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

  1. Yuto Yoshinari
  2. Tomotsune Ameku
  3. Shu Kondo
  4. Hiromu Tanimoto
  5. Takayuki Kuraishi
  6. Yuko Shimada-Niwa
  7. Ryusuke Niwa  Is a corresponding author
  1. Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan
  2. Invertebrate Genetics Laboratory, National Institute of Genetics, Japan
  3. Graduate School of Life Sciences, Tohoku University, Japan
  4. Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan
  5. AMED-PRIME, Japan Agency for Medical Research and Development, Japan
  6. Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Japan
  7. AMED-CREST, Japan Agency for Medical Research and Development, Japan
Research Article
Cite this article as: eLife 2020;9:e57101 doi: 10.7554/eLife.57101
7 figures, 2 videos, 1 table and 5 additional files

Figures

Figure 1 with 3 supplements
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 (w1118) 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.

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.

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 OambKI-T2A-GAL4. 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 OambKI-T2A-GAL4. 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 OambKI-T2A-GAL4. 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 OambKI-RD-T2A-GAL4. Scale bar, 20 µm.

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.

Figure 2 with 1 supplement
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 >LacZRNAi, n = 8) and c587 >OambRNAi (n = 8) female ovaries. Note that OA significantly increased the calcium response in escort cells, but OambRNAi 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.

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.

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 EcRA483T. 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.

Figure 4 with 1 supplement
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.

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.

Figure 5 with 1 supplement
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+ Tdc2+ neurons with the genotype indicated. (D–E) TrpA1-mediated activation of dsx+ Tdc2+ 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 OambΔ genetic background. (G) The inactivation of dsx+ Tdc2+ 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+ Tdc2+ 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.

Figure 5—figure supplement 1
dsx+ Tdc2+ 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+ Tdc2+ 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+ Tdc2+ 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 6 with 2 supplements
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+ Tdc2+ 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 shibirets (shits). (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.

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).

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 (nAChRα1WT) 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.

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+ Tdc2 neurons via acetylcholine signaling. The octopamine released from dsx+ Tdc2 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.

Videos

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.

Tables

Appendix 1—key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)c587-GAL4Manseau et al., 1997FBal0150629
RRID:BDSC_67747
A gift from Hiroko Sano, Kurume University, Japan
Genetic reagent (D. melanogaster)OambΔThis paperDetail described in Figure 1—figure supplement 1F
Genetic reagent (D. melanogaster)nAChRa1228This paperDetail described in Figure 6—figure supplement 2A
Genetic reagent (D. melanogaster)nAChRa1326This paperDetail described in Figure 6—figure supplement 2A
Genetic reagent (D. melanogaster)EcRA483TBloomington Drosophila Stock CenterBDSC: #5799
RRID:BDSC_5799
FBal0083501
Genetic reagent (D. melanogaster)EcRM554fsBloomington Drosophila Stock CenterBDSC: #4894
RRID:BDSC_4894
FBal0083490
Genetic reagent (D. melanogaster)Vkg::GFPKYOTO stock centerDGRC #110692
RRID:DGGR_110692
FBal0286156
Genetic reagent (D. melanogaster)Dad-LacZTsuneizumi et al., 1997FBal0065787
RRID:DGGR_118114
A gift from Yoshiki Hayashi, University of Tsukuba, Japan
Genetic reagent (D. melanogaster)R44E10-GAL4Deady and Sun, 2015FBal0252601
PMID:26473732
A gift from Jianjun Sun, University of Connecticut, USA
Genetic reagent (D. melanogaster)RS-GAL4Lee et al., 2009FBal0263794
PMID:19262750
A gift from Kyung-An Han, Pennsylvania State University, USA
Genetic reagent (D. melanogaster)nSyb-GAL4Bloomington Drosophila Stock CenterBDSC: #51941
RRID:BDSC_51941
FBti0154973
FACS (5 ul per test)
Genetic reagent (D. melanogaster)nSyb-GAL80Harris et al., 2015PMID:26193122A gift from James W. Truman, Janelia Research Campus, USA
Genetic reagent (D. melanogaster)tj-GAL4KYOTO stock centerDGRC: #104055
RRID:DGGR_104055
FBti0034540
Genetic reagent (D. melanogaster)R13C06-GAL4Bloomington Drosophila Stock CenterBDSC: #47860
RRID:BDSC_47860
FBal0249828
Genetic reagent (D. melanogaster)109–30 GAL4Bloomington Drosophila Stock CenterBDSC: #7023
RRID:BDSC_7023
FBti0027548
Genetic reagent (D. melanogaster)c355-GAL4Bloomington Drosophila Stock CenterBDSC: #3750
RRID:BDSC_3750
FBti0002591
Genetic reagent (D. melanogaster)c306-GAL4Bloomington Drosophila Stock CenterBDSC: #3743
RRID:BDSC_3743
FBal0048787
Genetic reagent (D. melanogaster)slbo-GAL4Bloomington Drosophila Stock CenterBDSC: #6458
RRID:BDSC_6458
FBst0006458
Genetic reagent (D. melanogaster)bab1-GAL4Bolívar et al., 2006FBal0242654
PMID:17013875
A gift from Satoru Kobayashi, University of Tsukuba, Japan
Genetic reagent (D. melanogaster)nos-GAL4KYOTO stock centerDGRC: #107748
RRID:DGGR_107748
FBst0306396
Genetic reagent (D. melanogaster)tub > FRT > GAL80>FRTBloomington Drosophila Stock CenterBDSC: #38879
RRID:BDSC_38879
FBti0147580
Genetic reagent (D. melanogaster)OambKI-RD-GAL4Deng et al., 2019
Bloomington Drosophila Stock Center
BDSC: #84677
RRID:BDSC_84677
FBti0209942
Genetic reagent (D. melanogaster)Oamb-KI-T2A-GAL4Kondo et al., 2020PMID:31914394
Genetic reagent (D. melanogaster)nAChRα1-T2A-GAL4Kondo et al., 2020PMID:31914394
Genetic reagent (D. melanogaster)nAChRα2-T2A-GAL4Kondo et al., 2020PMID:31914394
Genetic reagent (D. melanogaster)nAChRα3-T2A-GAL4Kondo et al., 2020PMID:31914394
Genetic reagent (D. melanogaster)nAChRβ1-T2A-GAL4Kondo et al., 2020PMID:31914394
Genetic reagent (D. melanogaster)nAChRβ2-T2A-GAL4Kondo et al., 2020PMID:31914394
Genetic reagent (D. melanogaster)ChaT-GAL4Bloomington Drosophila Stock CenterBDSC: #6793
RRID:BDSC_6793
FBst0006793
Genetic reagent (D. melanogaster)ppk-GAL4Grueber et al., 2007FBtp0039691
PMID:17164414
A gift from Hiroko Sano, Kurume University, Japan
Genetic reagent (D. melanogaster)SPSNs-LexAFeng et al., 2014FBtp0110869
PMID:24991958
A gift from Young-Joon Kim, Gwangju Institute of Science and Technology, South Korea
Genetic reagent (D. melanogaster)20xUAS-6xGFPBloomington Drosophila Stock CenterBDSC: #52261
RRID:BDSC_52261
FBst0052261
Genetic reagent (D. melanogaster)UAS-GFP;UAS-mCD8::GFPIto et al., 1997; Lee and Luo, 1999FBtp0002652
PMID:9043058
PMID:10457015
A gift from Kei Ito, University of Cologne, Germany
Genetic reagent (D. melanogaster)UAS-StingerBloomington Drosophila Stock CenterBDSC: #84277
RRID:BDSC_84277
Genetic reagent (D. melanogaster)UAS-mCD8::RFPBloomington Drosophila Stock CenterBDSC: #32219
RRID:BDSC_32219
FBti0131967
Genetic reagent (D. melanogaster)UAS-CsChrimsonBloomington Drosophila Stock CenterBDSC: #55134
RRID:BDSC_55134
FBti0160571
Genetic reagent (D. melanogaster)UAS-Insp3RBloomington Drosophila Stock CenterBDSC: #30742 RRID:BDSC_30742
FBti0129829
Genetic reagent (D. melanogaster)UAS-OambK3Lee et al., 2009FBtp0069415
PMID:19262750
A gift from Kyung-An Han, Pennsylvania State University, USA
Genetic reagent (D. melanogaster)UAS-TimpBloomington Drosophila Stock CenterBDSC: #58708
RRID:BDSC_58708
FBti0164930
A gift from Andrea Page-McCaw, Vanderbilt University, USA
Genetic reagent (D. melanogaster)UAS-nAChRα1This paperDetail described in Material and method
Genetic reagent (D. melanogaster)UAS > stop > dTrpA1mcherryvon Philipsborn et al., 2011FBtp0064577
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 > TNTvon Philipsborn et al., 2011FBtp0020863
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 > TNTinvon Philipsborn et al., 2011FBtp0020863
PMID:21315261
A gift from Daisuke Yamamoto, Advanced ICT Research Institute, National Institute of Information and Communications Technology, Japan
Genetic reagent (D. melanogaster)dsx-FLPRezával et al., 2014FBal0296301
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::CaMBloomington Drosophila Stock CenterBDSC: #61679
RRID:BDSC_61679
FBst0061679
Genetic reagent (D. melanogaster)ppk-eGFPGrueber et al., 2003FBtp0041053
PMID:12699617
A gift from Tadashi Uemura, Kyoto University, Japan
Genetic reagent (D. melanogaster)LexAop-Kir2.1Feng et al., 2014FBtp0110870
PMID:24991958
A gift from Young-Joon Kim, Gwangju Institute of Science and Technology, South Korea
Genetic reagent (D. melanogaster)UAS-LacZRNAiKennerdell and Carthew, 2000FBtp0016505
PMID:10932163
A gift from Masayuki Miura, The University of Tokyo, Japan
Genetic reagent (D. melanogaster)UAS-OambRNAi1Bloomington Drosophila Stock CenterBDSC: #31171
RRID:BDSC_31171
FBst0031171
Genetic reagent (D. melanogaster)UAS-OambRNAi2Bloomington Drosophila Stock CenterBDSC: #31233
RRID:BDSC_31233
FBst0031233
Genetic reagent (D. melanogaster)UAS-OambRNAi3Vienna Drosophila Resource CenterVDRC: #106511
FBst0478335
Genetic reagent (D. melanogaster)UAS-Octβ1RRNAiVienna Drosophila Resource CenterVDRC: #110537
FBst0482104
Genetic reagent (D. melanogaster)UAS-Octβ2RRNAiVienna Drosophila Resource CenterVDRC: #104524
FBst0476382
Genetic reagent (D. melanogaster)UAS-Octβ3RRNAiVienna Drosophila Resource CenterVDRC: #101189
FBst0473062
Genetic reagent (D. melanogaster)UAS-Insp3RRNAiBloomington Drosophila Stock CenterBDSC: #25937
FBst0025937
Genetic reagent (D. melanogaster)UAS-EcRRNAiVienna Drosophila Resource CenterVDRC: #37059
FBst0461818
Genetic reagent (D. melanogaster)UAS-Mmp2RNAi1Bloomington Drosophila Stock CenterBDSC: #31371
RRID:BDSC_31371
FBst0031371
Genetic reagent (D. melanogaster)UAS-Mmp2RNAi2Vienna Drosophila Resource CenterVDRC: #330203
FBst0490996
Genetic reagent (D. melanogaster)UAS-TimpRNAi1Bloomington Drosophila Stock CenterBDSC: #61294
RRID:BDSC_61294
FBst0061294
Genetic reagent (D. melanogaster)UAS-TimpRNAi2Vienna Drosophila Resource CenterVDRC: #109427
FBst0481116
Genetic reagent (D. melanogaster)UAS-Tdc2RNAi1Vienna Drosophila Resource CenterVDRC: #330541
FBst0492256
Genetic reagent (D. melanogaster)UAS-Tdc2RNAi2Bloomington Drosophila Stock CenterBDSC: #25871
RRID:BDSC_25871
FBst0025871
Genetic reagent (D. melanogaster)UAS-TβhRNAi1Vienna Drosophila Resource CenterVDRC: #107070
FBst0478893
Genetic reagent (D. melanogaster)UAS-TβhRNAi2Bloomington Drosophila Stock CenterBDSC: #67968
RRID:BDSC_67968
FBst0067968
Genetic reagent (D. melanogaster)UAS-ChATRNAi1Vienna Drosophila Resource CenterVDRC: #330291
FBst0490951
Genetic reagent (D. melanogaster)UAS-ChATRNAi2Bloomington Drosophila Stock CenterBDSC: #25856
RRID:BDSC_25856
FBst0025856
Genetic reagent (D. melanogaster)UAS-nAChRα1RNAiVienna Drosophila Resource CenterVDRC #48159
FBst0467755
Genetic reagent (D. melanogaster)UAS-nAChRα2RNAiVienna Drosophila Resource CenterVDRC: #101760
FBst0473633
Genetic reagent (D. melanogaster)UAS-nAChRα3RNAiVienna Drosophila Resource CenterVDRC: #101806
Genetic reagent (D. melanogaster)UAS-nAChRβ1RNAiVienna Drosophila Resource CenterVDRC: #106570
FBst0478394
Genetic reagent (D. melanogaster)UAS-nAChRβ2RNAiVienna Drosophila Resource CenterVDRC: #109450
FBst0481138
Genetic reagent (D. melanogaster)UAS-nvdRNAi1Yoshiyama et al., 2006FBal0193613
PMID:16763204
Genetic reagent (D. melanogaster)UAS-nvdRNAi2Yoshiyama et al., 2006FBal0193614
PMID:16763204
Chemical, compound, drugOctopamineSigma-Aldrich#O0250
Chemical, compound, drugSchneider’s Drosophila mediumThermo Fisher Scientific#21720024
Chemical, compound, drug20-hydroxyecdysoneEnzo Life SciencesALX-370–012
Antibodyanti-GFP (chicken polyclonal)Abcam#ab139701:4000 dilution
Antibodyanti-RFP (rabbit polyclonal)Medical and Biological Laboratories#PM0051:2000 dilution
Antibodyanti-Hts 1B1 (mouse monoclonal)Developmental Studies Hybridoma Bank1:50 dilution
Antibodyanti-DE-cadherin DCAD2 (rat monoclonal)Developmental Studies Hybridoma Bank1:50 dilution
Antibodyanti-pH3 (rabbit polyclonal)Merck Millipore#06–5701:2000 dilution
Antibodyanti-pMad (rabbit polyclonal)Abcam#ab529031:2000 dilution
Antibodyanti-Lamin C LC28.26 (mouse monoclonal)Developmental Studies Hybridoma Bank1:10 dilution
Antibodyanti-cleaved Dcp-1 (rabbit polyclonal)Cell Signaling Technology#95781:1000 dilution
Antibodyanti-Vasa (rat monoclonal)Developmental Studies Hybridoma Bank1:50 dilution
Antibodyanti-LacZ 40-1a (mouse monoclonal)Developmental Studies Hybridoma Bank1:50 dilution
Antibodyanti-Tdc2 (rabbit polyclonal)Abcam#ab1282251:2000 dilution
AntibodyAlexa Fluor 546 phalloidinThermo Fisher Scientific#A222831:200 dilution
AntibodyAlexa Fluor 633 phalloidinThermo Fisher Scientific#A222841:200 dilution
Chemical, compound, drugFluorSave reagentMerck Millipore#345789
Chemical, compound, drugall trans-RetinalSigma-Aldrich#R2500
Software, algorithmImageJhttps://imagej.nih.gov/ij; RRID:SCR_003070
PMID:22930834
Software, algorithmRRRID:SCR_001905

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