurons we identified that can sense the contractile status of the tract eliminates such attraction. Our work uncovers the circuit basis of an important reproductive need of Drosophila females and provides a simple model to dissect the neural mechanism that underlies a reproductive need-induced behavioral modification.

Keywords: Egg-laying, acetic acid attraction, mechanosensation, Drosophila, reproductive tract

Introduction

The need to care for offspring can alter behaviors of animal mothers significantly. Many commonly described maternal behaviors such as feeding and aggression against intruders serve the purpose of nurturing and protecting the newborns. But in some species, pregnancy alone is sufficient to induce changes in sensory processing and behaviors (J.S. and D.S., 1963). Pregnancy-induced hormonal changes are thought to play a role in activating “prenatal care behaviors” (Kristal, 2009), but the exact circuit mechanism by which the presence of a fetus in utero modifies the behaviors of expecting mothers remains not well understood.

The fruit flies Drosophila melanogaster have emerged as a suitable model to study the genetic and circuit basis of female reproductive behaviors. Similar to higher animals, virgin and mated/pregnant flies show significant differences in their behaviors and physiologies. For example, virgins are receptive to male courtship and lay very few eggs, whereas mated females are unreceptive to courtship, lay eggs frequently, and preferentially consume proteins over sugars (Carvalho et al., 2006; Kubli, 2003; Ribeiro and Dickson, 2010). Much progress has been made in recent years in elucidating the molecular and circuit basis by which the experience of mating modifies physiologies and behaviors of female flies (Bussell et al.; Feng et al., 2014; Häsemeyer et al., 2009; Rezával et al., 2012; Yang et al., 2009; Zhou et al., 2014). In contrast, whether and how egg-laying need influences how female flies interpret the valence of external stimuli – so as to guide their decision of whether to move towards or away from specific stimuli – remains little explored, despite the fact that female flies are known to be highly selective about where to lay eggs (Azanchi et al., 2013; Dweck et al., 2013; Joseph et al., 2009; Joseph and Heberlein, 2012; Rockwell and Grossfield, 1978; Schwartz et al., 2012; Yang et al., 2008).

Here we show that egg-laying need increases female flies’ attraction for acetic acid (AA) significantly. Behavioral analysis reveals that signs of AA attraction emerge prior to physical egg deposition. Manipulating the internal egg-delivery process (that precedes physical egg deposition) reveals that artificial distention of the internal reproductive tract is sufficient to activate AA attraction, and that mechanical stretch of the reproductive tract is sensed and relayed to CNS by a set of piezo-expressing sensory neurons. Our results suggest a model that Drosophila females modify their AA attraction by assessing – via mechanosensitive neurons on the tract – whether eggs are being pushed through their reproductive tract. We propose such activation of AA attraction in anticipation of impending physical egg-laying may be considered a rudimentary form of maternal care, and provides a suitable model to study the circuit mechanism by which reproductive needs modify females behaviors.

Results

Egg-laying but not mating correlates with mated females’ positional preference for AA

It has been shown that, as a population, mated females show stronger positional preference for AA than virgins do, and they also prefer to lay eggs on an AA-containing over an AA-free substrate (Joseph et al., 2009). But because mating induces several behavioral and physiological changes, we first ascertained that mated females’ increased AA preference is triggered by egg-laying need as opposed to other mating-induced changes. We custom-built an apparatus that contains several chambers, each of which can house one AA+ and one AA− substrate (Fig. S1B). We loaded females into this apparatus (one per chamber), recorded their behaviors for 4 hours (Fig. S1A and movie S1), and tracked their positions using Ctrax (Branson et al., 2009). Similar to what was reported previously (Joseph et al., 2009), we found that mated, egg-laying females show a clear preference to lay eggs and to spend time on AA site (Fig. 1A and Table S1).

Fig. 1 — **(A)** Mated and virgin flies that lay more eggs (with yeast, > 10 eggs in 4hrs) show positional preference for AA. Mated and virgin ones that lay few eggs (yeast deprived, ≤1 eggs in 4hrs) show positional avoidance of AA. The schematic shows the configuration of an egg-laying chamber. Shaded areas are “troughs” where we place substrates. Plain: 1% agarose. AA: 1% agarose with 3% (V/V) AA. Positional preference for AA is calculated as: (T_AA – T_plain)/(T_AA + T_plain), T_AA and T_plain are time-spent on the AA (red bracket) half and the plan half of the chamber (green bracket), respectively. See Fig. S1B for chamber picture. * p < 0.05, ** p < 0.01, *** p < 0.0001. n.s. = not significant, p > 0.05. Student’s t-test.

**(B)** Mated and virgin flies that lay many eggs show stronger “active” AA attraction than ones that lay few eggs. “Aversive return” and “attractive return” are as diagramed. The vertical bar in the right panel denotes the “return-point” of an attractive return. AA attraction index is calculated as: (R_attractive – R_aversive)/(R_attractive + R_aversive). R_attractive and R_aversive represent the # of attractive and aversive return in a given trajectory, respectively. * p < 0.05, ** p < 0.01, *** p < 0.0001. Student’s t test.

**(C)** Example of an on 1-hour, annotated trajectory of a single female. Y-axis: time. X-axis: animal position. The green and blue traces show the segments of the trajectory where the animal is in a high vs. low locomotion state. The two states can be separated using speed threshold of 0.7 mm/s (see **experimental procedure** and Fig S1C, D). Note that all the egg-laying events (short red ticks) occur in the low-locomotion state. Yellow circles: “attractive returns” in the trajectory.

**(D)** Individual females show stronger AA attraction when they are actively laying eggs trace than when they are not (blue vs. green trace). * p < 0.05. Student’s t test.

**(E-E′)** Return-points of attractive returns are closer to AA when animals are in high egg-laying (343 returns) than in low egg-laying state (1091 returns). Return point of an attractive return is where animal reverses its running direction (see also panel B). Distance between return point and AA substrate is calculated as % chamber-length. *** *p < 0.001*, Student’s t-test. **(E′)** Representative return points of 100 attractive returns in low vs. high egg-laying states.

**(F)** Raster of attractive returns surrounding 151 egg-laying events. There are 151 “invisible” horizontal lines, each of which represents an 8-minute trajectory where a single egg-laying event occurs at time 0. Each dot along the horizontal line denotes one attractive return. Light purple and gray highlights the 1-minute window before and after egg-laying, respectively.

**(F′)** Average # of attractive returns during each minute of the 8-minute timeline in **(F)**. * p < 0.05, ** p < 0.01, *** p < 0.0001. n.s. = not significant, p > 0.05. Student’s t test.

To disentangle the impacts of mating vs. egg-laying on positional preference for AA, we compared AA preference of mated females that lay very few eggs vs. virgins that lay many. (We have discovered that mated females reduce their egg-laying rate significantly if their diet were deprived of yeast paste, a protein source that boosts egg production. Conversely, virgins that have been continuously fed yeast paste after eclosion can sometimes lay many eggs). We found that mated females that lay very few eggs show no positional preference for AA, but virgins that lay many do (Fig. 1A). Importantly, neither yeast-fed males nor yeast-fed virgins that lay few eggs show any AA preference (Fig. S1F). Together, these results suggest egg-laying, not mating or yeast feeding, is responsible for mated females’ positional preference for AA.

Egg-laying females show “active attraction” for AA

We next determined whether egg-laying need triggers “active AA attraction” in addition to positional preference for AA. We noticed that females move constantly between AA+ and AA-free substrates, but occasionally reverse their running direction in the middle of the chamber. We labeled a reversal where females would switch from moving away to moving towards AA substrate an “attractive return,” and proposed that it indicates active AA attraction (Fig. 1B, right panel). A recent report uses a similar criterion to define behavioral attraction (Gao et al., 2013). Similarly, we proposed a reversal from moving towards to moving away from AA substrate signals active AA aversion and labeled it an “aversive return” (Fig. 1B, left panel). We can then determine net AA attraction by calculating an “AA attraction index” (Fig. 1B). Indeed, regardless of their mating status, females that lay many eggs show a significantly stronger AA attraction than ones that lay very few (Fig. 1B, Fig. S1G), suggesting increased egg-laying is sufficient to induce active AA attraction.

We have so far compared AA attraction of two types of females: one that lays many eggs and one that does not. But if egg-laying need is indeed the trigger for AA attraction, the same female should exhibit different levels of AA attraction depending on her egg-laying rate in a given time period. So we sought to correlate egg-laying rate and AA attraction in the same animals and found that temporal distribution of egg-laying events is non-uniform: there are clearly periods when a female lays eggs frequently and ones when she does not (Fig. 1C, blue and green traces). Moreover, females consistently move faster during “no egg-laying” periods than “high egg-laying” periods (Fig. 1C, blue and green traces), allowing us to use locomotion speed to separate their trajectories into high vs. low egg-laying states (Fig. 1C and Fig. S1C, D). We found that individuals show stronger AA attraction when they are actively laying eggs than when they are not (Fig. 1D). An examination of the “return-point” (Fig. 1B) further revealed that females in high egg-laying state tend to execute their attractive returns at positions closer to AA (Fig. 1E, E′), suggesting that when laying eggs actively, females are less tolerant about staying away from AA.

Finally, if egg-laying need is a trigger for AA attraction, signs of attraction might emerge prior to each egg-laying. We examined the trajectory in the 1-minute window immediately before and after 151 egg-laying events and found that, indeed, attractive returns tend to occur before but not after egg-laying (Fig. 1F, F′). Moreover, when we segmented the trajectory before each egg-laying into four consecutive 1-minute periods, we found that the number of attractive returns increases as females are nearing to laying an egg (Fig. 1F, F′). In contrast, aversive returns occur very rarely in these windows (Fig. S1E). Taken together, our analysis shows that Drosophila females are more attracted to AA when they are actively laying eggs and that such attraction emerges prior to physical egg-laying.

Persistent presence of egg(s) in the internal reproductive tract is sufficient to trigger AA attraction independent of egg-laying

Next, we wanted to uncover the neural basis by which egg-laying need activates AA attraction prior to physical egg-laying. We looked closer at the egg-delivery process that occurs in females’ internal reproductive tract. While egg-laying becomes obvious to observers only when females begin to display the ovipositor motor program (Yang et al., 2008), it has begun once an egg starts to descend from the ovaries into the internal reproductive tract – an epithelial tube that connects the two ovaries to the uterus, where eggs are fertilized (Fig. 2A). The reproductive tract has a small diameter and is encased by muscles that are innervated by sensory and motor neurons (Castellanos et al., 2013; Häsemeyer et al., 2009; Yang et al., 2009). Because egg-delivery in the tract precedes physical egg deposition, we hypothesize that perhaps females start to increase AA attraction once they sense eggs are being pushed through the tract.

Fig. 2 — **(A)** A diagram depicting the reproductive tract and its relative position to the CNS. Eggs (red arrow) are produced in the ovaries. To be deposited, they must be pushed out of the ovaries, squeezed through the lateral and common ducts, and fertilized in the uterus. In our manuscript, internal reproductive tract = lateral ducts + common duct. One *ILP7-*expressing motor neuron whose axon descends from the VNC to innervate the tract is shown in green.

**(B)** *ILP7* axons (magenta) form DLG-positive synapses (green) with tract muscles. *ILP7* axons are labeled by *mCD8-RFP*. DLG: Disc Large, an NMJ marker. Scale bar is 25 μm.

**(C, D)** Inhibiting *ILP7* neurons consistently (20/20) causes egg-jamming in the tract (white arrow) of yeast-fed mated females **(C)**. In contrast, inhibiting *ILP7* neurons did not cause egg-jamming consistently if females were yeast-deprived **(D)**. Note that ovaries from yeast-fed females are much larger – they contain many more eggs – than those from yeast-deprived ones. The two pictures were taken at the same magnification. Scale bar is 500 μm.

**(E)** Yeast-fed and yeast-deprived *ILP7-inhibited* mated females show strong and much-reduced AA attraction, respectively. 1-way ANOVA with Bonferroni’s post test. * p < 0.05. n.s. = not significant, *p > 0.05*.

To test this idea, we first assessed AA attraction of females who have eggs “persistently trapped in the tract”. It has been shown recently, and we confirmed, that some of the ILP7-Gal4-expressing neurons in the ventral nerve cord (VNC) are motor neurons that innervate the tract (Fig. 2B) (Castellanos et al., 2013). Importantly, inhibiting ILP7 neurons with Kir2.1 (Baines et al., 2001) consistently causes one or more eggs to be “jammed” in the tract (Castellanos et al., 2013; Yang et al., 2008) (Fig. 2C), providing us with the desired phenotype to test our hypothesis. Indeed, females with inhibited ILP7 neurons showed clear AA attraction, despite that they cannot physically lay any eggs (because their tract is jammed) (Fig. 2E). Importantly, females of the same genotype but were deprived of yeast paste showed much reduced egg jamming (Fig. 2D) as well as much reduced AA attraction (Fig. 2E), suggesting that persistent presence of egg(s) in the tract, not other changes induced by silencing ILP7 neurons, is what triggers AA attraction. Thus, we propose that during regular egg-laying, AA attraction is activated each time an egg is being squeezed through the tract.

A group of ppk1-expressing sensory neurons on the reproductive tract can sense contraction of the tract

How do females sense that an egg is being squeezed through their reproductive tract? We speculate some sensory neurons on the tract are mechanosensors. We have previously shown that the ppk-Gal4 labeled sensory neurons on the tract can be divided into at least two groups (Häsemeyer et al., 2009; Yang et al., 2009) (Fig. 3A). The first group expresses the fruitless transcripts, senses sex peptide (SP), and has relatively short dendrites (Häsemeyer et al., 2009; Rezával et al., 2012; Yang et al., 2009). We suspect ppk neurons of the second group, two on lateral ducts and 4–6 at the base of the common duct (Fig. 3B, Fig. S2H–I), are mechanosensors. They have large dendrites that “tile” the entire surface of tract (Fig. 3B) and appear to express the mechanosensitive channel Piezo (Coste et al., 2010; Coste et al., 2012; Kim et al., 2012) (Fig. 3C, Fig. S2E). However, there is no direct evidence showing that these “tract-tiling” ppk1 neurons are indeed capable of sensing tract contraction/distention.

We next devised a three-step approach to test whether these tract-tiling ppk1 neurons are mechanosensors (Fig. 3F). First, we used the calcium sensor GCaMP3 (Tian et al., 2009) to monitor activities of axonal termini of tract ppk1 neurons. (GCaMP3 signal in ppk axons in the VNC is less likely to move out of the focal plane when tract contraction occurs). To define the specific VNC region targeted by tract ppk1 neurons, we made use of a ppk1.0-Gal80 (Häsemeyer et al., 2009). This ppk1.0-Gal80 suppresses Gal4-dependent expression in nearly all ppk1-Gal4 neurons except ones that reside on the tract, revealing that axons of tract ppk1 neurons consistently target to the posterior tip of the VNC (Fig. 3D–E and Fig. S2A–B). This projection is further confirmed when we used the “FLP-out” approach (Gordon and Scott, 2009) to sparsely label ppk1-Gal4 neurons (Fig. S2F–G). Second, we used a chemical-genetic approach (Lima and Miesenböck, 2005; Yao et al., 2012) to stimulate contraction of tract muscles. We expressed the ATP-gated P₂X₂ channel in ILP7 neurons, and found that stimulating them with ATP induces robust contraction of the tract (Fig. 3G–H and movie S2). Finally, we used the LexA/LexAop2 and Gal4/UAS system to express P₂X₂ and GCaMP3 in ILP7 and ppk1 neurons, respectively (Fig. 3F), allowing us to record ppk1 neurons while activating tract muscle contraction.

CGaMP3 signal in the axonal termini of tract ppk1 neurons increased significantly when we stimulated ILP7 neurons (Fig. 3I–K, movie S3). To ensure that such GCaMP3 increase was not due to “local interaction” between ppk1 and ILP7 neurons – their processes overlap extensively in the VNC (Fig. S3A), we physically severed the connectives between the tract and the VNC. ILP7 neurons in such “severed” preparation still responded to ATP robustly (Fig. S3B–D), but their stimulation no longer induced a significant GCaMP3 increase in ppk1 axons (Fig. 3K), ruling out the possibility that ILP7 neurons locally activate ppk1 axons in the VNC. Thus, our results suggest that the tract-tiling ppk1 neurons are mechanosensors that can detect contraction/distention of the tract.

ppk1-expressing tract sensory neurons are required for egg-laying-induced AA attraction

To determine if the mechanosensitive tract ppk1 neurons play a role in promoting AA attraction, we next targeted them for inhibition. In our first “subtraction-based” approach, we used the ppk1-Gal4 to express Kir2.1 in the presence of ppk1-Gal80. This manipulation caused females to have egg(s) jammed in their tract (Fig. 4A), but unlike ILP7-inhibited females, they showed no AA attraction (Fig. 4A). In our second “intersection-based” approach, we used the 21-7-Gal4 that labels tract ppk1 neurons but not many of the none tract ppk1 neurons (Song et al., 2007) to express an UAS-FRT-stop-FRT-Kir2.1 (Yang et al., 2009) and introduced a ppk1-LexA and a source of flp into the same animals. Because 21-7-Gal4 and ppk1-LexA are co-active mostly only in the tract ppk1 neurons, animals bearing all 4 transgenes (ppk1-LexA, LexAop2-flp, 21-7-Gal4, UAS-FRT-stop-FRT-Kir2.1) should have mostly only their tract ppk1 neurons inhibited (Fig. S2C–D). Again, these “intersected” animals show an “egg-jamming” phenotype but no AA attraction (Fig. 4C). Lastly, restricting Kir2.1 expression to only adult stage still reduced AA attraction (Fig. 4B), ruling out potential developmental problems due to chronic silencing of ppk1 neurons as the cause for the lack of AA attraction we observed. Thus, active tract ppk1 sensory neurons are required to activate AA attraction induced by egg presence in the tract.

Fig. 4 — **(A)** Inhibiting *ppk1* neurons in the presence of *ppk1-Gal80* causes egg-jamming (white arrow) and reduces AA attraction. Scale bar = 500 μm. 1-way ANOVA with Bonferroni’s post test. * *p < 0.05.* n.s. = not significant, *p > 0.05*.

**(B)** Inhibiting *ppk1* neurons specifically in adult stage reduces AA attraction still. 1-way ANOVA with Bonferroni’s post test. n.s. = not significant. ****p < 0.001*

**(C)** Inhibiting tract *ppk1* neurons by the “intersection method” reduces AA attraction, as does reducing *piezo* expression in *21-7* neurons or tract *ppk1* neurons (2X *ppk1-Gal4* plus *ppk1-Gal80*).

**(D)** Expressing *NaChBac* in tract *ppk1* neurons fails to activate AA attraction.

To test whether the mechanosensitivity of the tract ppk1 neurons is important for AA attraction, we reduced their piezo expression. We first used 21-7-Gal4 to express an piezo-RNAi (Kim et al., 2012). In addition, we also used two copies of ppk1-Gal4 – in the presence of ppk1-Gal80 – to express the piezo-RNAi. Neither sets of animals showed AA attraction, suggesting that piezo expression in tract ppk1 neurons is important for AA attraction (Fig. 4C).

Finally, we asked whether artificial activation of tract ppk1 neurons in the absence of active egg delivery is sufficient to trigger AA attraction. We found that expressing in tract ppk1 neurons the sodium channel NaChBac (Luan et al., 2006), a commonly used effector for increasing membrane potential, failed to trigger AA attraction in yeast-deprived females (Fig. 4D). But because NaChBac overexpression may not be effective in stimulating ppk1 neurons, we also used the heat-gated dTRPA1 to stimulate ppk1 neurons. However, increasing temperature alone causes a significant alteration in AA attraction, making data interpretation difficult. Thus, we are unable to conclude whether stimulating ppk1 neurons is sufficient to induce AA attraction.

Discussion

In this report, we discovered that egg-laying need can activate AA attraction in Drosophila females, and that mechanical stretch of the tract – induced by egg-delivery through their internal reproductive tract – is one origin of the “egg-laying need.” To our knowledge, our study is the first to demonstrate that egg delivery in the internal reproductive tract is an important physiological signal that modulates how Drosophila females interpret the valence of sensory stimuli. The flexibility in egg-laying need-induced modification of sensory processing contrasts significantly to that triggered by sex peptide (SP): once SP gains control of female CNS, it keeps the mated females unreceptive to male courtship for days (Rezával et al., 2012; Ribeiro and Dickson, 2010; Yang et al., 2009). In contrast, mated females can readily turn on and off their AA attraction depending on their egg-laying need at a given moment. Perhaps this is because while there is no need for females to re-mate until the stored sperm is depleted, in between egg-laying, they have to tend to other needs and are better off not “too attached” to an AA site.

If tract ppk1 neurons play a critical role in AA attraction, why did our activation experiment fail? There are several possibilities. First, a temporally/spatially precise stimulation of ppk1 neurons is needed. Second, there are additional mechanosensors on the tract that act together with ppk1 neurons to promote AA attraction. Third, presence of eggs in the tract may signal via both a mechanical and a non-mechanical means. For example, passage of eggs through the tract may trigger release of hormones from the tract that then act together with ppk1 neurons to modulate behavior. Nevertheless, it is interesting to note that Drosophila is not the only species that relies on reproductive tract-generated mechanical stimuli to activate “maternal behaviors.” Mechanical stimulation of uterine wall has been shown to contribute to maternal behavior activation in sheep, dog, and rat (Hayes and De Vries, 2007; Kendrick et al., 1991; Keverne et al., 1983; Lévy et al., 2010; Poindron et al., 1989; Yeo and Keverne, 1986), raising the possibility that this feature of behavior control may be evolutionarily conserved.

Which sensory system is responsible for egg-laying induced AA attraction? Several reports suggest AA can promote attraction through the olfactory system (Ai et al., 2010; Root et al., 2011; Semmelhack and Wang, 2009), but Joseph et al showed that egg-laying preference for AA depends on taste but not olfaction (Joseph et al., 2009). Indeed, we have found that Ir64a and Or83b, two olfaction mutants with defective AA sensing (Ai et al., 2010; Semmelhack and Wang, 2009) both show strong egg-laying induced AA attraction (Fig. S4E–F). But if egg-laying induced AA attraction is indeed taste-driven, it may be partly driven by taste memory. This is because “attractive returns” occur in the middle of the chamber where females’ taste neurons are not in contact with AA. Mushroom body (MB) has been shown to regulate egg-laying-induced positional preference for AA (Joseph et al., 2009), but we did not observe a significant change in AA attraction when we ablated it (Fig. S4A–D), suggesting the taste memory that guides attractive returns in our paradigm is likely stored elsewhere. Determining the identity of the AA-sensing taste neurons that promote AA attraction will be an important next step.

Lastly, we also do not know which brain center(s) responds to tract ppk1 neurons to modify valence of AA signal. Pars intercerebralis, the neuroendocrine center, may be one potential (indirect) target, given that it was proposed recently to be a potential target of tract neurons that signal mating status change (Feng et al., 2014), and that hypothalamus, its vertebrate counterpart, is important for modulating reproductive behaviors also.

Experimental Procedure

Stocks

The following stocks were used in this work: ILP7-Gal4 (Yang et al., 2008), piezo-Gal4 (Kim et al., 2012), 21-7-Gal4 (Song et al., 2007), hs-FLP (Gordon and Scott, 2009), UAS-Kir2.1eGFP (Baines et al., 2001), ppk1-Gal4, UAS-mCD8-GFP, UAS-mCD8-RFP, UAS-NaChBac (Bloomington Stock Center), UAS-FRT-CD2-stop-FRT-Kir2.1eGFP (Yang et al., 2009), UAS-P₂X₂ (Lima and Miesenböck, 2005), UAS-GCaMP3 (Tian et al., 2009), UAS-piezoRNAi (Kim et al., 2012), ppk1-LexA (this work), ppk1.0-Gal80(Häsemeyer et al., 2009), ILP7-LexA (this work), LexAop2-FLP (Pan et al., 2012), LexAop2-P₂X₂ (this work), ppk1-GS-Gal4 (this work), LexAop2-GCaMP3 (this work), w¹¹¹⁸.

Transgenic animals

ILP7-LexA, ppk1-LexA constructs were produced by cloning the 1kb promoter upstream of ILP7 and ppk1 genes respectively into the recently modified LexA construct (Pfeiffer et al., 2010). LexAop2-GCaMP3 and LexAop2-P₂X₂ were constructed by cloning the GCaMP3 (Tian et al., 2009) and P₂X₂ (Lima and Miesenböck, 2005) genes into LexAop2 vectors (Pfeiffer et al., 2010). These constructs were then injected into attP-carrying animals following standard protocol.

Immunohistochemistry

Tissues were processed following the same protocol as previously described (Yang et al., 2008). Images were acquired using a Zeiss LSM 700 laser scanning confocal microscope. The following primary antibodies were used: mouse anti-Dlg (1:10, DHSB) and rat anti-mCD8 (1:100, Invitrogen), and mouse anti-GFP (1:100, Sigma) and rabbit anti-GFP (1:1000, Invitrogen). The following secondary antibodies were used: goat anti-mouse Alexa 488, goat anti-mouse Cy3, goat anti-rat Alexa 488, goat anti-rabbit Alexa 488, goat anti-rabbit Cy3.

Hydroxyurea (HU) treatment to ablate mushroom body

To ablate MB, 0–1 hour old larvae were collected and fed HU (sigma H8627) dissolved in inactive yeast paste (50 mg/ml) for 5 hours and then transferred into fresh HU-free food.

Egg-laying preference assay

Females were collected into groups of 20–30 with 15–20 males at the first day of eclosion and kept in fly incubator (25°C and 65% humidity) for 4–5 days. The vials where females were kept were supplemented with active yeast paste unless we wanted to reduce their egg production. Prior egg-laying/behavior assays, individual female flies were loaded into chambers in a customer-designed two-choice egg-laying apparatus (Fig. S1A, B). The 3% AA (V/V) substrate and plain substrate were made from 1% agarose that has been pre-heated in the 55°C water bath. The substrates were allowed to set for 30 minutes before experiments commence.

Behavior analysis

Flies were raised and placed into chambers as described above. A camera holder with 4 Microsoft LifeCam Cinema cameras was then attached to the top of the chambers-containing apparatus (Fig. S1A). Each camera was positioned above 2 egg-laying chambers to record 2 flies. Videos were acquired by CamUniversal and then converted (to allow faster tracking) using Avidemux. Egg-laying events were annotated manually by visually inspecting the videos. Ctrax (Branson et al., 2009) was used to track the fly positions. Custom MATLAB code was used to detect parameters we used to calculate positional preference index, attraction index, and to separate the trajectories into high and low egg-laying states based on the flies’ locomotion speed (Fig. S1C, D). The speed is calculated first by determining the path-length every 2 seconds (at 7.5 frames/seconds). We imposed a one-minute threshold when separating the low vs. high locomotion states. This is because flies sometimes increase their running speed suddenly and briefly – even though they have been consistently in a state of low locomotion – and they then immediately slow down and lay an egg (Fig. 1C). Thus, if flies do not maintain their increased speed for at least one minute, we consider such changes too transient to be labeled as “high locomotion state”.

Tract contraction assay

To determine if stimulating ILP7 can cause tract contraction (Fig. 3G, H), we isolated the tract from the rest of the body and pinned it onto a custom-made perfusion chamber that contains insect physiology buffer (Xiang et al., 2010). We then either perfused the chamber with buffer alone or with buffer with ATP (400 μM).

GCaMP3 recording

Intact VNC (with their connection to the tract carefully preserved) was dissected and mounted in a custom-built perfusion chamber and imaged through a water immersion 40X lens on our Zeiss LSM 700 confocal scope. To image neuronal activity, we recorded GCaMP3 fluorescence at 1 frame per second using the time-series function software. We normally waited for the baseline to stabilize before perfusing buffer alone for 3 minutes and then buffer with ATP to stimulate specific neurons and recorded changes in GCaMP3 signal. The acquired images were then analyzed using a custom MATLAB code. Change in fluorescence (ΔF/F) in a given ROI was averaged from signals from all focal planes.

Supplementary Material

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.

(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 < 0.0001. 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.

(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 P₂X₂ 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.

(D) Quantification of experiment in (B–C). As a control, ILP7 neurons that do not express P₂X₂ do not show any response to ATP. *** p < 0.001. 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 > 0.05.

(E, F) Olfactory mutants Or83b and Ir64 show egg-laying preference and active attraction for AA similar to WT.

NIHMS631474-supplement-1.docx^{(14.9KB, docx)}

2. 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.

Download video file^{(5.6MB, mov)}

3. Movie S2.

This movie shows that stimulating ILP7 axons can induce contraction of a reproductive tract severed from the CNS. P₂X₂ and GCaMP3 were driven by ILP7-Gal4 in this fly. ATP was added at 3 minute. Related to Fig. 3.

Download video file^{(960.7KB, mov)}

4. Movie S3.

This movie shows that ppk1 neurons responded to tract contraction induced by activation of ILP7 neurons. P₂X₂ 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.

Download video file^{(2.1MB, mov)}

5. Table S1.

This table lists the average # of eggs laid on AA vs. plain sides for each of our experiment.

NIHMS631474-supplement-5.pdf^{(1.7MB, pdf)}

Acknowledgments

We thank the Bloomington Stock Center, Drs. Y.N. Jan, B. Baker, G. Rubin, B. Pfeiffer, B. Dickson, and B. Cook for providing us with stocks. We also especially like to thank B. Pfeiffer for his advice on molecular cloning. We also like to thank Drs. R. Mooney, Y. Xiang, M. Gordon, F. Wang, Y. Yang, L. Liu, S. Lisberger and members of the Yang Lab for their helpful suggestions. This work is supported by the Klingenstein Fund and a NIH R01 GM100027 to C.H.Y.

Footnotes

Author Contributions

C.-H.Y. conceived the project with help from B.G. and Y.L. B.G. and Y.L. performed the experiments and analyzed the results with help from A.G, U.S. and C.-H.Y. U.S. designed the behavior setup. B.G, Y.L., and C.-H.Y. wrote the paper.

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