Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster.

Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. Here we report that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in female Drosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca2+ signaling. Moreover, we identified Matrix metalloproteinase-2 as a downstream component of the octopamine-Ca2+ signaling to induce GSC increase. Our study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling.

Introduction

Animal tissues are built from cells originally derived from stem cells (Spradling et al., 2001). During normal development and physiology, this robust stem cell system is precisely regulated (Drummond-Barbosa, 2008). Conversely, the dysregulation of these cells can result in abnormal tissue integrity and lead to deleterious diseases. Previous studies have revealed that many types of stem cells reside in a specialized microenvironment, or niche, where they are exposed to local signals required for their function and identity (Morrison and Spradling, 2008; Spradling et al., 2001). Recently, researchers have demonstrated how stem cell activity is regulated by tissue-extrinsic signals, such as hormones and neurotransmitters. For instance, in mammals, hematopoietic stem cells, mammary stem cells, muscle stem cells, and neural stem cells are influenced by sex hormones such as estrogen (Asselin-Labat et al., 2010; Bramble et al., 2019; Kim et al., 2016; Nakada et al., 2014). Retinoic acid and thyroid hormone play essential roles in the differentiation of testicular stem cells and neural stem cells, respectively (Gothié et al., 2017; Ikami et al., 2015). In addition, mesenchymal stem cell proliferation is stimulated by adrenaline (Wu et al., 2014). However, the when, how, and why these humoral factors are produced, circulated, and received during stem cell regulation remain to be elucidated.

The ovaries of the fruit fly Drosophila melanogaster are an excellent model system on how stem cell lineages are shaped by both local niche signals and tissue-extrinsic signals (Drummond-Barbosa, 2019). D. melanogaster ovary is composed of 16–20 chains of developing egg chambers called ovarioles. The anterior-most region of which, known as the germarium, contains germline stem cells (GSCs) that give rise to the eggs (Figure 1A and B). GSCs are adjacent to the somatic niche cells, which comprises cap cells, escort cells, and terminal filament cells (Figure 1A). After GSC divides, one daughter cell that remains attached to the niche cells retains its GSC identity, whereas the remaining daughter cells are displaced away from the niche cells and differentiate into cystoblast (CB). Each CB then undergoes differentiation into 15 nurse cells and one oocyte in each egg chamber, which is surrounded by somatic follicle cells.

Figure 1.

Post-mating GSC increase requires Oamb in the escort cells.

(A) A schematic representation of Drosophila germarium. GSCs reside in a niche consisting of somatic cells such as cap cells, terminal filament cells, and escort cells and are identifiable by their stereotypical spectrosome morphology and location (adjacent to cap cells). GSC division produces one self-renewing daughter and one cystoblast (CB) that differentiates into a germline cyst. (B) Representative images of wild-type (w) female adult germariums, containing 1, 2 and 3 GSCs from top to bottom. The samples were stained with monoclonal antibody 1B1 (green) and anti-DE-cadherin (magenta), which stain the spectrosome and overall cell membranes, respectively. GSCs are indicated by asterisk. Scale bar, 20 µm. (C–D) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. c587>+ flies were used as the control in D. (E) The ratio of pH3+ GSCs per total GSCs. (F) The ratio of apoptotic (Dcp-1+) somatic cells and germ cells per germarium. c587>+ flies were used as the control. (G) Representative images of adult female germaria immunostained with anti-pMad antibody (green) and DAPI (blue) are shown. GSCs are outlined with dotted lines. Scale bar, 10 µm. (H) Quantification of relative pMad intensity levels in the GSCs (i.e. virgin (V), mated (M)) as normalized to the pMad intensity in CBs. Each sample number was at least 25. The three horizontal lines for each sample indicate lower, median, and upper quartiles. (I) The number of cap cells per germarium in the control and Oamb RNAi driven by c587-GAL4. Values on the y-axis are presented as the mean with standard error of the mean. c587>+ flies were used as the control. For C-F, and I the number of germaria analyzed is indicated inside the bars. Wilcoxon rank sum test with Holm’s correction was used for C, D, H, and I. Fisher’s exact test with Holm’s correction was used for E and F. ***p≤0.001, **p≤0.01, and *p≤0.05; NS, nonsignificant (p>0.05). All source data are available in Source data 1 and 2.

(A–E) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies in (A) Oamb RNAi in mature follicle cells by (R44E10-GAL4); (B) Oamb RNAi in the oviduct (by RS-GAL4); (C) Oamb RNAi by tj-GAL4; (D) Oamb RNAi in cap cells (by bab-GAL4), nervous system (by nSyb-GAL4), and germ cells (by nos-GAL4); and (E) Oamb RNAi in escort cells (by R13C06-GAL4), follicle cells in germarium (by 109–30 GAL4), and stage 9–10 follicle cells (by c355-GAL4 and c306-GAL4), late stage follicle cells and border cells (slbo-GAL4); The number of germaria analyzed is indicated inside the bars. (F) A schematic representation of gRNA target sites (cleavage sites: gray arrowhead) and premature stop codon (red arrowhead) in coding sequences of Oamb genes. Regions of the putative transmembrane domains of Oamb are highlighted in blue. The target locus in Cas9-induced mutant was PCR-amplified and sequenced. The WT sequence is shown on the top of sequences as reference. The Cas9-gRNA target sequence is underlined with the PAM indicated in red. Inserted nucleotides are indicated in light blue lowercase letters. The indel size is shown next to the sequence. The indel mutation results in a premature stop codon. Wilcoxon rank sum test was used for A-E. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

(A–B) Immunofluorescence of germarium in adult female flies expressing 20xUAS-6xGFP reporter under Oamb. The GSCs are indicated by asterisk. Note that 20xUAS-6xGFP has leak signal in the germarium even in the control (+ > 20xUAS-6xGFP). Scale bar, 20 µm. (B) Immunofluorescence of stage 14 egg chamber expressing 20xUAS-6xGFP reporter under Oamb. Note that GFP expression was not observed in the stage 14 egg chamber. Scale bar, 100 µm. (C) Immunofluorescence of germarium (left) and posterior follicle cells of stage 14 egg chamber (right) in adult female flies expressing UAS-Stinger reporter under Oamb. Note that GFP signal is not detected in the germarium and stage 14 egg chamber. Scale bar, 20 µm. (D) Immunofluorescence of germarium in adult female flies expressing 20xUAS-6xGFP reporter under Oamb. Scale bar, 20 µm.

(A) Representative images of adult female germaria immunostained with anti-LacZ antibody (magenta) and DAPI (blue) are shown. GSCs are outlined with dotted lines. Scale bar, 10 µm. (B) Quantification of relative Dad-LacZ intensity levels in the GSCs (i.e. virgin (V), mated (M)) as normalized to the Dad-LacZ intensity in CBs. Each sample number was at least 25. The three horizontal lines for each sample indicate lower, median, and upper quartiles. Wilcoxon rank sum test was used for B. **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 2.

GSC niche produces and secretes several local niche signals that regulate the balance between GSC self-renewal and differentiation (Hayashi et al., 2020; Kirilly and Xie, 2007; Spradling et al., 2011). For example, bone morphogenetic protein (BMP) ligands Decapentaplegic (Dpp) and Glass bottom boat (Gbb) are produced from the niche cells and directly activate BMP receptors in GSCs, leading to the repression of the differentiation inducer, bag-of-marbles (bam) (Morrison and Spradling, 2008; Zhang and Cai, 2020). Recent D. melanogaster GSC studies have also contributed to understanding of the systemic regulation of stem cell proliferation and maintenance in response to external environmental cues (Ables and Drummond-Barbosa, 2017; Drummond-Barbosa, 2019; Lin and Hsu, 2020; Yoshinari et al., 2019). For example, protein restriction results in a reduction in GSC division, which is mediated by Drosophila insulin-like peptides (DILPs) (LaFever, 2005). In addition, nutrients influence GSC maintenance via the adipocyte metabolic pathway (Armstrong and Drummond-Barbosa, 2018; Matsuoka et al., 2017).

Besides nutrients, we have recently found out that mating is another external cue that significantly affects D. melanogaster GSC increase. Mated females show a dramatic increase in egg production, as well as GSC, which is induced by a male-derived peptide from the seminal fluid called sex peptide (SP) (Kubli, 2003; Yoshinari et al., 2019). SP is received by its specific receptor, sex peptide receptor (SPR), in a small subset of SPR-positive sensory neurons (SPSNs), which are located in the uterine lumen and send afferent axons into the tip of the abdominal ganglion (Häsemeyer et al., 2009; Yapici et al., 2008). The SP-SPR signaling in the SPSNs stimulates the biosynthesis of the ovarian insect steroid hormones (ecdysteroids), which play an essential role in mating-induced GSC increase (Ameku et al., 2017; Ameku and Niwa, 2016; Uryu et al., 2015). Because SPSNs do not directly innervate into the ovary, it is hypothesized that a signal and its signaling pathway are involved in bridging the gap between SPSNs and GSCs. However, it is still unclear how mating information is transmitted from SPSNs to GSCs at the molecular and cellular levels.

Here, we present a series of new findings that reveal a novel and fundamental neuronal mechanism connecting SPSNs and GSCs to regulate mating-induced GSC increase. We demonstrate that a small subset of neurons directly innervating into the ovary plays an indispensable role in regulating mating-induced GSC increase. These neurons produce the monoamine neurotransmitter, octopamine (OA), the insect equivalent of noradrenaline (Roeder, 2005). We also show that the neuronal activity of the OA-producing neurons is required for mating-induced GSC increase. Moreover, we find that the OA directory activates GSC increase through its receptor, octopamine receptor in mushroom body (Oamb), followed by Ca2+ signaling in the ovarian escort cells. Furthermore, OA/Oamb signaling requires Matrix metalloproteinase 2 (Mmp2) to activate GSC increase in the ovarian escort cells. Finally, we show that SPSNs relay the mating signal to the ovary-projecting OA neurons via nicotinic acetylcholine receptor signaling. Taken together, we propose a novel efferent neuronal pathway that transmits mating stimulus to the GSC to control stem cell number. Our study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues, such as mating, through stem cell niche signaling.

Results

Mating-induced GSC increase requires the octopamine receptor Oamb in ovarian escort cells

As a candidate signal that bridges between SPSNs and GSC increase, we focused on the biogenic amine, OA, because a part of the octopaminergic neurons innervate to the ovary and the oviduct (Heifetz et al., 2014; Rezával et al., 2014). Moreover, it has been reported that OA and Oamb signaling regulate ovulation process and ovarian-muscle contraction (Deady and Sun, 2015; Monastirioti, 2003; Rezával et al., 2014). We first conducted transgenic RNAi screen against 4 OA receptor genes with c587-GAL4, which is active in the ovarian-somatic cells, including the escort cells of the germarium (Manseau et al., 1997). In control females, the mated ones exhibited an increase in GSC number as we have reported previously (Ameku and Niwa, 2016; Figure 1C). In contrast, c587-GAL4–mediated Oamb knock-down (c587 >Oamb showed significantly impaired mating-induced GSC increase (Figure 1C). This phenotype was observed with two independent UAS-Oamb-RNAi strains (Oamb and Oamb) (Figure 1C), each of which targeted a different region in the Oamb mRNA. The specificity of Oamb was also confirmed by the fact that the c587-GAL4–driven transgenic RNAi of other octopamine receptor genes (Octβ1R, Octβ2R and Octβ3R) (Ohhara et al., 2012) had no significant effect on the GSC number between virgin and mated females (Figure 1C). Therefore, Oamb has a pivotal role in mating-induced GSC increase.

We next examined in which ovarian-somatic cells Oamb regulates mating-induced GSC increase with several GAL4 lines that are active in the specific ovarian-somatic cells. Previous studies have demonstrated that Oamb in mature follicle cells and in the oviduct has a significant role in ovulation (Deady and Sun, 2015; Lee et al., 2009; Lee et al., 2003). Therefore, it is possible that Oamb may indirectly induce GSC increase via Oamb-mediated ovulation processes. However, mating-induced GSC increase was not impaired by Oamb RNAi in the stage-14 follicle cells by R44E10-GAL4 (Deady and Sun, 2015) (R44E10 >Oambi), in the stage 9–10 follicle cells by c355-GAL4, c306-GAL4, and slbo-GAL4 (Barth et al., 2012), or in the common oviduct by RS-GAL4 (Lee et al., 2003) (RS >Oambi) (Figure 1—figure supplement 1A,B and E). These data suggest that mating-induced GSC increase is independent from the ovulation process.

Figure 1—figure supplement 1.

Oamb acts in the escort cells for post-mating GSC increase.

(A–E) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies in (A) Oamb RNAi in mature follicle cells by (R44E10-GAL4); (B) Oamb RNAi in the oviduct (by RS-GAL4); (C) Oamb RNAi by tj-GAL4; (D) Oamb RNAi in cap cells (by bab-GAL4), nervous system (by nSyb-GAL4), and germ cells (by nos-GAL4); and (E) Oamb RNAi in escort cells (by R13C06-GAL4), follicle cells in germarium (by 109–30 GAL4), and stage 9–10 follicle cells (by c355-GAL4 and c306-GAL4), late stage follicle cells and border cells (slbo-GAL4); The number of germaria analyzed is indicated inside the bars. (F) A schematic representation of gRNA target sites (cleavage sites: gray arrowhead) and premature stop codon (red arrowhead) in coding sequences of Oamb genes. Regions of the putative transmembrane domains of Oamb are highlighted in blue. The target locus in Cas9-induced mutant was PCR-amplified and sequenced. The WT sequence is shown on the top of sequences as reference. The Cas9-gRNA target sequence is underlined with the PAM indicated in red. Inserted nucleotides are indicated in light blue lowercase letters. The indel size is shown next to the sequence. The indel mutation results in a premature stop codon. Wilcoxon rank sum test was used for A-E. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

Consistent with the observation using c587-GAL4, Oamb RNAi by Traffic jam (Tj)-GAL4 (Olivieri et al., 2010) (Tj >Oamb), R13C06-GAL4, and 109–30-GAL4 (Sahai-Hernandez and Nystul, 2013), which are active in the pan-ovarian-somatic cells, the escort cells, and the germarium follicle cells, respectively, also resulted in the failure of mating-induced GSC increase (Figure 1—figure supplement 1C and E). On the other hand, Oamb RNAi in the cap cells (bab >Oamb) and germ cells (nos >Oamb) had no effect on GSC increase (Figure 1—figure supplement 1D). These results suggest that Oamb in the escort cells or the follicle cells of the germarium plays an essential role in mating-induced GSC increase.

It must be noted that c587-GAL4 and Tj-GAL4 are expressed not only in the ovarian-somatic cells but also in the nervous system (Ameku et al., 2018). Moreover, Oamb is expressed in the nervous system (Han et al., 1998). However, Oamb RNAi in the nervous system (nSyb >Oamb) did not affect the mating-induced GSC increase (Figure 1—figure supplement 1D), suggesting that the impairment of GSC increase of c587 >Oamb or tj >Oamb is not due to gene knock-down in neuronal cells but rather in the ovarian-somatic cells. These data also support our idea that Oamb in the ovarian-somatic cells regulate mating-induced GSC increase.

To confirm the role of Oamb in mating-induced GSC increase, we generated a Oamb complete loss-of-function genetic allele by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology (Kondo and Ueda, 2013; Figure 1—figure supplement 1F). Similar to Oamb RNAi females, Oamb homozygous mutant females (c587>+; Oamb) did not exhibit mating-induced GSC increase (Figure 1D). In addition, the GSC increase of Oamb was restored by overexpression of Oamb in the escort cells (c587 >Oamb). These findings are all consistent with the idea that Oamb in escort cells modulates GSC increase after mating.

We also examined Oamb expression in the ovarian-somatic cells by two Oamb-knock-in GAL4 lines (Deng et al., 2019; Kondo et al., 2020). However, we could not detect any reliable signals in the germarium or the mature follicle cells via these GAL4 lines with UAS-GFP and UAS-Stinger lines (Figure 1—figure supplement 2A,B,C and D). We speculate that this may be due to lower amounts of Oamb transcript in the germarium.

Figure 1—figure supplement 2.

Expression of Oamb knock-in GAL4.

(A–B) Immunofluorescence of germarium in adult female flies expressing 20xUAS-6xGFP reporter under Oamb. The GSCs are indicated by asterisk. Note that 20xUAS-6xGFP has leak signal in the germarium even in the control (+ > 20xUAS-6xGFP). Scale bar, 20 µm. (B) Immunofluorescence of stage 14 egg chamber expressing 20xUAS-6xGFP reporter under Oamb. Note that GFP expression was not observed in the stage 14 egg chamber. Scale bar, 100 µm. (C) Immunofluorescence of germarium (left) and posterior follicle cells of stage 14 egg chamber (right) in adult female flies expressing UAS-Stinger reporter under Oamb. Note that GFP signal is not detected in the germarium and stage 14 egg chamber. Scale bar, 20 µm. (D) Immunofluorescence of germarium in adult female flies expressing 20xUAS-6xGFP reporter under Oamb. Scale bar, 20 µm.

Oamb in escort cells is required for GSC increase after mating

Because mating-induced GSC increase is accompanied by GSC division (Ameku and Niwa, 2016), We next examined whether Oamb in the escort cells is involved in GSC division after mating. We determined the number of GSCs during the M phase by staining using anti-phospho-Histone H3 (pH3) in control and c587 >Oamb females. In control female flies, mating increased the frequency of GSCs in the M phase (Figure 1E), whereas in c587 >Oamb flies, this was not observed. We also monitored the fraction of apoptotic cells in the germarium by staining with anti-cleaved death caspase-1 (Dcp-1), a marker for apoptotic cells (Song et al., 1997). The number of apoptotic cells in the germarium did not change in c587 >Oamb female flies compared with controls (Figure 1F), suggesting that Oamb activates GSC increase by pushing the cell cycle of GSCs and that the lack of mating-induced GSC increase in Oamb RNAi is not due to the enhancement of cell death.

Our previous studies revealed that mating-induced GSC increase is mediated by GSC niche signals (Ameku et al., 2018; Ameku and Niwa, 2016). In particular, Decapentaplegic (Dpp), the fly counterpart to bone morphogenetic protein (BMP), is the essential niche signal (Spradling et al., 2011; Xie and Spradling, 1998). We therefore examined whether Oamb knock-down affects Dpp signaling in GSCs by measuring the level of phosphorylated Mad (pMad), a readout of Dpp signaling activation (Chen and McKearin, 2003; Raftery and Sutherland, 1999). We confirmed that mating induced the increase in pMad level in GSCs, whereas mating did not increase pMad levels in c587 >Oamb animals (Figure 1G and H). We also confirmed that mating increased the signal intensity of Daughters against dpp (Dad)-LacZ, a reporter gene that reflect an expression of the BMP target gene Dad, while Oamb knock-down in the escort cells impaired the increase of Dad-LacZ signal after mating (Figure 1—figure supplement 3A and B). These results suggest that mating activates the BMP signal in GSCs through Oamb in the escort cells, thereby resulting in the increase in GSCs.

Figure 1—figure supplement 3.

Oamb in the escort cells is necessary on mating-induced BMP signaling increase.

(A) Representative images of adult female germaria immunostained with anti-LacZ antibody (magenta) and DAPI (blue) are shown. GSCs are outlined with dotted lines. Scale bar, 10 µm. (B) Quantification of relative Dad-LacZ intensity levels in the GSCs (i.e. virgin (V), mated (M)) as normalized to the Dad-LacZ intensity in CBs. Each sample number was at least 25. The three horizontal lines for each sample indicate lower, median, and upper quartiles. Wilcoxon rank sum test was used for B. **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 2.

Further, we determined the number of cap cells, which are critical components of the GSC niche (Xie and Spradling, 1998). c587 >Oamb did not change the number of cap cells in virgin nor mated female flies, suggesting that Oamb knock-down does not affect the overall architecture of the niche (Figure 1I). Overall, Oamb in the escort cells plays a pivotal role in mating-induced GSC increase.

OA administration is sufficient to induce GSC increase and BMP signaling in GSCs in an Oamb-dependent manner

To examine whether OA is received in the ovary but not in other organs to induce GSC increase, we cultured dissected virgin ovaries ex vivo with or without purified OA in the culture medium. After incubation for 12 hr, the ovaries cultured with OA had more GSCs as compared to those without OA (Figure 2—figure supplement 1A). Whereas the minimal OA concentration to induce ex vivo GSC increase was 1 μM, hereafter we used 100 μM OA because the GSC number plateaued with this concentration (Figure 2—figure supplement 1A). This OA-mediated ex vivo GSC increase was not observed in c587 >Oamb virgin ovaries (Figure 2A).

Figure 2—figure supplement 1.

OA treatment induces GSC increase.

(A) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin female flies. The addition of OA to the medium is sufficient to induce GSC increase. (B) Representative images of adult female germaria in response to OA in tj >GCaMP6s; mCD8::RFP. Note that calcium response was observed in the escort cells (arrowheads) and follicle cells (arrow) of the germarium. Scale bar, 10 µm. Wilcoxon rank sum test with Holm’s correction was used for statistical analysis. ***p≤0.001. All source data are available in Source data 1.

Figure 2.

Ca2+ signaling is necessary for mating-induced GSC increase.

(A) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis). The ovaries were dissected from virgin (V), mated (M), and virgin ovaries cultured with OA (+OA). c587>+ flies were used as the control. The number of germaria analyzed is indicated inside the bars. (B) Quantification of relative pMad intensity levels in the GSCs of ex vivo cultured ovaries (i.e. virgin (V), mated (M), and virgin cultured with OA (+OA)) as normalized to the pMad intensity in CBs. For the quantification of pMad intensity, the cell boundaries of GSCs and CBs were determined using anti-Vasa staining. Each sample number was at least 25. The three horizontal lines for each sample indicate lower, median, and upper quartiles. (C) A schematic representation of ex vivo calcium imaging. The dissected ovariole was incubated in Schneider’s Drosophila medium with or without OA. (D) Changes in the relative fluorescence intensity of GCaMP6s after 200 s without stimulation (n = 8) or with stimulation (n = 10) with 100 μM OA, and (E) with 100 μM OA as control (c587 >LacZ, n = 8) and c587 >Oamb (n = 8) female ovaries. Note that OA significantly increased the calcium response in escort cells, but Oamb impaired the calcium response. Statistical analysis was done at 120 s. (F) Equipment setup for optogenetic activation of ChR. Flies were placed under the light for 16 hr before dissection. (G) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) with light, with light and all trans-retinal (ATR) or with dark and ATR. Germarium was dissected from virgin females. nSyb-GAL80; c587 >GFP flies were used as control. The number of germaria analyzed is indicated inside the bars. (H) The ratio of pH3+ GSCs and total GSCs. The number of GSCs analyzed is indicated inside the bars. (I, left) Representative images of adult female germaria immunostained with anti-pMad antibody (green), anti-1B1 antibody (red), and anti-Vasa antibody (germ cell marker; blue) are shown. GSCs are outlined with dotted lines. (I, right) Quantification of the relative pMad intensity in GSCs, which was normalized to that in CBs. For the quantification of pMad intensity, the cell boundaries of GSCs and CBs were determined using anti-Vasa staining. Each sample number is at least 30. The three horizontal lines for each data sample indicate lower, median, and upper quartiles. (J) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. c587>+ flies were used as the control. The number of germaria analyzed is indicated inside the bars. Wilcoxon rank sum test with Holm’s correction was used for A, B, D, E, G, I, and J. Fisher’s exact test was used for H. ***p≤0.001, **p≤0.01, and *p≤0.05; NS, non-significant (p>0.05). All source data are available in Source data 1, 2, and 4.

(A) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin female flies. The addition of OA to the medium is sufficient to induce GSC increase. (B) Representative images of adult female germaria in response to OA in tj >GCaMP6s; mCD8::RFP. Note that calcium response was observed in the escort cells (arrowheads) and follicle cells (arrow) of the germarium. Scale bar, 10 µm. Wilcoxon rank sum test with Holm’s correction was used for statistical analysis. ***p≤0.001. All source data are available in Source data 1.

We also examined whether OA treatment affects Dpp signaling in GSCs ex vivo by measuring the level of pMad. We confirmed that OA treatment was sufficient to induce the increase in pMad level in GSCs, even in the ex vivo culture system (Figure 2B). On the other hand, OA treatment did not increase pMad levels in c587 >Oamb ovaries (Figure 2B). These results suggest that OA activates the BMP signal in GSCs through Oamb in the escort cells.

Ca2+ signaling in escort cells is essential for OA-dependent GSC increase

Upon OA binding, Oamb evokes Ca2+ release from the endoplasmic reticulum (ER) into the cytosol, leading to a transient increase in intercellular Ca2+ concentration ([Ca2+]i) (Han et al., 1998). To determine whether OA induces GSC increase via affecting [Ca2+]i in escort cells, we first monitored [Ca2+]i using a genetically encoded calcium sensor, GCaMP6s (Nakai et al., 2001; Ohkura et al., 2012). We dissected the virgin ovaries, in which GCaMP6s transgene was expressed driven by Tj-GAL4 or c587-GAL4, cultured them ex vivo, and then observed GCaMP6s fluorescence (Figure 2C). We found that 100 μM of OA treatment evoked an increase in GCaMP6s fluorescence in the escort cells and follicle cells, whereas the control medium treatment (0 μM of OA) did not show any increase in fluorescence (Figure 2D and Figure 2—figure supplement 1B; also see Videos 1 and 2). In addition, the OA-mediated increase in GCaMP6s fluorescence intensity was not observed in the germarium of Oamb RNAi flies (Figure 2E), suggesting that the OA-dependent increase in [Ca2+]i in the germarium is required for Oamb. Notably, the timeline of signal increase was very slow as this fluorescence increases progressively. This response to OA treatment was similar to a report featuring mature follicle cells of stage-14 oocytes (Deady and Sun, 2015).

Video 1.

A video image of the GCaMP6 signal in the ex vivo-cultured germarium without OA administration.

A genotype of the germarium was Tj-GAL4 >UAS-GCaMP6s UAS-mCD8::RFP.

Video 2.

A video image of the GCaMP6 signal in the ex vivo-cultured germarium with 100 mM OA administration.

A genotype of the germarium was Tj-GAL4 >UAS-GCaMP6s UAS-mCD8::RFP.

We also employed another approach utilizing the light-gated cation channel, CsChrimsonn (Klapoetke et al., 2014). We prepared c587-GAL4 >CsChrimson flies in combination with nSyb-GAL80 (Harris et al., 2015), allowing the expression of CsChrimson gene only in the germarium but not in the nervous system. Because CsChrimson requires all trans-retinal (ATR) to form its proper protein conformation (Wang et al., 2012), we utilized the flies fed with and without ATR supplement as experimental and control groups, respectively. When we irradiated an orange-light to c587-GAL4, nSyb-GAL80 >CsChrimson flies to induce Ca2+ flux in the germarium (Figure 2F), GSC increased in the virgin females (Figure 2G). In addition, the ratio of GSC in the M phase increased by CsChrimson activation (Figure 2H). Moreover, the pMad level in GSCs was increased by CsChrimson activation, suggesting that the forced Ca2+ flux is sufficient to induce GSC increase through the upregulation of BMP signaling (Figure 2I).

We next confirmed whether the downstream component of Ca2+ signaling is involved in the mating-induced GSC increase. The knock-down of Inositol 3-receptor (c587 >Insp3R) encoding a protein that releases the stored Ca2+ from ER suppressed GSC increase in mated females (Figure 2J). Conversely, the overexpression of Insp3R in the escort cells increased the GSC number in virgin females (Figure 2J). Furthermore, OA-mediated ex vivo GSC increase was not observed in c587 >Insp3R virgin ovaries (Figure 2A). Overall, we demonstrated that OA signaling regulates the mating-induced GSC increase by controlling Ca2+ signaling in escort cells, which is thereby necessary and sufficient to induce GSC increase.

Ovarian ecdysteroid signaling is required in the OA-Oamb-Ca2+-dependent GSC increase

The biosynthesis and signaling of ecdysteroid in the ovary are required for the mating-induced GSC increase (Ameku et al., 2017; Ameku and Niwa, 2016). Therefore, we next examined whether ecdysteroid signaling has a pivotal role in the OA-Oamb-Ca2+-dependent GSC increase. As we have previously reported, the RNAi of neverland (nvd), which encodes an ecdysteroidogenic enzyme (Yoshiyama-Yanagawa et al., 2011; Yoshiyama et al., 2006) in the escort cells, suppressed the mating-induced GSC increase (Figure 3A; Ameku et al., 2017; Ameku and Niwa, 2016). We also found a similar phenotype in the RNAi of ecdysone receptor (EcR) in the escort cells (Figure 3A). To assess the requirement of ecdysteroid biosynthesis and signaling in OA-induced GSC increase, we employed an ex vivo experiment. Interestingly, the OA-mediated GSC increase was not observed in nvd RNAi ovaries (c587 >nvd) (Figure 3B). Moreover, this impairment of GSC increase in c587 >nvd was restored with the administration of 20-hydroxyecdysone (20E) in the culture media. On the other hand, 20E treatment without OA did not induce GSC increase in the control ovaries (Figure 3B). This observation is consistent with our previous study showing that wild-type virgin females fed with 20E did not exhibit any increase in GSCs (Ameku and Niwa, 2016). Therefore, 20E is required for the OA-mediated increase in GSCs; however, by itself, 20E is not sufficient to induce GSC increase.

Figure 3.

Ecdysteroid signaling is necessary for OA-mediated GSC increase.

(A–D) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. c587>+ flies were used as the control. The number of germaria analyzed is indicated inside the bars. (A) GSC number of nvd and EcR RNAi flies in vivo. (B) Virgin ovaries were cultured ex vivo with or without OA and 20E (+OA, +20E, −), and then the GSC number was determined. (C–D) Experiments using a temperature-sensitive allele EcR. 21°C and 31°C were used as the permissive and restrictive temperatures, respectively. Flies were cultured at 21°C and transferred to 31°C 1 d prior to the assays (L; light, D; dark). (C) GSC number in vivo. (D) Virgin ovaries were cultured ex vivo with or without OA (+OA, −). The number of germaria analyzed is indicated inside the bars. Wilcoxon rank sum test with Holm’s correction was used for statistical analysis. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

To assess the role of EcR in downstream OA signaling, we utilized the temperature-sensitive and null alleles of EcR (EcR and EcR, respectively) (Bender et al., 1997). At a restrictive temperature of 31°C, the mating-induced GSC increase was suppressed in EcR/EcR flies, consistent with our previous report (Ameku and Niwa, 2016; Figure 3C). In the ex vivo experiment, the OA-dependent GSC increase was also suppressed in EcR/EcR ovaries (Figure 3D). These results suggest that OA-Oamb-Ca2+ signaling requires ovarian ecdysteroid signaling.

Matrix metalloproteinase two acts downstream of OA-Oamb-Ca2+ signaling

So far, we have identified four components with indispensable roles in mating-induced GSC increase, namely OA, Oamb, Ca2+, and ecdysteroids. Interestingly, recent studies have reported that they are also essential in the follicle rupture in D. melanogaster ovary (Deady and Sun, 2015; Knapp and Sun, 2017). In this process, Matrix metalloproteinase 2 (Mmp2), a membrane-conjugated proteinase, acts downstream of the OA-Oamb-Ca2+ signaling pathway in mature follicle cells (Deady et al., 2015). Because recent studies have indicated the expression of Mmp2 in niche cells, including escort cells (Pearson et al., 2016; Wang and Page-McCaw, 2014), we examined Mmp2 function in the escort cells using RNAi of Mmp2 with c587-GAL4. Similar to c587 >Oamb, c587 >Mmp2 impaired mating-induced GSC increase (Figure 4A). Notably, Mmp2 knock-down in cap cells by bab-GAL4 also suppressed GSC increase, suggesting that Mmp2 acts in both escort cells and cap cells to induce GSC increase (Figure 4—figure supplement 1A). We also found that Mmp2 RNAi in the ovarian-somatic cells by another GAL4 (Tj >Mmp2) resulted in the failure of mating-induced GSC increase, whereas Mmp2 RNAi in the nervous system (nSyb >Mmp2) and in the follicle cells of stage 14 oocytes (R44E10 >Mmp2) had no effect on GSC increase (Figure 4—figure supplement 1B). These results suggest that Mmp2 in the escort cells and cap cells are necessary to induce post-mating GSC increase.

Figure 4.

Mmp2 is necessary for OA-mediated GSC increase.

(A–C, E) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. c587>+ flies were used as the control. The number of germaria analyzed is indicated inside the bars. (A) Mmp2 RNAi by c587-GAL4 driver. (B) RNAi and the overexpression of Timp by c587-GAL4 driver. (C) Ex vivo culture experiment using c587 >Mmp2 RNAi. OA was added into the ex vivo culture medium. Cultured with or without OA (+OA, -, respectively) is indicated under each bar. (D) Quantification of the relative pMad intensity in GSCs of the ex vivo cultured ovaries normalized to pMad intensity in CBs. Cultured with or without OA (+OA, −) is indicated under each bar. For the quantification of pMad intensity, the cell boundaries of GSCs and CBs were determined using anti-Vasa staining (n > 15). The three horizontal lines for each data sample indicate lower, median, and upper quartiles. (E) Oamb, nvd, or Mmp2 RNAi in the genetic background of c587 >Insp3R overexpression. (F) A model of signaling in the escort cell to induce the mating-induced GSC increase. Oamb in the escort cells receives OA, and induce [Ca2+]i in the cells. The [Ca2+]i induces GSC increase via Mmp2. Ecdysteroid signaling is also involved in this process. Wilcoxon rank sum test with Holm’s correction was used. ***p≤0.001, **p≤0.01, and *p≤0.05; NS, non-significant (p>0.05). All source data are available in Source data 1 and 2.

(A–C) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) female flies. Mmp2 RNAi in the (A) cap cells by bab-GAL4 driver and in the (B) nervous system by nSyb-GAL4, mature follicle cells by R44E10-GAL4, and ovarian-somatic cells by tj-GAL4. (C) Timp RNAi in the escort cells by c587-GAL4, cap cells by bab-GAL4, and mature follicle cells by R44E10-GAL4. (D) The number of cap cells in the control and Mmp2 RNAi driven by c587-GAL4. Values on y-axis are presented as the mean with standard error of the mean. (E) Representative images of Vkg::GFP adult female germaria immunostained with anti-GFP antibody (green), anti-Lamin C antibody (red; cap cells, asterisk), and DAPI. Note that the Vkg::GFP signal around cap cells is not affected even in Mmp2 RNAi flies. Scale bar, 10 µm. Wilcoxon rank sum test with Holm’s correction was used for A, B, C and D. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

Figure 4—figure supplement 1.

Mmp2 is necessary in the escort cells to induce GSC increase.

(A–C) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) female flies. Mmp2 RNAi in the (A) cap cells by bab-GAL4 driver and in the (B) nervous system by nSyb-GAL4, mature follicle cells by R44E10-GAL4, and ovarian-somatic cells by tj-GAL4. (C) Timp RNAi in the escort cells by c587-GAL4, cap cells by bab-GAL4, and mature follicle cells by R44E10-GAL4. (D) The number of cap cells in the control and Mmp2 RNAi driven by c587-GAL4. Values on y-axis are presented as the mean with standard error of the mean. (E) Representative images of Vkg::GFP adult female germaria immunostained with anti-GFP antibody (green), anti-Lamin C antibody (red; cap cells, asterisk), and DAPI. Note that the Vkg::GFP signal around cap cells is not affected even in Mmp2 RNAi flies. Scale bar, 10 µm. Wilcoxon rank sum test with Holm’s correction was used for A, B, C and D. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

In the GSC niche cells in D. melanogaster, Tissue inhibitors of metalloproteinases (Timp) gene encoding an endogenous proteinase inhibitor of Mmp2 is expressed (Gomis-Rüth et al., 1997; Page-McCaw et al., 2003; Pearson et al., 2016). The knock-down of Timp in escort cells induced GSC increase even in virgin females, whereas its overexpression suppressed mating-induced GSC increase (Figure 4B). Consistent with Mmp2 knock-down, Timp knock-down in cap cells (bab >TimpRNAi) increased the GSC number in virgin females, whereas its knock-down in the follicle cells of stage 14 oocyte (R44E10 >TimpRNAi) had no effect (Figure 4—figure supplement 1C). These data suggest that Mmp2 activity in the GSC niche cells is necessary for mating-induced GSC increase.

We next examined whether Mmp2 is necessary for OA-induced GSC increase. Our ex vivo culture experiment revealed that OA-induced GSC increase was suppressed in c587 >Mmp2 RNAi flies, suggesting that Mmp2 acts downstream of OA signaling (Figure 4C). Moreover, the OA-dependent upregulation of pMad level in GSCs was suppressed in c587 >Mmp2 RNAi flies (Figure 4D). Notably, in virgin females, Mmp2 RNAi affected neither the GSC number nor the cap cell number, indicating that Mmp2 RNAi does not influence the overall niche architecture (Figure 4—figure supplement 1D). Mmp2 in the mature follicle cells cleaves and downregulates collagen VI, also known as Viking (Vkg) and is the major component of the basement membrane (Deady et al., 2017; Wang et al., 2008). However, Mmp2 RNAi had no effect on Vkg::GFP level around the cap cells (Figure 4—figure supplement 1E). Therefore, the suppression of mating-induced GSC increase in Mmp2 RNAi does not likely depend on the collagen VI level.

To examine the epistasis of OA/Oamb-Ca2+ signaling, ecdysteroid signaling, and Mmp2, we knocked down Oamb, nvd, or Mmp2 in c587 >Insp3R genetic background, where Ca2+ signaling was forcedly upregulated. Whereas the Oamb RNAi did not suppress GSC increase in virgin female (c587 >Insp3R), the RNAi of nvd or Mmp2 suppressed GSC increase even when Ca2+ signaling were activated (c587 >Insp3R or c587 >Insp3R) (Figure 4E). These results suggest that ecdysteroid signaling and Mmp2 act downstream of Ca2+ signaling in OA-induced GSC increase. Taken together, both the OA-Oamb signaling and the downstream Ca2+ signaling regulate the mating-induced GSC increase via Mmp2 and ecdysteroid signaling (Figure 4F).

Octopamine from dsx neurons regulates the mating-induced GSC increase

To examine the in vivo role of OA in mating-induced GSC increase, we silenced the expression of Tyrosine decarboxylase 2 (Tdc2) and Tyramine β hydroxylase (TβH) genes, which code for enzymes responsible for OA biosynthesis (Cole et al., 2005; Monastirioti et al., 1996; Figure 5A), with Tdc2-GAL4 driver-mediated RNAi (Tdc2 >Tdc2). Similar to the phenotype of Oamb RNAi, Tdc2 or TbH RNAi with Tdc2-GAL4 or nSyb-GAL4 impaired the mating-induced GSC increase (Figure 5A and Figure 5—figure supplement 1A–B). Moreover, the impairment of GSC increase was restored when Tdc2 or TbH RNAi flies were fed with food supplemented with OA (Figure 5A), supporting our hypothesis that OA is responsible for the mating-induced GSC increase in vivo.

Figure 5.

Ovary-projecting OA neurons control the GSC increase.

(A, C–D, F–G) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. The number of germaria analyzed is indicated inside the bars. (A) RNAi of Tdc2 and TβH by Tdc2-GAL4. OA was added into the standard food. (B) A schematic drawing of Drosophila central nervous system and the ovary-projecting OA neurons with the dsx OA neurons projecting to the ovary. (C) Tdc2 RNAi in dsx neurons with the genotype indicated. (D–E) TrpA1-mediated activation of dsx neurons. 17°C and 29°C were used as the permissive and restrictive temperatures, respectively, of TrpA1 channel. (D) GSC number. (E) The ratio of pH3+ GSCs and total GSCs. (F) The activation of Tdc2 neurons with Oamb genetic background. (G) The inactivation of dsx neurons. (H) Illustration showing the location of three clusters of Tdc2 neurons in the caudal part of the abdominal ganglion (I–K, I’–K’). Negative images of TRIC labeling (anti-GFP) in the abdominal ganglions of virgin (I–K) and mated females (I’–K’) of TRIC (Tdc2 >UAS-mCD8::RFP, UAS-p65AD::CaM LexAop2-mCD8::GFP; nSyb-MKII::nlsLexADBDo;UAS-p65AD::CaM) flies, indicating intracellular Ca2+ transients. Scale bars, 20 μm. (L) The GFP intensities from the Tdc2 median cluster, Tdc2 dorsal cluster, and dsx cluster of TRIC females show Ca2+ activity in virgin (gray) and mated females (red). Wilcoxon rank sum test was used for A, C, D, F, G, and L. Fisher’s exact test with Holm’s correction was used for E. ***p≤0.001, **p≤0.01, and *p≤0.05; NS, non-significant (p>0.05). All source data are available in Source data 1 and 3.

(A–B) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies using nSyb-GAL4 (A) and Tdc2-GAL4 (B). (C–D) Images of dsx neurons expressing the UAS > stop >mCD8::GFP reporter under Tdc2-GAL4 with dsx-FLP. GFP expression was detected only in the abdominal ganglion neurons projecting to the ovary. Images of the abdominal ganglion and the reproductive system are shown in C and D, respectively. (E–G) Immunofluorescence of dsx neurons expressing UAS >mCD8::GFP reporter under Tdc2-GAL4 with tub >FRT >GAL80>FRT. GFP expression was only observed in the anti-Tdc2 positive neurons in the abdominal ganglion that projected to the ovary (E). Scale bars, 100 µm in C, D, E, and G; 10 µm in F. Wilcoxon rank sum test with Holm’s correction was used. ***p≤0.001; NS, non-significant (p>0.05). All source data are available in Source data 1.

Figure 5—figure supplement 1.

dsx neurons control GSC increase.

(A–B) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies using nSyb-GAL4 (A) and Tdc2-GAL4 (B). (C–D) Images of dsx neurons expressing the UAS > stop >mCD8::GFP reporter under Tdc2-GAL4 with dsx-FLP. GFP expression was detected only in the abdominal ganglion neurons projecting to the ovary. Images of the abdominal ganglion and the reproductive system are shown in C and D, respectively. (E–G) Immunofluorescence of dsx neurons expressing UAS >mCD8::GFP reporter under Tdc2-GAL4 with tub >FRT >GAL80>FRT. GFP expression was only observed in the anti-Tdc2 positive neurons in the abdominal ganglion that projected to the ovary (E). Scale bars, 100 µm in C, D, E, and G; 10 µm in F. Wilcoxon rank sum test with Holm’s correction was used. ***p≤0.001; NS, non-significant (p>0.05). All source data are available in Source data 1.

Because Tdc2-GAL4 is active in the nervous system (Busch et al., 2009; Pauls et al., 2018), we then identified which neurons secrete OA to the escort cells. D. melanogaster has more than 70–100 OAergic neurons dispersed throughout the nervous system (Monastirioti, 2003; Schwaerzel et al., 2003; Zhou et al., 2008). Among them, we were particularly interested in a small subset innervating the reproductive system (Figure 5B) as several recent studies have revealed that these neurons regulate mating behavior, egg laying, and ovarian-muscle contraction (Heifetz et al., 2014; Lee et al., 2003; Middleton et al., 2006; Rezával et al., 2014; Rubinstein and Wolfner, 2013). The ovary-projecting OAergic neurons are doublesex (dsx) and Tdc2 double-positive (Rezával et al., 2014; Figure 5B). Therefore, to manipulate the gene expression of dsx neurons only, we implemented a FLP/FRT intersectional strategy using dsx-FLP (Rezával et al., 2014). We could detect GFP expression only in the dsx neurons innervating to the ovary, whose cell bodies are located on a caudal part of the abdominal ganglion (Rezával et al., 2014; Figure 5—figure supplement 1C–E). We next knocked down Tdc2 in dsx neurons by RNAi (tub >GAL80>Tdc2) and found that these RNAi flies failed to increase GSC number after mating. Given the fact that the intersectional strategy using dsx-FLP does not label any neurons in the central nervous system (Rezával et al., 2014), these data suggest that only a small subset of dsx neurons controls the mating-induced GSC increase.

To assess whether the activity of dsx neurons affects the GSC number, we overexpressed TrpA1, a temperature-sensitive cation channel gene, in the dsx neurons only. In Tdc2 >stop >TrpA1;dsx-FLP flies, we can tightly control the TrpA1 expression in the dsx neurons only (Rezával et al., 2014). Both the control flies and TrpA1-overexpressing flies at permissive temperature (17°C) had the normal GSC number in virgin and mated females. On the other hand, at the restrictive temperature (29°C), the TrpA1-overexpressing flies, even the virgin ones, had more GSCs (Figure 5D). We also found that Tdc2 >stop >TrpA1; dsx-FLP virgin females at the restrictive temperature had increased GSC frequency in the M phase (Figure 5E). Importantly, the TrpA1-mediated activation of Tdc2 neurons did not induce the GSC increase in loss-of -Oamb-function females (Tdc2 >TrpA1; Oamb) (Figure 5F), suggesting that the TrpA1-mediated GSC increase requires Oamb.

Furthermore, we employed the Tetanus toxin light chain (TNT) to inhibit neuronal activity (Sweeney et al., 1995). When we overexpressed TNT in dsx neurons only, the mating-induced GSC increase was suppressed in mated females as compared with the control, whose inactivated TNT was overexpressed (Figure 5G). Taken together, these findings suggest that the mating-induced GSC increase is mediated by the neuronal activity of dsx neurons innervating to the ovary.

dsx neurons are activated after mating

Because the dsx neuronal activity has a significant role in mating-induced GSC increase, we next examined whether these neurons change their activity before and after mating. We monitored the neuronal activity using an end-point Ca2+ reporting system, the transcriptional reporter of intracellular Ca2+ (TRIC) (Gao et al., 2015). TRIC is designed to increase the GFP expression in proportion to [Ca2+]i. We classified female Tdc2 neurons in the caudal part of the abdominal ganglion into three clusters based on their location and morphology. We designated the three clusters of these Tdc2 neurons as the Tdc2 median, Tdc2 dorsal, and Tdc2 caudal clusters (Figure 5H). Among them, the position of first two clusters are not similar to that of dsx neurons, whereas that of the Tdc2 caudal cluster is similar (Rezával et al., 2014). In virgin females, we detected robust TRIC signals in the Tdc2 median and Tdc2 dorsal clusters but not in the Tdc2 caudal cluster (Figure 5I–L). In contrast, 24 hr after mating, we observed a significant increase in the TRIC signal in the Tdc2 caudal cluster, whereas those in the Tdc2 median and Tdc2 dorsal clusters were not changed in virgin and mated females (Figure 5I–L). This result suggests that the Tdc2 caudal cluster, which are likely dsx neurons, is significantly activated after mating.

The activity switch of dsx neurons is regulated by the sex peptide sensory neurons via acetylcholine signaling

Our previous study revealed that the mating-induced GSC increase is mediated by the male seminal fluid protein SP (Ameku and Niwa, 2016). SP is received by SPR in a small number of SPSNs, followed by a neural silencing of SPSNs (Häsemeyer et al., 2009; Yapici et al., 2008). Notably, SPSNs project their arbors into a caudal part of the abdominal ganglion, where the cell bodies of the dsx cluster neurons are located (Rezával et al., 2014; Rezával et al., 2012). Therefore, we examined whether SPSNs physically interact with Tdc2 neurons in the abdominal ganglion by performing the GFP Reconstitution Across Synaptic Partners (GRASP) analysis (Feinberg et al., 2008; Gordon and Scott, 2009), in which two complementary fragments of GFP were expressed in SPSNs and Tdc2 neurons. GRASP signals were detected in the abdominal ganglion (Figure 6A), suggesting that the axon termini of SPSNs and the cell bodies and/or dendrites of Tdc2 neurons contact each other likely through synaptic connections.

Figure 6.

SPSNs control GSC increase through OA neurons.

(A) Neuronal proximity of SPSNs and Tdc2 neurons in the abdominal ganglion of female flies stained with anti-Tdc2 (magenta). Note that reconstituted GFP (GRASP) signal was detected in the caudal part of the abdominal ganglion surrounded by broken white lines. Scale bar, 25 µm. (B) Cell bodies of SPSNs (yellow arrows) of ChAT-GAL4; UAS-mCD8::RFP; ppk-EGFP virgin females. Note that mCD8::RFP and EGFP signals overlapped in the cell bodies (yellow arrow) of SPSNs. White broken lines outline the oviduct. Scale bar, 25 µm. (C–F) Frequencies of germaria containing 1, 2, 3, and 4 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. The number of germaria analyzed is indicated inside the bars. (C) ChaT RNAi by ppk-GAL4. (D) RNAi of nAChRs in dsx neurons. (E) RNAi of Tdc2 by Tdc2-GAL4 along with the silencing of SPSNs. 21 and 31°C were used as the permissive and restrictive temperatures, respectively, of shibire. (F) RNAi of Oamb and Insp3R by c587-GAL4 along with the silencing of SPSNs. Kir2.1 was used in this experiment. Note that frequencies of germaria containing 4 GSCs increased. Wilcoxon rank sum test with Holm’s correction was used for C, D, E and F. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

(A) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (v) female flies. (B–F) Representative images of the ovaries stained by anti-GFP (B–F) and anti-Tdc2 (B’–F’). Both signals merged on the surface of the ovary (B’’–F’’). Scale bar, 50 µm. Wilcoxon rank sum test was used. ***p≤0.001; NS, non-significant (p>0.05).

(A) Schematic representation of gRNA target sites (cleavage sites: gray arrowhead) and premature stop codon (red arrowhead) in coding sequences of nAChRα1 genes. Regions of the putative transmembrane domains of nAChRα1 are highlighted in blue. The target locus in Cas9-induced mutant was PCR-amplified and sequenced. The WT sequence (nAChRα1) is shown as reference. The Cas9-gRNA target sequence is underlined with the PAM indicated in red. Inserted nucleotides are indicated in light blue lowercase letters. The indel size is shown next to the sequence. The indel mutation results in a premature stop codon at the 228th and 326th amino acid sequence. (B–C) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. (B) GSC numbers in nAChRα1 genetic mutants. (C) nAChRα1 overexpression in the Tdc2 neurons was sufficient to restore increased GSC in nAChRα1 mutants. Wilcoxon rank sum test with Holm’s correction was used. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

Because SPSNs have been implied as cholinergic neurons (Rezával et al., 2012), we next examined the expression of Choline acetyltransferase (ChaT)-GAL4 in SPSNs. ChaT encodes an acetylcholine biogenic enzyme (Greenspan, 1980). The SPSNs located on the oviduct, which also co-express pickpocket (ppk) and fruitless (fru), are particularly crucial for inducing the major behavioral changes in female flies after mating (Ameku and Niwa, 2016; Rezával et al., 2012). By using UAS-mCD8::RFP with ChaT-GAL4 alongside ppk-EGFP, we confirmed that the ppk-EGFP–positive population near the oviduct were co-labeled by RFP (Figure 6B), consistent with the speculation that SPSNs are cholinergic.

We next counted the GSC number in ChAT RNAi flies using ppk-GAL4. ppk >ChAT virgin flies had more GSCs compared with the control (Figure 6C). In addition, mating did not induce GSC increase in ppk >ChAT flies, suggesting that the acetylcholine released from SPSNs is responsible for suppressing the GSC increase.

To further ascertain whether the acetylcholine released from SPSNs is received by dsx neurons to mediate mating-induced GSC increase, we focused on the fast-ionotropic nicotinic acetylcholine receptors (nAChR), which belong to the Cys-loop receptor subfamily of ligand-gated ion channels (Breer and Sattelle, 1987; Gundelfinger and Hess, 1992; Lee and O’Dowd, 1999). In D. melanogaster, 10 genes coding nAChR subunits have been identified. Among these, we focused on nAChRα1, nAChRα2, nAChRα3, nAChRβ1 and nAChRβ2 because the knock-down of these genes in dsx (tub >GAL80>, dsx-FLP; Tdc2-GAL4) or Tdc2 neurons (Tdc2-GAL4) increased the GSC number in virgin females similar to ppk >ChAT (Figure 6D and Figure 6—figure supplement 1A). We then confirmed the expression of these acetylcholine receptor genes in Tdc2 neurons by generating a knock-in T2A-GAL4 line as previously described (Kondo et al., 2020; Ihara et al., 2020) for each 5 nAChR subunits and observed their expression with UAS-mCD8::GFP. All of the five knock-in-GAL4 expressions were detected in anti-Tdc2 positive neurons around the ovary, suggesting that the ovary-projecting dsx neurons expresses these nAChRs (Figure 6—figure supplement 1B–F).

Figure 6—figure supplement 1.

nAChRs are expressed in the ovary-projecting Tdc2 neurons.

(A) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (v) female flies. (B–F) Representative images of the ovaries stained by anti-GFP (B–F) and anti-Tdc2 (B’–F’). Both signals merged on the surface of the ovary (B’’–F’’). Scale bar, 50 µm. Wilcoxon rank sum test was used. ***p≤0.001; NS, non-significant (p>0.05).

To confirm the role of nAChR in mating-induced GSC increase, we generated nAChRα1 complete loss-of-function genetic alleles by CRISPR/Cas9 technology (Kondo and Ueda, 2013; Figure 6—figure supplement 2A). Similar to nAChRα1 RNAi females, the nAChRα1 transheterozygous mutant virgin females (nAChRα1) had more GSCs compared with the controls (Figure 6—figure supplement 2B). In addition, the GSC increase of nAChRα1 was restored by the overexpression of nAChRa1 in Tdc2 neurons (Tdc2 >nAChRα1; nAChRα1) (Figure 6—figure supplement 2C). These data support our hypothesis that acetylcholine signaling in Tdc2 neurons has a negative role in mating-induced GSC increase.

Figure 6—figure supplement 2.

nAChRα1 in the Tdc2 neurons regulates GSC increase.

(A) Schematic representation of gRNA target sites (cleavage sites: gray arrowhead) and premature stop codon (red arrowhead) in coding sequences of nAChRα1 genes. Regions of the putative transmembrane domains of nAChRα1 are highlighted in blue. The target locus in Cas9-induced mutant was PCR-amplified and sequenced. The WT sequence (nAChRα1) is shown as reference. The Cas9-gRNA target sequence is underlined with the PAM indicated in red. Inserted nucleotides are indicated in light blue lowercase letters. The indel size is shown next to the sequence. The indel mutation results in a premature stop codon at the 228th and 326th amino acid sequence. (B–C) Frequencies of germaria containing 1, 2, and 3 GSCs (left vertical axis) and the average number of GSCs per germarium (right vertical axis) in virgin (V) and mated (M) female flies. (B) GSC numbers in nAChRα1 genetic mutants. (C) nAChRα1 overexpression in the Tdc2 neurons was sufficient to restore increased GSC in nAChRα1 mutants. Wilcoxon rank sum test with Holm’s correction was used. ***p≤0.001 and **p≤0.01; NS, non-significant (p>0.05). All source data are available in Source data 1.

We next assessed relationship between SPSNs, Tdc2 neurons, and OA-Oamb-Ca2+ signaling in ovarian cells. The silencing of SPSNs neuronal activity (SPSNs-LexA and LexAop-shi) increased the GSC number in virgin females (Figure 6E), consistent with our previous study (Ameku and Niwa, 2016). Upon SPSNs silencing, Tdc2 RNAi by Tdc2-GAL4 reduced the GSC number (Figure 6E), suggesting that Tdc2 neurons act downstream of SPSNs. In addition, the GSC increase through the silencing of SPSNs (SPSNs-LexA >LexAop-kir2.1) was suppressed by Oamb or Insp3R RNAi in the escort cells (Figure 6F), suggesting that OA-Oamb-Ca2+ signaling in ovarian cells acts downstream of SPSNs. Overall, our findings revealed a novel neuronal relay in response to mating that regulates the female GSC increase in the ovary before/after mating (Figure 7).

Figure 7.

Neuronal octopamine signaling, followed by Oamb-Ca2+-Mmp2 signaling, regulates the mating-induced GSC increase.

The illustration is the proposed working model from our findings here. SP signaling and SP sensory neurons activate dsx neurons via acetylcholine signaling. The octopamine released from dsx neurons is received by the Oamb in escort cells and then activates intracellular Ca2+ flux. The OA-mediated signaling increases pMad levels in GSCs to evoke mating-induced GSC increase via Mmp2.

Discussion

In this study, we report that the mating-induced GSC increase in female D. melanogaster is regulated by OAergic neurons directly projecting to the ovary. From our in vivo and ex vivo experiments, we propose the following model to explain the mating-induced GSC increase. After mating, the male seminal fluid SP is transferred into the female uterus, stimulating SPR-positive neurons. As the liganded SPR silences the neuronal activity of SPSNs (Häsemeyer et al., 2009), the acetylcholine released from SPSNs is suppressed. As SPSNs and dsx neurons are directly connected, this suppression directly modulates dsx neuronal activity. Because we have shown that nAChRs in dsx neurons exhibit an inhibitory effect with an unknown mechanism (to be discussed later), the inactivation of nAChRs in the absence of acetylcholine results in the activation of dsx neurons in mated females. As a consequence, OA is released from dsx neurons, received by Oamb, induces [Ca2+]i in the escort cells, and finally activates the Mmp2 enzymatic activity. The activity of Mmp2 positively regulates the Dpp-mediated niche signaling, thereby leading to mating-induced GSC increase (Figure 7).

The octopamine- and its receptor-dependent GSC control

Our proposed model is that the OA from dsx neurons is directly received by the escort and follicle cells in the germarium. Our model is supported by two of our observations. First, mating-induced GSC increase is impaired by Oamb RNAi using a GAL4 driver that is active specifically in the germarium cells but not mature follicle cells. Second, OA treatment evokes [Ca2+]i elevation in these germarium cells in an Oamb-dependent manner. However, in this study, we did not address whether the escort cells and/or the follicle cells in the germarium express Oamb, as we failed to observe any clear GAL4 expression in two independent Oamb-T2A-GAL4 drivers (Figure 1—figure supplement 2A,B,C and D). We surmise that this may be due to lower amounts of Oamb transcript in the germarium.

We have shown that the activation of the ovary-projecting dsx neurons is necessary and sufficient to induce GSC increase. However, from an anatomical point of view, the dsx neurons project to the distal half of the ovary but not to the germarium (Figure 5—figure supplement 1E). Considering our model described above, this disagreement can be attributed to the characteristic volume transmission of monoamine neurotransmitters. In other words, neurotransmitters act at a distance well beyond their release sites from cells or synapses (Fuxe et al., 2010). Therefore, the OA secreted from the terminals of dsx neurons could reach the germarium located at the most proximal part of the ovary.

Several previous studies have revealed that OA signaling has a pivotal role in reproductive tissues other than germarium, such as mature follicle cells, oviduct, and ovarian muscle, to promote ovulation, oviduct remodeling, and ovarian-muscle contraction, respectively (Deady and Sun, 2015; Heifetz et al., 2014; Lee et al., 2009; Middleton et al., 2006; Rezával et al., 2014). Therefore, it is likely that the dsx neurons orchestrate multiple different events during oogenesis in response to mating stimulus. Because a mated female needs to activate oogenesis to continuously produce eggs in concert with sperm availability, it is reasonable that the ovary-projecting neurons switch on the activity of the entire process of reproduction.

The role of Mmp2 in mating-induced GSC increase

Based on our present study and several previous studies (Deady et al., 2015; Deady and Sun, 2015; Knapp and Sun, 2017), the OA-Oamb-Ca2+-Mmp2 axis is required for GSC increase and follicle rupture, both of which are induced by mating stimuli in D. melanogaster. In both cases, Mmp2 enzymatic activity is likely to be essential, as the overexpression of Timp encoding a protein inhibitor of Mmp2 suppresses GSC increase, as well as follicle rupture. Mmp2 in mature follicle cells cleaves and downregulates Viking/collagen VI (Deady et al., 2017; Wang et al., 2008). In fact, several previous studies have revealed that Viking/collagen VI is required for GSC maintenance in female D. melanogaster (Van De Bor et al., 2015; Wang et al., 2008). However, we observed no significant change in Viking/Collagen VI levels in the germarium between the control and Mmp2 RNAi flies (Figure 4—figure supplement 1E). Therefore, we concluded that Viking/collagen VI is not a substrate of Mmp2 in the regulation of mating-induced GSC increase. Besides Viking/Collagen VI, Dally-like (Dlp) is another basement membrane protein associated with extracellular matrix and known as the Mmp2 substrate (Wang and Page-McCaw, 2014). Interestingly, dlp is expressed in the escort cells (Wang and Page-McCaw, 2014). Moreover, Dlp controls the distribution of Dpp and Wnts, both of which significantly affect GSC self-renewal and differentiation (Wang et al., 2015; Xie and Spradling, 1998). Future research should decipher the exact substrate by which Mmp2 controls Dpp and/or Wnts to modulate GSC behavior in response to mating stimulus.

Another remaining question to be addressed is how Mmp2 function is regulated in GSC increase. Ecdysteroid biosynthesis and signaling in the ovary are necessary but not sufficient for the OA-Oamb-Ca2+–mediated GSC increase and follicle rupture (Ameku and Niwa, 2016; Knapp and Sun, 2017). We found that in the regulation of mating-induced GSC increase, ecdysteroid signaling acts downstream of Ca2+ signaling (Figure 4F). On the other hand, in the follicle rupture process, ecdysteroid signaling either acts downstream, upstream, or both, of Ca2+ signaling. Further, the precise action of ecdysteroid has yet to be elucidated (Knapp and Sun, 2017). The Mmp2-GFP fusion protein level in the follicle cells is not changed in the loss-of-Ecdysone receptor-function flies, implying that ecdysteroid signaling might regulate Mmp2 enzymatic activity by an unknown mechanism (Knapp and Sun, 2017). Considering the involvement of both the OA-Oamb-Ca2+-Mmp2 axis and ecdysteroid biosynthesis, it is very likely that the Mmp2 enzymatic activity is also regulated by the same, unknown mechanism not only in the mature follicle cells to control follicle rupture, but also in the germarium to control mating-induced GSC increase.

SPSN-mediated suppression of dsx neurons

In many animals, reproduction involves significant behavioral and physiological shifts in response to mating. In female D. melanogaster, several post-mating responses are coordinated by SPSNs and their downstream afferent neuronal circuit (Wang et al., 2020), including Stato-Acoustic Ganglion neurons, the ventral abdominal lateral Myoinhibitory peptide neurons, and the efferent dsx neurons (Feng et al., 2014; Häsemeyer et al., 2009; Jang et al., 2017; Rezával et al., 2014). Our GRASP analysis indicates a direct synaptic connection between cholinergic SPSNs and OAergic neurons. Moreover, we demonstrated that nAChRs in dsx neurons are responsible for the suppression of their neuronal activity in virgin females. However, nAChRs are the cation channels leading to depolarization upon acetylcholine binding, and therefore usually activate neurons (Corringer et al., 2000; Lee and O’Dowd, 1999; Perry et al., 2012). How is the opposite role of nAChRs in dsx neuronal activity achieved? One possibility is that acetylcholine-nAChR signaling does not evoke a simple depolarization but rather generates a virgin-specific temporal spike pattern in dsx neurons. Interestingly, recent studies demonstrated that the pattern, instead of the frequency, of neuronal firing is significant in adjusting the neuronal activity of clock neurons in D. melanogaster (Tabuchi et al., 2018). The firing pattern relies on control of ionic flux by the modulation of Ca2+-activated potassium channel and Na+/K+ ATPase activity. Because whether mating changes the firing pattern of dsx neurons remains to be examined, the neuronal activity in SPSNs and the dsx neuronal circuit between virgin and mated females are future research areas.

Interorgan communication among multiple organs to regulate the increase and maintenance of female GSCs

In the last decades, there is growing evidence that GSCs and their niche are influenced by multiple humoral factors (Drummond-Barbosa, 2019; Yoshinari et al., 2019). Based on the data from our current study and previous studies, there are at least four crucial humoral factors for regulating the increase and/or maintenance of D. melanogaster female GSCs, including DILPs (Hsu et al., 2008; Hsu and Drummond-Barbosa, 2009; LaFever, 2005), ecdysteroids (Ables and Drummond-Barbosa, 2010; Ameku et al., 2017; Ameku and Niwa, 2016; König et al., 2011), Neuropeptide F (NPF) (Ameku et al., 2018), and OA (this study). Notably, all of these come from different sources: DILPs are from the insulin-producing cells located in the pars intercerebralis of the central brain; ecdysteroids from the ovary; NPF from the midgut; and OA from the neurons located in the abdominal ganglion. In addition to these identified humoral factors, recent studies also imply that adiponectin and unknown adipocyte-derived factor(s) are essential for GSC maintenance (Armstrong and Drummond-Barbosa, 2018; Laws et al., 2015; Matsuoka et al., 2017). These data clearly indicate that D. melanogaster female GSCs are systemically regulated by interorgan communication involving multiple organs. The additional interorgan communication mechanisms that ensure the faithful coupling of the increase and maintenance of GSC to the organism’s external and physiological environments are essential to be investigated in future studies.

To modulate the increase and maintenance of GSC, ecdysteroids are received by both GSCs and niche cells (Ables and Drummond-Barbosa, 2010; König et al., 2011), whereas DILPs, NPF, and OA are received by niche cells. A major signal transduction mechanism of each of these humoral factors have been well characterized, namely phosphoinositide 3-kinase pathway for DILPs-InR signaling, EcR/Ultraspiracle-mediated pathway for ecdysteroid signaling, cAMP pathway for NPF-NPFR signaling (Garczynski et al., 2002), and Ca2+ pathway for OA-Oamb signaling. However, it remains unclear whether and how each of these signaling pathways control the production and secretion of the niche signal, as well as its distribution and transduction. In addition, it is important to understand whether and how the multiple system signals are integrated to control the mating-induced increase and maintenance of GSCs.

Evolutionarily conservation of monoamine-steroid hormone axis to control female reproduction

In recent years, many studies have revealed that not only local niche signals but also systemic and neuronal factors play indispensable roles in regulating GSC behavior (Ables and Drummond-Barbosa, 2017; Drummond-Barbosa, 2019; Yoshinari et al., 2019). In D. melanogaster, ecdysteroid signaling is essential for the proliferation and maintenance of GSCs and neural stem cells (Ables and Drummond-Barbosa, 2010; Homem et al., 2014; König et al., 2011). In this study, we have identified the ovary-projecting OAergic neurons as new regulators of stem cell homeostasis. Both steroid hormones and OA-like monoamines, such as noradrenaline, are also involved in stem cell regulation in mammals. For example, the mammalian steroid hormone, estrogen, is important in regulating cell division and/or maintenance of hematopoietic stem cells, mammary stem cell, neural stem cells, and hematopoietic stem cells (Asselin-Labat et al., 2010; Bramble et al., 2019; Kim et al., 2016; Nakada et al., 2014). Moreover, noradrenergic neurons, which directly project to the bone marrow, regulate the remodeling of hematopoietic stem cells niche (Ho et al., 2019; Méndez-Ferrer et al., 2010; Méndez-Ferrer et al., 2008). Therefore, the steroid hormone- and noradrenergic nerve-dependent control of stem cell homeostasis are likely conserved across animal species. In this regard, the D. melanogaster reproductive system will further serve as a powerful model to unravel the conserved systemic and neuronal regulatory mechanisms for stem cell homeostasis in animals.

Materials and methods

Drosophila strains

Flies were raised on cornmeal-yeast-agar medium at 25°C. EcR, temperature-sensitive mutants, were cultured at 31°C for 1 d prior to the assays. w was used as the control strain.

The genetic mutant stocks used were EcR (Bloomington Drosophila Stock Center [BDSC] #5799) and EcR (BDSC #4894). The protein-trap GFP line of Vkg (Vkg::GFP) was obtained from Kyoto Stock Center (DGRC #110692). Dad-LacZ (Tsuneizumi et al., 1997) (a gift from Yoshiki Hayashi, University of Tsukuba, Japan).

The following GAL4 and LexA strains were used: c587-GAL4 (Manseau et al., 1997) (gift from Hiroko Sano, Kurume University, Japan), R44E10-GAL4 (Deady and Sun, 2015) (a gift from Jianjun Sun, University of Connecticut, USA), RS-GAL4 (Lee et al., 2009) (a gift from Kyung-An Han, Pennsylvania State University, USA), nSyb-GAL4 (BDSC #51941), nSyb-GAL80 (Harris et al., 2015) (a gift from James W. Truman, Janelia Research Campus, USA), tj-GAL4 (DGRC #104055), R13C06-GAL4 (BDSC #47860), 109–30 GAL4 (BDSC #7023), c355-GAL4 (BDSC #3750), c306-GAL4 (BDSC #3743), slbo-GAL4 (BDSC #6458), bab1-GAL4 (Bolívar et al., 2006) (a gift from Satoru Kobayashi, University of Tsukuba, Japan), nos-GAL4 (DGRC #107748), tub >FRT >GAL80>FRT (BDSC #38879), Oamb (BDSC#84677) (Deng et al., 2019), Oamb-KI-T2A-GAL4, nAChRα1-T2A-GAL4, nAChRα2-T2A-GAL4, nAChRα3-T2A-GAL4, nAChRβ1-T2A-GAL4, nAChRβ2-T2A-GAL4 (Kondo et al., 2020; Ihara et al., 2020), ChaT-GAL4 (BDSC #6793), ppk-GAL4 (Grueber et al., 2007) (a gift from Hiroko Sano, Kurume University, Japan), and SPSNs-LexA (Feng et al., 2014) (a gift from Young-Joon Kim, Gwangju Institute of Science and Technology, South Korea).

The following UAS and LexAop strains were used: 20xUAS-6xGFP (BDSC #52261), UAS-GFP;UAS-mCD8::GFP (Ito et al., 1997; Lee and Luo, 1999) (a gift from Kei Ito, University of Cologne, Germany), UAS-Stingar (BDSC #84277), UAS-mCD8::RFP (BDSC #32219), UAS-CsChrimson (BDSC #55134), UAS-Insp3R (BDSC #30742), UAS-Oamb (Lee et al., 2009) (a gift from Kyung-An Han, Pennsylvania State University, USA), UAS-Timp (BDSC #58708) (a gift from Andrea Page-McCaw, Vanderbilt University, USA), UAS > stop >dTrpA1, UAS > stop >TNT, UAS > stop >TNT (von Philipsborn et al., 2011; Yu et al., 2010), dsx-FLP (Rezával et al., 2014) (a gift from Daisuke Yamamoto, Advanced ICT Research Institute, National Institute of Information and Communications Technology, Japan) TRiC; UAS-mCD8::RFP, LexAop2-mCD8::GFP;nSyb-MKII::nlsLexADBDo;UAS-p65AD::CaM (BDSC:61679), ppk-eGFP (Grueber et al., 2003) (a gift from Tadashi Uemura, Kyoto University, Japan), and LexAop-Kir2.1 (Feng et al., 2014) (a gift from Young-Joon Kim, Gwangju Institute of Science and Technology, South Korea).

The RNAi transgenic lines used were as follows: UAS-LacZ (a gift from Masayuki Miura, The University of Tokyo, Japan), UAS-Oamb(BDSC #31171), UAS-Oamb(BDSC #31233), UAS-Oamb (Vienna Drosophila Resource Center [VDRC] #106511), UAS-Octβ1R(VDRC #110537), UAS-Octβ2R(VDRC #104524), UAS-Octβ3R (VDRC #101189), UAS-Insp3R (BDSC #25937), UAS-EcR (VDRC #37059), UAS-Mmp2 (BDSC #31371), UAS-Mmp2 (VDRC #330303), UAS-Timp (BDSC #61294), UAS-Timp (VDRC #109427), UAS-Tdc2 (VDRC #330541), UAS-Tdc2 (BDSC #25871), UAS-Tβh (VDRC #107070), UAS-Tβh (BDSC #67968), UAS-ChAT (VDRC #330291), UAS-ChAT (BDSC #25856), UAS-nAChRα1 (VDRC #48159), UAS-nAChRα2 (VDRC #101760), UAS-nAChRα3 (VDRC #101806), UAS-nAChRβ1 (VDRC #106570), UAS-nAChRβ2 (VDRC #109450), UAS-nvd, and UAS-nvd (Yoshiyama et al., 2006).

Generation of Oamb and nAChRα1 genetic loss-of-function mutant strains

The mutant alleles Oamb (Figure 1—figure supplement 1F), nAChRα1, and nAChRα1 (Figure 6—figure supplement 2A) were created in a white (w) background using CRISPR/Cas9 as previously described (Kondo and Ueda, 2013). The following guide RNA (gRNA) sequences were used: Oamb, 5ʹ-GATGAACTCGAGTACGGCCA-3ʹ, and 5ʹ-GCGATCTCTGGTGCCGCATT-3ʹ; nAChRα1, 5ʹ-GGACATCATGCGTGTGCCGG-3ʹ; nAChRα1, 5ʹ-GGGCAGGTAGAAGACCAGAA-3ʹ. The breakpoint detail of Oamb is described in Figure 1—figure supplement 1F, whereas those of nAChRα1 and nAChRα1 are described in Figure 6—figure supplement 2A.

Generation of UAS-nAChRα1 transgenic line

The pcDNA3.1 plasmid containing the wild-type D. melanogaster nAChRα1 coding sequences (nAChRα1-pcDNA3.1) was synthesized previously described (Ihara et al., 2018). Briefly, nAChRα1-pcDNA3.1 was digested with EcoRI and NotI, and then the digested nAChRα1 fragment was ligated with a EcoRI-NotI–digested pWALIUM10-moe plasmid (Perkins et al., 2015). Transformants were generated using the phiC31 integrase system in the P{CaryP}attP40 strain (Groth et al., 2004). The w transformants of pWALIUM10-moe were established using standard protocols.

Behavioral assays

Flies were reared at 25°C and aged for 5–6 d. Virgin female flies were mated overnight to w male flies at 25°C (10 males and 5–8 females per vial). For the thermal activation assays, flies were first reared at 17°C for 6 d and transferred to 29°C. In the case of EcR mutant assays, flies were transferred to 31°C for 24 hr before mating or ex vivo culture.

For OA feeding, newly eclosed virgin females were aged for 4 d in vials with standard food containing 7.5 mg/mL of OA (Monastirioti et al., 1996; Rubinstein and Wolfner, 2013).

Immunohistochemistry

Tissues were dissected in phosphor buffer serine (PBS) and fixed in 4% paraformaldehyde in PBS for 30 to 60 min at room temperature (RT). The fixed samples were washed three times in PBS supplemented with 0.2% Triton X-100, blocked in blocking solution (PBS with 0.3% Triton X-100% and 0.2% bovine serum albumin [BSA]) for 1 hr at RT, and incubated with a primary antibody in the blocking solution at 4°C overnight. The primary antibodies used were chicken anti-GFP (Abcam #ab13970; 1:4,000), rabbit anti-RFP (Medical and Biological Laboratories PM005; 1:2,000), mouse anti-Hts 1B1 (Developmental Studies Hybridoma Bank [DSHB]; 1:50), rat anti-DE-cadherin DCAD2 (DSHB; 1:50), rabbit anti-pH3 (Merck Millipore #06–570; 1:1000), rabbit monoclonal anti-pMad (Abcam #ab52903; 1:1000), mouse anti-Lamin C LC28.26 (DSHB; 1:10), rabbit cleaved Dcp-1 (Cell Signaling Technology #9578; 1:100), rat anti-Vasa (DSHB; 1:50), mouse anti-LacZ (β-galactosidase) (DSHB#40-1a; 1:50), rabbit anti-Tdc2 (Abcam #ab128225; 1:2000), Alexa Fluor 546 phalloidin (Thermo Fisher Scientific #A22283; 1:200), and Alexa Fluor 633 phalloidin (Thermo Fisher Scientific #A22284; 1:200). After washing, fluorophore (Alexa Fluor 488, 546 or 633)-conjugated secondary antibodies (Thermo Fisher Scientific) were used at a 1:200 dilution, and the samples were incubated for 2 hr at RT in the blocking solution. After another washing step, all samples were mounted in FluorSave reagent (Merck Millipore #345789). GSC numbers were determined based on the morphology and position of their anteriorly anchored spherical spectrosome (Ables and Drummond-Barbosa, 2010; Ameku et al., 2018; Ameku and Niwa, 2016). Cap cells were identified by immunostaining with anti-Lamin C antibody as previously described (Ables and Drummond-Barbosa, 2010).

Ex vivo ovary culture

We used 5–6-day-old females. The ovaries were dissected in Schneider’s Drosophila medium (Thermo Fisher Scientific #21720024) and isolated from oviduct using forceps. Approximately 5–6 ovaries were immediately transferred to a dish containing 3 mL of Schneider’s Drosophila medium supplemented with 15% fetal calf serum and 0.6% penicillin-streptomycin with/without the addition of OA (Sigma, final concentration of OA is 0–1000 μM) and 20E (Enzo Life Sciences; final concentration of 20 nM). The cultures were incubated at RT (except for EcR mutant flies, Figure 3D) for 16 hr, and the samples were immunostained to determine the GSC number.

Ex vivo calcium imaging

We employed the previously described imaging methods to visualize GSC behavior (Morris and Spradling, 2011; Reilein et al., 2018). For the live imaging, the ovaries dissected from adult virgin female flies were placed on a glass bottom dish (IWAKI #4970–041) with 3 mL of Schneider’s Drosophila medium and 100 µL of the test reagent (Schneider’s Drosophila medium containing 300 mM OA) placed directly at the center of each dish. The images were obtained with a × 40 objective lens (water-immersion) using a Zeiss LSM 700 confocal microscope and were recorded every 4 s. The GCaMP6s fluorescence intensity in the escort cell was then calculated for each time point. The ratio of fluorescence (ΔF) at each time point was calculated by normalizing the fluorescence with the initial fluorescence (F0). The initial fluorescence (F0) is the average GCaMP6s fluorescence intensity before adding the test reagent.

Optogenetic activation of the escort cells

Red-shifted channelrhodopsin CsChrimson (Klapoetke et al., 2014) was used to increase the [Ca2+]i in the escort cells by light. UAS-CsChrimson was expressed using c587-GAL4 with nSyb-GAL80. All crosses and the early development of flies were performed under dark conditions. The experiment was done at 25°C. Adult flies were raised with standard food for 3 d after eclosion and then with standard food with 1 mM all-trans-retinal (ATR) for 3 d. Subsequently, they were kept in the presence of orange–red light from LED for 24 hr. LED light was shone from the outside of the plastic chamber covered by aluminum foil to enhance light intensity.

Statistical analysis

All experiments were performed independently at least twice. Fluorescence intensity in confocal sections was measured via ImageJ. For pMad quantification, signal intensity was calculated by measuring the fluorescence intensity in GSCs and CBs, which were co-stained with anti-Vasa antibody to visualize their cell boundaries. Sample sizes were chosen based on the number of independent experiments required for statistical significance and technical feasibility. The experiments were not randomized, and the investigators were not blinded. All statistical analyses were carried out using the ‘R’ software environment. The P value is provided in comparison with the control and indicated as * for p≤0.05, ** for p≤0.01, *** for p≤0.001, and ‘NS’ for non-significant (p>0.05).

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

In this manuscript, Niwa and colleagues investigated the mechanism of octopaminergic system in regulating mating-induced GSC increase, a phenomenon previously identified by the same group. A fundamental question remaining in the field is how SPR-positive sensory neurons (SPSNs) relays the mating signal to GSC to increase their numbers after mating. These authors found that Oamb, one of the octopamine (OA) receptors, is expressed in escort cells in the germarium. Using sophisticated genetic tools, they illustrated that OA/Oamb signaling in escort cells is required and sufficient for mating induced GSC increase. OA/Oamb-induced GSC increase depends on Ca2+ increase, ecdysone signaling, and Mmp2, similar to the OA-induced follicle rupture in mature follicles. They went on to identify a subset of OA neurons (dsx+Tdc2+) in the abdominal ganglion responsible for OA signaling in escort cells and GSC increase. Furthermore, they illustrated that SPSNs directly innervate dsx+Tdc2+ neurons and that acetylcholine released from SPSNs inhibits OA release from dsx+Tdc2+ neurons, which could be overridden by mating. Therefore, they established a clear relay mechanism from mating to SPSNs, dsx+Tdc2+ neurons, Oamb signaling in escort cells, and GSC increase.

Overall, all experiments were well designed and included all the controls. The data were interpreted accurately and support their conclusions in general. The work is a nice addition to our understanding of extrinsic cues to stem cell regulation.

Decision letter after peer review:

Thank you for submitting your article "Neuronal octopamine signaling regulates mating-induced germline stem cell proliferation in female Drosophila" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Yukiko M Yamashita as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Utpal Banerjee as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Jianjun Sun (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

In this manuscript, Niwa and colleagues investigated the mechanism of octopaminergic system in regulating mating-induced GSC increase, a phenomenon previously identified by the same group. A fundamental question remaining in the field is how SPR-positive sensory neurons (SPSNs) relays the mating signal to GSC to increase their numbers after mating. These authors found that Oamb, one of the octopamine (OA) receptors, is expressed in escort cells in the germarium. Using sophisticated genetic tools, they illustrated that OA/Oamb signaling in escort cells is required and sufficient for mating induced GSC increase. OA/Oamb-induced GSC increase depends on Ca2+ increase, ecdysone signaling, and Mmp2, similar to the OA-induced follicle rupture in mature follicles. They went on to identify a subset of OA neurons (dsx+Tdc2+) in the abdominal ganglion responsible for OA signaling in escort cells and GSC increase. Furthermore, they illustrated that SPSNs directly innervate dsx+Tdc2+ neurons and that acetylcholine released from SPSNs inhibits OA release from dsx+Tdc2+ neurons, which could be overridden by mating. Therefore, they established a clear relay mechanism from mating to SPSNs, dsx+Tdc2+ neurons, Oamb signaling in escort cells, and GSC increase.

Overall, all experiments were well designed and included all the controls. The data were interpreted accurately and support their conclusions in general. The work is a nice addition to our understanding of extrinsic cues to stem cell regulation. The reviewers identified the following major concerns, which should be addressed in the revision. Given the pandemic, the reviewers feel it to be appropriate if the statements are weakened, acknowledging the caveats in interpretation.

Essential revisions (which can be done with textual changes, unless they do not already have the data):

1) The evidence that the relevant target cells of the octopaminergic neurons are escort cells is rather weak; The authors cannot rule out contributions from follicle cells based on the current data. The authors use drivers expressed in both cell types (C587-Gal4, Tj-gal4) for most knockdown/overexpression experiments. It will be important to exclude this possibility by using a Gal4 driver only expressed in early follicle cells. (R44E10-Gal4 used to exclude other cell types is expressed in follicle cells of stage 14 oocytes---which is too late to assess the involvement of follicle cells). Alternatively, the authors should weaken the statement to be precise on this point.

2) Inferred from Timp experiments, authors conclude that Mmp2 activity in the GSC niche cells is necessary for mating-induced GSC increase (subsection “Matrix metalloproteinase 2 acts downstream of OA-Oamb-Ca2+ signaling”, second paragraph). It will be important to examine the Mmp2 expression and activity in both virgin and mated female to see whether Mmp2 activity is indeed regulated by mating. The authors seem to alternate between two models. Throughout most of the manuscript, the mating signal received by the ovary seems to be the sex peptide-regulated octopamine release. However, in the section where the Mmp2/Timp1 data is presented, the implication is that Timp1 expression would go down after mating, resulting in increased Mmp2 activity. Do Timp1 or any other members of the OA-to-Mmp2 signalling axis change after mating? How would the authors reconcile/integrate the two models? Related to this, the epistasis between ecdysone signalling and Mmp2 activity is unclear.

Essential revisions (which can be done with textual changes, unless they do not already have the data):

1) The evidence that the relevant target cells of the octopaminergic neurons are escort cells is rather weak; The authors cannot rule out contributions from follicle cells based on the current data. The authors use drivers expressed in both cell types (C587-Gal4, Tj-gal4) for most knockdown/overexpression experiments. It will be important to exclude this possibility by using a Gal4 driver only expressed in early follicle cells. (R44E10-Gal4 used to exclude other cell types is expressed in follicle cells of stage 14 oocytes---which is too late to assess the involvement of follicle cells). Alternatively, the authors should weaken the statement to be precise on this point.

According to the reviewers’ suggestion, we did additional RNAi experiments using c355-GAL4, c306-GAL4, slbo-GAL4, R13C06-GAL4, and 109-30-GAL4. Each of these strains expresses GAL4 in distinct cell types of ovarian somatic cells. We found that Oamb RNAi in the stage-14 follicle cells by R44E10-GAL4, or in the stage 9-10 follicle cells by c355-GAL4, c306-GAL4, and slbo-GAL4, had no significant effect on mating-induced GSC increase. These results suggest that Oamb in the mature follicle cells is not involved in mating-induced GSC increase. Therefore, it is quite unlikely that Oamb in the follicle cells after stage 9 has a large contribution to regulating mating-induced GSC increase.

On the other hand, we found that Oamb RNAi by R13C06-GAL4, and 109-30-GAL4, which are active in the escort cells, and the germarium follicle cells, respectively, resulted in the failure of mating-induced GSC increase. Along with the data obtained from Tj-GAL4 and c587-GAL4 drivers, we conclude that Oamb in the in the escort cells or the follicle cells of the germarium, plays an essential role in mating-induced GSC increase.

These additional data are described in the subsection “Mating-induced GSC increase requires the octopamine receptor Oamb in ovarian escort cells” and represented in Figure 1—figure supplement 1C, D and E of the revised manuscript.

2) Inferred from Timp experiments, authors conclude that Mmp2 activity in the GSC niche cells is necessary for mating-induced GSC increase (subsection “Matrix metalloproteinase 2 acts downstream of OA-Oamb-Ca2+ signaling”, second paragraph). It will be important to examine the Mmp2 expression and activity in both virgin and mated female to see whether Mmp2 activity is indeed regulated by mating. The authors seem to alternate between two models. Throughout most of the manuscript, the mating signal received by the ovary seems to be the sex peptide-regulated octopamine release. However, in the section where the Mmp2/Timp1 data is presented, the implication is that Timp1 expression would go down after mating, resulting in increased Mmp2 activity. Do Timp1 or any other members of the OA-to-Mmp2 signalling axis change after mating? How would the authors reconcile/integrate the two models? Related to this, the epistasis between ecdysone signalling and Mmp2 activity is unclear.

We appreciate the reviewer’s comments and wish to allay any reservations. First, we did not succeed in obtaining any solid data to support the idea that Timp expression is changed before and after mating. Nevertheless, we admittedly overstated the role of Timp in mating-induced GSC increase in our original manuscript. Therefore, we have deleted this sentence from the revised manuscript. We apologize for any confusion or overstatements.

Second, regarding Mmp2 activity, we tried to examine whether Mmp2 protein levels were changed before and after mating with Mmp2-EGFP strain (Deady et al., 2015) and also with anti-Mmp2 antibody (Dear et al. Development 2016). However, unfortunately, we could not detect any obvious signals in the germarium region of either virgin or mated females in our hands. It must be noted, however, that previous studies detected Mmp2 expression in the germarium (Pearson et al., 2016 and Wang and Page-McCaw, 2014). Currently we are unsure as to the reason why we could not detect these signals in our experimental conditions, but the COVID-19 situation prevents us from promptly solving this problem during this revision process. Therefore, we did not include our negative data in the revised manuscript, but just cited the previous studies (Pearson et al., 2016 and Wang and Page-McCaw, 2014) in the subsection “Matrix metalloproteinase 2 acts downstream of OA-Oamb-Ca2+ signaling”.

Regarding another question about the epistasis between ecdysone signaling and Mmp2 activity, we did not have a clear answer but faithfully discussed this point in the subsection “The role of Mmp2 in mating-induced GSC increase”.

We would like to emphasize again that Timp overexpression significantly suppressed mating-induced GSC increase in our model system (Figure 4B), strongly suggesting that Mmp2 is involved in this process.

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (D. melanogaster)	c587-GAL4	Manseau et al., 1997	FBal0150629 RRID:BDSC_67747	A gift from Hiroko Sano, Kurume University, Japan
Genetic reagent (D. melanogaster)	OambΔ	This paper		Detail described in Figure 1—figure supplement 1F
Genetic reagent (D. melanogaster)	nAChRa1228	This paper		Detail described in Figure 6—figure supplement 2A
Genetic reagent (D. melanogaster)	nAChRa1326	This paper		Detail described in Figure 6—figure supplement 2A
Genetic reagent (D. melanogaster)	EcRA483T	Bloomington Drosophila Stock Center	BDSC: #5799 RRID:BDSC_5799 FBal0083501
Genetic reagent (D. melanogaster)	EcRM554fs	Bloomington Drosophila Stock Center	BDSC: #4894 RRID:BDSC_4894 FBal0083490
Genetic reagent (D. melanogaster)	Vkg::GFP	KYOTO stock center	DGRC #110692 RRID:DGGR_110692 FBal0286156
Genetic reagent (D. melanogaster)	Dad-LacZ	Tsuneizumi et al., 1997	FBal0065787 RRID:DGGR_118114	A gift from Yoshiki Hayashi, University of Tsukuba, Japan
Genetic reagent (D. melanogaster)	R44E10-GAL4	Deady and Sun, 2015	FBal0252601 PMID:26473732	A gift from Jianjun Sun, University of Connecticut, USA
Genetic reagent (D. melanogaster)	RS-GAL4	Lee et al., 2009	FBal0263794 PMID:19262750	A gift from Kyung-An Han, Pennsylvania State University, USA
Genetic reagent (D. melanogaster)	nSyb-GAL4	Bloomington Drosophila Stock Center	BDSC: #51941 RRID:BDSC_51941 FBti0154973	FACS (5 ul per test)
Genetic reagent (D. melanogaster)	nSyb-GAL80	Harris et al., 2015	PMID:26193122	A gift from James W. Truman, Janelia Research Campus, USA
Genetic reagent (D. melanogaster)	tj-GAL4	KYOTO stock center	DGRC: #104055 RRID:DGGR_104055 FBti0034540
Genetic reagent (D. melanogaster)	R13C06-GAL4	Bloomington Drosophila Stock Center	BDSC: #47860 RRID:BDSC_47860 FBal0249828
Genetic reagent (D. melanogaster)	109–30 GAL4	Bloomington Drosophila Stock Center	BDSC: #7023 RRID:BDSC_7023 FBti0027548
Genetic reagent (D. melanogaster)	c355-GAL4	Bloomington Drosophila Stock Center	BDSC: #3750 RRID:BDSC_3750 FBti0002591
Genetic reagent (D. melanogaster)	c306-GAL4	Bloomington Drosophila Stock Center	BDSC: #3743 RRID:BDSC_3743 FBal0048787
Genetic reagent (D. melanogaster)	slbo-GAL4	Bloomington Drosophila Stock Center	BDSC: #6458 RRID:BDSC_6458 FBst0006458
Genetic reagent (D. melanogaster)	bab1-GAL4	Bolívar et al., 2006	FBal0242654 PMID:17013875	A gift from Satoru Kobayashi, University of Tsukuba, Japan
Genetic reagent (D. melanogaster)	nos-GAL4	KYOTO stock center	DGRC: #107748 RRID:DGGR_107748 FBst0306396
Genetic reagent (D. melanogaster)	tub > FRT > GAL80>FRT	Bloomington Drosophila Stock Center	BDSC: #38879 RRID:BDSC_38879 FBti0147580
Genetic reagent (D. melanogaster)	OambKI-RD-GAL4	Deng et al., 2019 Bloomington Drosophila Stock Center	BDSC: #84677 RRID:BDSC_84677 FBti0209942
Genetic reagent (D. melanogaster)	Oamb-KI-T2A-GAL4	Kondo et al., 2020	PMID:31914394
Genetic reagent (D. melanogaster)	nAChRα1-T2A-GAL4	Kondo et al., 2020	PMID:31914394
Genetic reagent (D. melanogaster)	nAChRα2-T2A-GAL4	Kondo et al., 2020	PMID:31914394
Genetic reagent (D. melanogaster)	nAChRα3-T2A-GAL4	Kondo et al., 2020	PMID:31914394
Genetic reagent (D. melanogaster)	nAChRβ1-T2A-GAL4	Kondo et al., 2020	PMID:31914394
Genetic reagent (D. melanogaster)	nAChRβ2-T2A-GAL4	Kondo et al., 2020	PMID:31914394
Genetic reagent (D. melanogaster)	ChaT-GAL4	Bloomington Drosophila Stock Center	BDSC: #6793 RRID:BDSC_6793 FBst0006793
Genetic reagent (D. melanogaster)	ppk-GAL4	Grueber et al., 2007	FBtp0039691 PMID:17164414	A gift from Hiroko Sano, Kurume University, Japan
Genetic reagent (D. melanogaster)	SPSNs-LexA	Feng et al., 2014	FBtp0110869 PMID:24991958	A gift from Young-Joon Kim, Gwangju Institute of Science and Technology, South Korea
Genetic reagent (D. melanogaster)	20xUAS-6xGFP	Bloomington Drosophila Stock Center	BDSC: #52261 RRID:BDSC_52261 FBst0052261
Genetic reagent (D. melanogaster)	UAS-GFP;UAS-mCD8::GFP	Ito et al., 1997; Lee and Luo, 1999	FBtp0002652 PMID:9043058 PMID:10457015	A gift from Kei Ito, University of Cologne, Germany
Genetic reagent (D. melanogaster)	UAS-Stinger	Bloomington Drosophila Stock Center	BDSC: #84277 RRID:BDSC_84277
Genetic reagent (D. melanogaster)	UAS-mCD8::RFP	Bloomington Drosophila Stock Center	BDSC: #32219 RRID:BDSC_32219 FBti0131967
Genetic reagent (D. melanogaster)	UAS-CsChrimson	Bloomington Drosophila Stock Center	BDSC: #55134 RRID:BDSC_55134 FBti0160571
Genetic reagent (D. melanogaster)	UAS-Insp3R	Bloomington Drosophila Stock Center	BDSC: #30742 RRID:BDSC_30742 FBti0129829
Genetic reagent (D. melanogaster)	UAS-OambK3	Lee et al., 2009	FBtp0069415 PMID:19262750	A gift from Kyung-An Han, Pennsylvania State University, USA
Genetic reagent (D. melanogaster)	UAS-Timp	Bloomington Drosophila Stock Center	BDSC: #58708 RRID:BDSC_58708 FBti0164930	A gift from Andrea Page-McCaw, Vanderbilt University, USA
Genetic reagent (D. melanogaster)	UAS-nAChRα1	This paper		Detail described in Material and method
Genetic reagent (D. melanogaster)	UAS > stop > dTrpA1mcherry	von Philipsborn et al., 2011	FBtp0064577 PMID:21315261	A gift from Daisuke Yamamoto, Advanced ICT Research Institute, National Institute of Information and Communications Technology, Japan
Genetic reagent (D. melanogaster)	UAS > stop > TNT	von Philipsborn et al., 2011	FBtp0020863 PMID:21315261	A gift from Daisuke Yamamoto, Advanced ICT Research Institute, National Institute of Information and Communications Technology, Japan
Genetic reagent (D. melanogaster)	UAS > stop > TNTin	von Philipsborn et al., 2011	FBtp0020863 PMID:21315261	A gift from Daisuke Yamamoto, Advanced ICT Research Institute, National Institute of Information and Communications Technology, Japan
Genetic reagent (D. melanogaster)	dsx-FLP	Rezával et al., 2014	FBal0296301 PMID:24631243	A gift from Daisuke Yamamoto, Advanced ICT Research Institute, National Institute of Information and Communications Technology, Japan
Genetic reagent (D. melanogaster)	TRiC; UAS-mCD8::RFP, LexAop2-mCD8::GFP;nSyb-MKII::nlsLexADBDo;UAS-p65AD::CaM	Bloomington Drosophila Stock Center	BDSC: #61679 RRID:BDSC_61679 FBst0061679
Genetic reagent (D. melanogaster)	ppk-eGFP	Grueber et al., 2003	FBtp0041053 PMID:12699617	A gift from Tadashi Uemura, Kyoto University, Japan
Genetic reagent (D. melanogaster)	LexAop-Kir2.1	Feng et al., 2014	FBtp0110870 PMID:24991958	A gift from Young-Joon Kim, Gwangju Institute of Science and Technology, South Korea
Genetic reagent (D. melanogaster)	UAS-LacZRNAi	Kennerdell and Carthew, 2000	FBtp0016505 PMID:10932163	A gift from Masayuki Miura, The University of Tokyo, Japan
Genetic reagent (D. melanogaster)	UAS-OambRNAi1	Bloomington Drosophila Stock Center	BDSC: #31171 RRID:BDSC_31171 FBst0031171
Genetic reagent (D. melanogaster)	UAS-OambRNAi2	Bloomington Drosophila Stock Center	BDSC: #31233 RRID:BDSC_31233 FBst0031233
Genetic reagent (D. melanogaster)	UAS-OambRNAi3	Vienna Drosophila Resource Center	VDRC: #106511 FBst0478335
Genetic reagent (D. melanogaster)	UAS-Octβ1RRNAi	Vienna Drosophila Resource Center	VDRC: #110537 FBst0482104
Genetic reagent (D. melanogaster)	UAS-Octβ2RRNAi	Vienna Drosophila Resource Center	VDRC: #104524 FBst0476382
Genetic reagent (D. melanogaster)	UAS-Octβ3RRNAi	Vienna Drosophila Resource Center	VDRC: #101189 FBst0473062
Genetic reagent (D. melanogaster)	UAS-Insp3RRNAi	Bloomington Drosophila Stock Center	BDSC: #25937 FBst0025937
Genetic reagent (D. melanogaster)	UAS-EcRRNAi	Vienna Drosophila Resource Center	VDRC: #37059 FBst0461818
Genetic reagent (D. melanogaster)	UAS-Mmp2RNAi1	Bloomington Drosophila Stock Center	BDSC: #31371 RRID:BDSC_31371 FBst0031371
Genetic reagent (D. melanogaster)	UAS-Mmp2RNAi2	Vienna Drosophila Resource Center	VDRC: #330203 FBst0490996
Genetic reagent (D. melanogaster)	UAS-TimpRNAi1	Bloomington Drosophila Stock Center	BDSC: #61294 RRID:BDSC_61294 FBst0061294
Genetic reagent (D. melanogaster)	UAS-TimpRNAi2	Vienna Drosophila Resource Center	VDRC: #109427 FBst0481116
Genetic reagent (D. melanogaster)	UAS-Tdc2RNAi1	Vienna Drosophila Resource Center	VDRC: #330541 FBst0492256
Genetic reagent (D. melanogaster)	UAS-Tdc2RNAi2	Bloomington Drosophila Stock Center	BDSC: #25871 RRID:BDSC_25871 FBst0025871
Genetic reagent (D. melanogaster)	UAS-TβhRNAi1	Vienna Drosophila Resource Center	VDRC: #107070 FBst0478893
Genetic reagent (D. melanogaster)	UAS-TβhRNAi2	Bloomington Drosophila Stock Center	BDSC: #67968 RRID:BDSC_67968 FBst0067968
Genetic reagent (D. melanogaster)	UAS-ChATRNAi1	Vienna Drosophila Resource Center	VDRC: #330291 FBst0490951
Genetic reagent (D. melanogaster)	UAS-ChATRNAi2	Bloomington Drosophila Stock Center	BDSC: #25856 RRID:BDSC_25856 FBst0025856
Genetic reagent (D. melanogaster)	UAS-nAChRα1RNAi	Vienna Drosophila Resource Center	VDRC #48159 FBst0467755
Genetic reagent (D. melanogaster)	UAS-nAChRα2RNAi	Vienna Drosophila Resource Center	VDRC: #101760 FBst0473633
Genetic reagent (D. melanogaster)	UAS-nAChRα3RNAi	Vienna Drosophila Resource Center	VDRC: #101806
Genetic reagent (D. melanogaster)	UAS-nAChRβ1RNAi	Vienna Drosophila Resource Center	VDRC: #106570 FBst0478394
Genetic reagent (D. melanogaster)	UAS-nAChRβ2RNAi	Vienna Drosophila Resource Center	VDRC: #109450 FBst0481138
Genetic reagent (D. melanogaster)	UAS-nvdRNAi1	Yoshiyama et al., 2006	FBal0193613 PMID:16763204
Genetic reagent (D. melanogaster)	UAS-nvdRNAi2	Yoshiyama et al., 2006	FBal0193614 PMID:16763204
Chemical, compound, drug	Octopamine	Sigma-Aldrich	#O0250
Chemical, compound, drug	Schneider’s Drosophila medium	Thermo Fisher Scientific	#21720024
Chemical, compound, drug	20-hydroxyecdysone	Enzo Life Sciences	ALX-370–012
Antibody	anti-GFP (chicken polyclonal)	Abcam	#ab13970	1:4000 dilution
Antibody	anti-RFP (rabbit polyclonal)	Medical and Biological Laboratories	#PM005	1:2000 dilution
Antibody	anti-Hts 1B1 (mouse monoclonal)	Developmental Studies Hybridoma Bank		1:50 dilution
Antibody	anti-DE-cadherin DCAD2 (rat monoclonal)	Developmental Studies Hybridoma Bank		1:50 dilution
Antibody	anti-pH3 (rabbit polyclonal)	Merck Millipore	#06–570	1:2000 dilution
Antibody	anti-pMad (rabbit polyclonal)	Abcam	#ab52903	1:2000 dilution
Antibody	anti-Lamin C LC28.26 (mouse monoclonal)	Developmental Studies Hybridoma Bank		1:10 dilution
Antibody	anti-cleaved Dcp-1 (rabbit polyclonal)	Cell Signaling Technology	#9578	1:1000 dilution
Antibody	anti-Vasa (rat monoclonal)	Developmental Studies Hybridoma Bank		1:50 dilution
Antibody	anti-LacZ 40-1a (mouse monoclonal)	Developmental Studies Hybridoma Bank		1:50 dilution
Antibody	anti-Tdc2 (rabbit polyclonal)	Abcam	#ab128225	1:2000 dilution
Antibody	Alexa Fluor 546 phalloidin	Thermo Fisher Scientific	#A22283	1:200 dilution
Antibody	Alexa Fluor 633 phalloidin	Thermo Fisher Scientific	#A22284	1:200 dilution
Chemical, compound, drug	FluorSave reagent	Merck Millipore	#345789
Chemical, compound, drug	all trans-Retinal	Sigma-Aldrich	#R2500
Software, algorithm	ImageJ		https://imagej.nih.gov/ij; RRID:SCR_003070 PMID:22930834
Software, algorithm	R		RRID:SCR_001905