Two classes of amine/glutamate multi-transmitter neurons innervate Drosophila internal male reproductive organs
- Montana State University, Department of Microbiology and Cell Biology, United States
- The University of Montana, Division of Biological Sciences, United States
Figures
Figure 1
The Drosophila male reproductive system.
(A) Schematic diagram showing paired testes (uncolored), SVs (green), and AGs (purple), Sph (red), ED (gray), and EB (uncolored). Modified version of Figure 38A page 508 (Demerec, 1950). Used with kind permission from Cold Spring Harbor Laboratory (CSHL) Press. (B) Actual male reproductive system. Te-testes, SV-seminal vesicle, AG-accessory gland, Sph-sphincter, ED-ejaculatory duct, EB-ejaculatory bulb. Scale bar: 200 μm.
Figure 2
Gal4 and Split-GAL4 expression patterns in the Drosophila male reproductive system.
(A) Tdc2-GAL4; (B) TRH-GAL4; (C) vGlut-GAL4; (D) Tdc2-AD ∩ vGlut-GAL4-DBD; (E) vGlut-AD Ç Tdc2-GAL4-DBD; (F) TRH-AD Ç vGlut-GAL4-DBD; (G) vGlut-AD Ç TRH-GAL4-DBD; (H) Tdc2-AD Ç TRH-GAL4-DBD. Scale bar: 200 μm.
Figure 3 with 3 supplements
Expression of TRH-GAL4 and Tdc2-LexA in the Drosophila male reproductive system.
(A, D, G, J, M, P) TRH-GAL4, UAS-6XGFP; (B, E, H, K, N, Q) Tdc2-LexA, LexAop-6XmCherry. (C, F, I, L, O, R) overlay. Scale bars: O-50 μm; R-10 μm.
Figure 3—figure supplement 1
Expression of vGlut-LexA and TRH-GAL4 in the Drosophila male reproductive system.
(A, D, G, J, M, P) vGlut-LexA, LexAop-6XmCherry; (B, E, H, K, N, Q) TRH-GAL4, UAS-6XGFP (C, F, I, L, O, R) overlay. Scale bars: O-50 μm; R-10 μm.
Figure 3—figure supplement 2
Expression of vGlut-LexA and Tdc2-GAL4 in the Drosophila male reproductive system.
(A, D, G, J, M, P) vGlut-LexA, LexAop-6XmCherry; (B, E, H, K, N, Q) Tdc2-GAL4, UAS-6XGFP (C, F, I, L, O, R) overlay. Scale bars: O-50 μm; R-10 μm.
Figure 3—figure supplement 3
Fruitless and Doublesex expression in the Drosophila male reproductive system.
(A) fru-GAL4, UAS-6XGFP; (B) vGlut-LexA, LexAop-6XmCherry; (C) overlay; (D) Tdc2-AD ∩ dsx-GAL4-DBD; (E) TRH-AD ∩ dsx-GAL4-DBD. Scale bar: 200 μm.
Figure 4 with 4 supplements
Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the Drosophila male reproductive system.
(A) vGlut-40XV5; (B) TβH-GFP; (C) 5-HT; (D) vGlut-40XV5, TβH-GFP overlay; (E) vGlut-40XV5, 5-HT overlay; (F) TβH-GFP, 5-HT overlay; (G) vGlut-40XV5; (H) Tdc2; (I) 5-HT; (J) vGlut-40XV5, Tdc2 overlay; (K) vGlut-40XV5, 5-HT overlay; (L) Tdc2, 5-HT overlay. Scale bar: 200 μm.
Figure 4—figure supplement 1
Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the seminal vesicle (SV) of the Drosophila male reproductive system.
(A, G) vGlut-40XV5; (B, H) TβH-GFP; (C, I) 5-HT; (D, J) vGlut-40XV5, TβH-GFP overlay; (E, K) vGlut-40XV5, 5-HT overlay; (F, L) TβH-GFP, 5-HT overlay; (M, S) vGlut-40XV5; (N, T) Tdc2; (O, U) 5-HT; (P, V) vGlut-40XV5, Tdc2 overlay; (Q, W) vGlut-40XV5, 5-HT overlay; (R, X) Tdc2, 5-HT overlay. Scale bars: R-50 μm; X-10 μm.
Figure 4—figure supplement 2
Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the ejaculatory duct (ED) of the Drosophila male reproductive system.
(A, G) vGlut-40XV5; (B, H) TβH-GFP; (C, I) 5-HT; (D, J) vGlut-40XV5, TβH-GFP overlay; (E, K) vGlut-40XV5, 5-HT overlay; (F, L) TβH-GFP, 5-HT overlay; (M, S) vGlut-40XV5; (N, T) Tdc2; (O, U) 5-HT; (P, V) vGlut-40XV5, Tdc2 overlay; (Q, W) vGlut-40XV5, 5-HT overlay; (R, X) Tdc2, 5-HT overlay. Scale bars: R-50 μm; X-10 μm.
Figure 4—figure supplement 3
Expression of vGlut, TβH-GFP, Tdc2, and 5-HT in the accessory gland (AG) of the Drosophila male reproductive system.
(A, G) vGlut-40XV5; (B, H) TβH-GFP; (C, I) 5-HT; (D, J) vGlut-40XV5, TβH-GFP overlay; (E, K) vGlut-40XV5, 5-HT overlay; (F, L) TβH-GFP, 5-HT overlay; (M, S) vGlut-40XV5; (N, T) Tdc2; (O, U) 5-HT; (P, V) vGlut-40XV5, Tdc2 overlay; (Q, W) vGlut-40XV5, 5-HT overlay; (R, X) Tdc2, 5-HT overlay. Scale bars: R-50 μm; X-10 μm.
Figure 4—figure supplement 4
Expression of vGlut, TβH-GFP, and 5-HT at the junction of the seminal vesicle (SV) and accessory glands (AGs) with the ejaculatory duct (ED) of the Drosophila male reproductive system.
(A) vGlut-40XV5; (B) TβH-GFP; (C) 5-HT; (D) vGlut-40XV5, TβH-GFP overlay; (E) vGlut-40XV5, 5-HT overlay; (F) TβH-GFP, 5-HT overlay. Scale bar: 50 μm.
Figure 5 with 5 supplements
Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in the seminal vesicle (SV) of serotonergic (TRH) or octopaminergic (Tdc2) neurons of the Drosophila male reproductive system.
(A–D) 63X. (A) vGlut-40XMYC; (B) 6XV5-vMAT; (C) Tdc2; (D) TRH >CD8-mCherry. (E–L) 63X zoom 4X. (E) vGlut-40XMYC; (F) 6XV5-vMAT; (G) Tdc2; (H) TRH >CD8-mCherry; (I) vGlut-40XMYC, 6XV5-vMAT overlay; (J) vGlut-40XMYC, 6XV5-vMAT, Tdc2 overlay; (K) 6XV5-vMAT, Tdc2 overlay; (L) Tdc2, TRH >CD8-mCherry overlay. (M–P) 63X. (M) vGlut-40XMYC; (N) 6XV5-vMAT; (O) 5-HT; (P) Tdc2>CD8-mCherry. (Q–X) 63X zoom 4X. (Q) vGlut-40XMYC; (R) 6XV5-vMAT; (S) 5-HT; (T) Tdc2>CD8-mCherry; (U) vGlut-40XMYC, 6XV5-vMAT overlay; (V) vGlut-40XMYC, 6XV5-vMAT, 5-HT overlay; (W) 6XV5-vMAT, 5-HT overlay; (X) 5-HT, Tdc2>CD8-mCherry overlay. Scale bars: P-50 μm; X-10 μm.
Figure 5—figure supplement 1
Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in the ejaculatory duct (ED) of serotonergic (TRH) or octopaminergic (Tdc2) neurons of the Drosophila male reproductive system.
(A–D) 63X. (A) vGlut-40XMYC; (B) 6XV5-vMAT; (C) Tdc2; (D) TRH >CD8-mCherry. (E–L) 63X zoom 4X. (E) vGlut-40XMYC; (F) 6XV5-vMAT; (G) Tdc2; (H) TRH >CD8-mCherry; (I) vGlut-40XMYC, 6XV5-vMAT overlay; (J) vGlut-40XMYC, 6XV5-vMAT, Tdc2 overlay; (K) 6XV5-vMAT, Tdc2 overlay; (L) Tdc2, TRH >CD8-mCherry overlay. (M–P) 63X. (M) vGlut-40XMYC; (N) 6XV5-vMAT; (O) 5-HT; (P) Tdc2>CD8-mCherry. (Q–X) 63X zoom 4X. (Q) vGlut-40XMYC; (R) 6XV5-vMAT; (S) 5-HT; (T) Tdc2>CD8-mCherry; (U) vGlut-40XMYC, 6XV5-vMAT overlay; (V) vGlut-40XMYC, 6XV5-vMAT, 5-HT overlay; (W) 6XV5-vMAT, 5-HT overlay; (X) Tdc2, Tdc2>CD8-mCherry overlay. Scale bars: P-50 μm; X-10 μm.
Figure 5—figure supplement 2
Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in the accessory gland (AG) of serotonergic (TRH) or octopaminergic (Tdc2) neurons of the Drosophila male reproductive system.
(A–D) 63X. (A) vGlut-40XMYC; (B) 6XV5-vMAT; (C) Tdc2; (D) TRH >CD8-mCherry. (E–L) 63X zoom 4X. (E) vGlut-40XMYC; (F) 6XV5-vMAT; (G) Tdc2; (H) TRH >CD8-mCherry; (I) vGlut-40XMYC, 6XV5-vMAT overlay; (J) vGlut-40XMYC, 6XV5-vMAT, Tdc2 overlay; (K) 6XV5-vMAT, Tdc2 overlay; (L) Tdc2, TRH >CD8-mCherry overlay. (M–P) 63X. (M) vGlut-40XMYC; (N) 6XV5-vMAT; (O) 5-HT; (P) Tdc2>CD8-mCherry. (Q–X) 63X zoom 4X. (Q) vGlut-40XMYC; (R) 6XV5-vMAT; (S) 5-HT; (T) Tdc2>CD8-mCherry; (U) vGlut-40XMYC, 6XV5-vMAT overlay; (V) vGlut-40XMYC, 6XV5-vMAT, 5-HT overlay; (W) 6XV5-vMAT, 5-HT overlay; (X) Tdc2, Tdc2>CD8-mCherry overlay. Scale bars: P-50 μm; X-10 μm.
Figure 5—figure supplement 3
Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in serotonergic neurons of the Drosophila ejaculatory duct (ED) using expansion microscopy.
(A, D) 6XV5-vMAT; (B, E) vGlut-40XMYC; (C, F) overlay. (A–C) 6XV5-vMAT and vGlut-40XMYC expression above the threshold intensity values. (D–F) Complete 6XV5-vMAT and vGlut-40XMYC expression. (G) Mander’s plot of 6XV5-vMAT and vGlut-40XMYC. Vertical line indicates intensity threshold value for 6XV5-vMAT. Horizontal line indicates intensity threshold value for vGlut-40XMYC. (H) Mander’s coefficients. Grayscale signal in A–C denotes the CD8-mCherry plasma membrane marker. Scale bars: 500 nm. Scale bars have been corrected by 10X to reflect the empirically determined 10.5X expansion factor.
Figure 5—figure supplement 4
Co-conditional expression of vGlut-40XMYC and 6XV5-vMAT in octopaminergic neurons of the Drosophila ejaculatory duct (ED) using expansion microscopy.
(A, D) 6XV5-vMAT; (B, E) vGlut-40XMYC; (C, F) overlay. (A–C) 6XV5-vMAT and vGlut-40XMYC expression above the threshold intensity values. (D–F) Complete 6XV5-vMAT and vGlut-40XMYC expression. (G) Mander’s plot of 6XV5-vMAT and vGlut-40XMYC. Vertical line indicates intensity threshold value for 6XV5-vMAT. Horizontal line indicates intensity threshold value for vGlut-40XMYC. (H) Mander’s coefficients. Grayscale signal in A-C denotes the CD8-mCherry plasma membrane marker. Scale bars: 500 nm. Scale bars have been corrected by 10X to reflect the empirically determined 10.5X expansion factor.
Figure 5—figure supplement 5
Nuclei of unexpanded and expanded ejaculatory ducts (EDs).
(A) DAPI-stained nuclei from an unexpanded ED. (B) DAPI-stained nuclei of an expanded ED. (C) Bar graph plot of nuclei area (mean ± SE-black bars). For the experiment shown, the calculated expansion factor (post-ExM nuclear area/pre-ExM nuclear area) was 10.47, p-value <0.00001. Fij/ImageJ open-source software was used to segment images and calculate nuclear area. Scale bars: 25 μm.
Figure 6 with 5 supplements
Expression of the synaptic vesicle marker Synapsin in combination with vGlut-40XMYC and 6XV5-vMAT in the seminal vesicle (SV), ejaculatory duct (ED), and accessory gland (AG) of the Drosophila male reproductive system.
(A–I) SV. (A) vGlut-40XMYC; (B) 6XV5-vMAT; (C) Synapsin; (D) vGlut-40XMYC; (E) 6XV5-vMAT; (F) Synapsin; (G) vGlut-40XMYC, 6XV5-vMAT overlay; (H) vGlut-40XMYC, Synapsin overlay; (I) 6XV5-vMAT, Synapsin overlay. (A’-I’) ED. (A’) vGlut-40XMYC; (B’) 6XV5-vMAT; (C’) Synapsin; (D’) vGlut-40XMYC; (E’) 6XV5-vMAT; (F’) Synapsin; (G’) vGlut-40XMYC, 6XV5-vMAT overlay; (H’) vGlut-40XMYC, Synapsin overlay; (I’) 6XV5-vMAT, Synapsin overlay. (A’’-I’’) AG. (A’’) vGlut-40XMYC; (B’’) 6XV5-vMAT; (C’’) Synapsin; (D’’) vGlut-40XMYC; (E’’) 6XV5-vMAT; (F’’) Synapsin; (G’’) vGlut-40XMYC, 6XV5-vMAT overlay; (H’’) vGlut-40XMYC, Synapsin overlay; (I’’) 6XV5-vMAT, Synapsin overlay. Scale bars: C’’–50 μm; I’’–10 μm.
Figure 6—figure supplement 1
Expression of the active zone marker Brp in combination with vGlut-40XMYC and 6XV5-vMAT in the seminal vesicle (SV), ejaculatory duct (ED), and accessory gland (AG) of the Drosophila male reproductive system.
(A–H) SV. (A) vGlut-40XMYC; (B) 6XV5-vMAT; (C) Brp; (D) 6XV5-vMAT, Brp overlay; (E) vGlut-40XMYC; (F) 6XV5-vMAT; (G) Brp; (H) 6XV5-vMAT, Brp overlay. (I–P) ED. (I) vGlut-40XMYC; (J) 6XV5-vMAT; (K) Brp; (L) 6XV5-vMAT, Brp; (M) vGlut-40XMYC; (N) 6XV5-vMAT; (O) Brp; (P) 6XV5-vMAT, Brp overlay. (I–P) AG. (Q) vGlut-40XMYC; (R) 6XV5-vMAT; (S) Brp; (T) 6XV5-vMAT, Brp; (U) vGlut-40XMYC; (V) 6XV5-vMAT; (W) Brp; (X) 6XV5-vMAT, Brp overlay. Scale bars: P-50 μm; X-10 μm.
Figure 6—figure supplement 2
Expression of the post-synaptic density marker Dlg in combination with vGlut-40XMYC and 6XV5-vMAT in the seminal vesicle (SV), ejaculatory duct (ED), and accessory gland (AG) of the Drosophila male reproductive system.
(A–D) Complete reproductive system. (A) vGlut-40XMYC; (B) 6XV5-vMAT; (C) Dlg; (D) 6XV5-vMAT, Dlg overlay; (E–L) SV. (E) vGlut-40XMYC; (F) 6XV5-vMAT; (G) Dlg; (H) 6XV5-vMAT, Dlg overlay; (I) vGlut-40XMYC; (J) 6XV5-vMAT; (G) Dlg; (H) 6XV5-vMAT, Dlg overlay. (I–P) ED. (I) vGlut-40XMYC; (K) 6XV5-vMAT; (L) Dlg; (M–P) ED muscle surface. (M) vGlut-40XMYC; (N) 6XV5-vMAT; (O) Dlg; (P) 6XV5-vMAT, Dlg overlay (Q) Dlg in the ED epithelial cells. (R–U) AG muscle surface. (R) vGlut-40XMYC; (S) 6XV5-vMAT; (T) Dlg; (U) 6XV5-vMAT, Dlg overlay; (V) Dlg in AG epithelial cells. Scale bars: D-200 μm; H-50 μm; V-10 μm.
Figure 6—figure supplement 3
Expression of the large core dense vesicle marker IA2-GFP in combination with vGlut-40XMYC and 6XV5-vMAT in the seminal vesicle (SV) of the Drosophila male reproductive system.
(A–C) Complete reproductive system. (A) vGlut-40XMYC; (B) 6XV5-vMAT; (C) IA2-GFP; (D–O) SV. (D, J) vGlut-40XMYC; (E, K) 6XV5-vMAT; (F, L) IA2-GFP; (G, M) vGlut-40XMYC, 6XV5-vMAT overlay; (H, N) vGlut-40XMYC, IA2-GFP overlay; (I, O) 6XV5-vMAT, IA2 overlay. Scale bars: C-200 μm; F-50 μm; O-10 μm.
Figure 6—figure supplement 4
Expression of the large core dense vesicle marker IA2-GFP in combination with vGlut-40XMYC and 6XV5-vMAT in the ejaculatory duct (ED) and accessory gland s (AGs) of the Drosophila male reproductive system.
(A–L) ED. (A, G) vGlut-40XMYC; (B, H) 6XV5-vMAT; (C, I) IA2-GFP; (D, J) vGlut-40XMYC, 6XV5-vMAT overlay; (E, K) vGlut-40XMYC, IA2-GFP overlay; (F, L) 6XV5-vMAT, IA2 overlay. (M–X) AG. (M, S) vGlut-40XMYC; (N, T) 6XV5-vMAT; (O, U) IA2-GFP; (P, V) vGlut-40XMYC, 6XV5-vMAT overlay; (Q, W) vGlut-40XMYC, IA2-GFP overlay; (R, X) 6XV5-vMAT, IA2 overlay. Scale bars: R-50 μm; X-10 μm.
Figure 6—figure supplement 5
Expression of vGlut-40XMYC, 6XV5-vMAT, 7XMYC-vAChT, and 9XV5-vGAT in the seminal vesicle (SV), ejaculatory duct (ED), and accessory gland (AG) of the Drosophila male reproductive system.
(A–D) SV. (A) vGlut-40XV5; (B) 6XV5-vMAT; (C) 7XMYC-vAChT; (D) 9XV5-vGAT. (E–H) ED. (E) vGlut-40XV5; (F) 6XV5-vMAT; (G) 7XMYC-vAChT; (H) 9XV5-vGAT. (I–L) AG. (I) vGlut-40XV5; (J) 6XV5-vMAT; (K) 7XMYC-vAChT; (L) 9XV5-vGAT. Scale bar: 50 μm.
Figure 7 with 2 supplements
Glutamate receptor GAL4 expression patterns in Drosophila male reproductive system.
(A) GluRIIB; (B) GluRIIC. Scale bar: 200 μm.
Figure 7—figure supplement 1
Octopamine (OA) receptor GAL4 expression patterns in Drosophila male reproductive system.
(A) OAMB; (B) OAα2R; (C) Oct-TyrR; (D) OAβ1R; (E) OAβ2R; (F) OAβ3R. Scale bar: 200 μm.
Figure 7—figure supplement 2
5-HT receptor GAL4 expression patterns in Drosophila male reproductive system.
(A) 5-HT1A; (B) 5-HT2A; (C) 5-HT1B; (D) 5-HT2B; (E) 5-HT7. Scale bar: 200 μm.
Figure 8 with 10 supplements
GluRIIA-GFP expression in the seminal vesicle (SV), ejaculatory duct (ED), and accessory gland (AG) of the Drosophila male reproductive system.
(A–F) SV. (A, D) vGlut-40XV5; (B, E) GluRIIA-GFP; (C, F) vGlut-40XV5, GluRIIA-GFP overlay. (G–L) ED. (G, J) vGlut-40XV5; (H, K) GluRIIA-GFP; (I, L) vGlut-40XV5, GluRIIA-GFP overlay. (M–R) AG. (M, P) vGlut-40XV5; (N, Q) GluRIIA-GFP; (O, R) vGlut-40XV5, GluRIIA-GFP overlay. Scale bars: O-50 μm; R-10 μm.
Figure 8—figure supplement 1
OAMB expression in the Drosophila male reproductive system.
(A–C) Complete male reproductive system. (A) OAMB-10XV5; (B) Syn; (C) OAMB-10XV5, Syn overlay. (D–I) SV. (D, G) OAMB-10XV5; (E, H) Syn; (F, I) OAMB-10XV5, Syn overlay. (J–R) ED. (J–L) muscle surface. (J) OAMB-10XV5; (K) Syn; (L) OAMB-10XV5, Syn overlay. (M–O) muscles and epithelial layers. (M) OAMB-10XV5; (N) Syn; (O) OAMB-10XV5, Syn overlay. (P–R) epithelial layer. (P) OAMB-10XV5; (Q) Syn; (R) OAMB-10XV5, Syn overlay. Scale bars: C-200 μm; F, O-50 μm; I, L, R-10 μm.
Figure 8—figure supplement 2
OAMB expression in the Drosophila male reproductive system.
(A–C) AG. (A) OAMB-10XV5; (B) Syn; (C) OAMB-10XV5, Syn overlay. (D–F) Epithelial layer of AG. (D) OAMB-10XV5; (E) Syn; (F) OAMB-10XV5, Syn overlay. (G–I) Muscle layer of AG. (G) OAMB-10XV5; (H) Syn; (I) OAMB-10XV5, Syn overlay. (J–L) Ejaculatory bulb. (J) OAMB-10XV5; (K) Syn; (L) OAMB-10XV5, Syn overlay. (M–O) Muscle layer of ejaculatory bulb. (M) OAMB-10XV5; (N) Syn; (O) OAMB-10XV5, Syn overlay. (P–Q) Epithelial layer of ejaculatory bulb. (P) OAMB-10XV5; (Q) Syn; (R) OAMB-10XV5, Syn overlay. Scale bars: C, L-50 μm; F, I, R-10 μm.
Figure 8—figure supplement 3
OAα2R-20XV5 expression in the Drosophila male reproductive system.
(A–C) Complete male reproductive system. (A) OAα2R-20XV5; (B) Syn; (C) OAα2R-20XV5, Syn overlay. (D–I) SV. (D, G) OAα2R-20XV5; (E, H) Syn; (F, I) OAα2R-20XV5, Syn overlay. Cross-section of SV. (J) OAα2R-20XV5: (K) Syn; (L) OAα2R-20XV5, Syn overlay. (M–R) AG. (M, P) OAα2R-20XV5; (N, Q) Syn; (O, R) OAα2R-20XV5, Syn overlay. Scale bars: C-200 μm; O-50 μm; R-10 μm.
Figure 8—figure supplement 4
OAα2R-20XV5 expression in the Drosophila male reproductive system.
(A–C) SV/ED junction. (A, D) OAα2R-20XV5; (B, E) Syn; (C, F) OAα2R-20XV5, Syn overlay. (G–L) Proximal anterior ED. (G, J) OAα2R-20XV5; (H, K) Syn; (I, L) OAα2R-20XV5, Syn overlay. (M–U) Distal anterior ED. (M, P, S) OAα2R-20XV5; (N, Q, T) Syn; (O, R, U) OAα2R-20XV5, Syn overlay. Scale bars: C, I, O-50 μm; F-25 μm; L, U-10 μm.
Figure 8—figure supplement 5
OAβ2R-40XV5 expression in the Drosophila male reproductive system.
(A–C) Complete male reproductive system. (A) OAβ2R-40XV5; (B) Syn; (C) OAβ2R-40XV5, Syn overlay. (D–I) seminal vesicle (SV). (D, G) OAβ2R; (E, H) Syn; (F, I) OAβ2R-40XV5, Syn overlay. (J–L) Cross section of SV. (J) OAβ2R-40XV5; (K) Syn; (L) OAβ2R-40XV5, Syn overlay. (M–R) ED. (M, P) OAβ2R; (N, Q) Syn; (O, R) OAβ2R-40XV5, Syn overlay. Scale bars: C-200 μm; O-50 μm; R-10 μm.
Figure 8—figure supplement 6
OAβ2R-40XV5 expression in the Drosophila male reproductive system.
(A–C) Junction of seminal vesicle (SV) and ejaculatory duct (ED). (A) OAβ2R-40XV5; (B) Syn; (C) OAβ2R-40XV5, Syn overlay. (D–F) Cross section of ED. (D) OAβ2R-40XV5; (E) Syn; (F) OAβ2R-40XV5, Syn overlay. (G–I) Junction of SV and ED. (G) OAβ2R-40XV5; (H) Syn; (I) OAβ2R-40XV5, Syn overlay. (J–L) Distal SV. (J) OAβ2R-40XV5; (E) Syn; (F) OAβ2R-40XV5, Syn overlay. (M–O) Cross section of distal SV. (M) OAβ2R-40XV5; (N) Syn; (O) OAβ2R-40XV5, Syn overlay. Scale bars: C, I-50 μm; O-10 μm.
Figure 8—figure supplement 7
OAβ2R-40XV5 expression in the Drosophila male reproductive system.
(A–C) Distal anterior ejaculatory duct (ED). (A) OAβ2R-40XV5; (B) Syn; (C) OAβ2R-40XV5, Syn overlay. (D–F) Cross section of distal anterior ED. (D) OAβ2R-40XV5; (E) Syn; (F) OAβ2R-40XV5, Syn overlay. (G–L) AG. (G, J) OAβ2R-40XV5; (H, K) Syn; (I, L) OAβ2R-40XV5, Syn overlay. (M–O) Ejaculatory bulb. (M, O) OAβ2R-40XV5; (N, Q) Syn; (O, R) OAβ2R-40XV5, Syn overlay. Scale bars: O-50 μm; R-10 μm.
Figure 8—figure supplement 8
5-HT7-20XV5 expression in the Drosophila male reproductive system.
(A–C) Complete male reproductive system. (A) 5-HT7-20XV5; (B) Syn; (C) 5-HT7-20XV5, Syn overlay. (D–I) SV. (D, G) 5-HT7-20XV5; (E, H) Syn; (F, I) 5-HT7-20XV5, Syn overlay. (J–O) ED. (J, M) 5-HT7-20XV5; (K, N) Syn; (L, O) 5-HT7-20XV5, Syn overlay. Scale bars: C-200 μm; L-50 μm; O-10 μm.
Figure 8—figure supplement 9
5-HT7-20XV5 expression in the Drosophila male reproductive system.
(A–I) Seminal vesicle (SV). (A, D, G) 5-HT7-20XV5; (B, E, H) Syn; (C, F, I) 5-HT7-20XV5, Syn overlay. (J–L) Cross section of SV. (J) 5-HT7-20XV5; (K) Syn; (L) 5-HT7-20XV5, Syn overlay. (M–O) SV terminus. (M) 5-HT7-20XV5; (N) Syn; (O) 5-HT7-20XV5, Syn overlay. (P–R) SV/ED junction. (P) 5-HT7-20XV5; (Q) Syn; (R) 5-HT7-20XV5, Syn overlay. Scale bars: C, G-50 μm; F, O-10 μm; R-25 μm.
Figure 8—figure supplement 10
5-HT7-20XV5 expression in the Drosophila male reproductive system.
(A–C) Accessory gland (AG). (A) 5-HT7-20XV5; (B) Syn; (C) 5-HT7-20XV5, Syn overlay. (D–F) Epithelial layer of AG. (D) 5-HT7-20XV5; (E) Syn; (F) 5-HT7-20XV5, Syn overlay. (G–I) Cross-section of epithelial layer of AG. (G) 5-HT7-20XV5; (H) Syn; (I) 5-HT7-20XV5, Syn overlay. (J–L) Muscle layer of AG. (J) 5-HT7-20XV5; (K) Syn; (L) 5-HT7-20XV5, Syn overlay. (M–O) Ejaculatory bulb. (M) 5-HT7-20XV5.; (N) Syn; (O) 5-HT7-20XV5, Syn overlay. Scale bars: C, O-50 μm; L-10 μm.
Figure 9 with 3 supplements
Low-resolution images of nuclear expression of VT019028-GAL4-DBD in combination with TRH-AD and AbdB-AD in Drosophila male and female adult nervous system relative to 5-HT and Tdc2.
(A–D) nuclear expression using HIS2A-GFP marker. (A) male VT019028-GAL4-DBD/TRH-AD; (B) female VT019028-GAL4-DBD/TRH-AD; (C) male VT019028-GAL4-DBD/AbdB-AD; (B) female VT019028-GAL4-DBD/AbdB-AD. (E–H) 5-HT expression. (E) male VT019028-GAL4-DBD/TRH-AD; (F) female VT019028-GAL4-DBD/TRH-AD; (G) male VT019028-GAL4-DBD/AbdB-AD; (H) female VT019028-GAL4-DBD/AbdB-AD. Tdc2 expression. (I) male VT019028-GAL4-DBD/TRH-AD; (J) female VT019028-GAL4-DBD/TRH-AD; (K) male VT019028-GAL4-DBD/AbdB-AD; (L) female VT019028-GAL4-DBD/AbdB-AD. Scale bar: 200 μm.
Figure 9—figure supplement 1
High-resolution images of nuclear expression of VT019028-GAL4-DBD in combination with TRH-AD in posterior ventral nerve cord of Drosophila male adult nervous system relative to 5-HT and Tdc2.
(A–E) Dorsal view of complete stack of confocal images. (A) His2A-GFP; (B) 5-HT; (C) Tdc2; (D) His2A-GFP, 5-HT overlay; (E) His2A-GFP, Tdc2 overlay. (F) Ventral view of His2A-GFP/5-HT overlay of subset of slices containing the dorsal cluster of 5-HT neurons; (G–K) Dorsal view of subset of slices containing the dorsal cluster of 5-HT neurons. (G) His2A-GFP; (H) 5-HT; (I) Tdc2; (J) His2A-GFP, 5-HT overlay; (K) His2A-GFP, Tdc2 overlay. (L–P) Dorsal view of subset of slices containing the ventral cluster of 5-HT neurons. (L) His2A-GFP; (M) 5-HT; (N) Tdc2; (O) His2A-GFP, 5-HT overlay; (P) His2A-GFP, Tdc2 overlay. Scale bar: 50 μm.
Figure 9—figure supplement 2
High-resolution images of nuclear expression of VT019028-GAL4-DBD in combination with TRH-AD in posterior ventral nerve cord of Drosophila male adult nervous system relative to 5-HT and Tdc2.
(A–E) Dorsal view of complete stack of confocal images. (A) His2A-GFP; (B) 5-HT; (C) Tdc2; (D) His2A-GFP, 5-HT overlay; (E) His2A-GFP, Tdc2 overlay. (F) Ventral view of His2A-GFP/5-HT overlay of subset of slices containing the dorsal cluster of 5-HT neurons; (G–K) Dorsal view of subset of slices containing the dorsal cluster of 5-HT neurons. (G) His2A-GFP; (H) 5-HT; (I) Tdc2; (J) His2A-GFP, 5-HT overlay; (K) His2A-GFP, Tdc2 overlay. (L–P) Dorsal view of subset of slices containing the ventral cluster of 5-HT neurons. (L) His2A-GFP; (M) 5-HT; (N) Tdc2; (O) His2A-GFP, 5-HT overlay; (P) His2A-GFP, Tdc2 overlay. Scale bar: 50 μm.
Figure 9—figure supplement 3
High-resolution images of nuclear expression of VT019028-GAL4-DBD in combination with AbdB-AD in posterior ventral nerve cord of Drosophila male adult nervous system relative to 5-HT and Tdc2.
(A–E) Dorsal view of complete stack of confocal images. (A) His2A-GFP; (B) 5-HT; (C) Tdc2; (D) His2A-GFP, 5-HT overlay; (E) His2A-GFP, Tdc2 overlay. (F–J) Dorsal view of subset of slices containing the dorsal cluster of 5-HT neurons. (F) His2A-GFP; (G) 5-HT; (H) Tdc2; (I) His2A-GFP, 5-HT overlay; (J) His2A-GFP, Tdc2 overlay. (K–O) Dorsal view of subset of slices containing the ventral cluster of 5-HT neurons. (K) His2A-GFP; (L) 5-HT; (M) Tdc2; (N) His2A-GFP, 5-HT overlay; (O) His2A-GFP, Tdc2 overlay. Scale bar: 50 μm.
Figure 10 with 3 supplements
Comparison of mating duration between genotypes.
Bar plots show the mean ± SD for each group with individual data points overlaid on each bar. Statistical comparisons were made using unpaired two-tailed t tests where appropriate. Asterisks above indicate significance levels: p<0.001 (**), p<0.0001 (***), ns = not significant. (A) Columns from left to right: TRH ∩ AbdB >BONT-C: n=10, mean = 19.11, SD = 4.251. Tdc2 ∩ AbdB >BONT-C: n=14, mean = 21.80, SD = 3.155. AbdB ∩ VT019028>BONT-C: n=12, mean = 24.75, SD = 4.247. TRH ∩ VT019028>BONT-C: n=11, mean = 28.74, SD = 2.155. AbdB; BONT-C: n=8, mean = 27.07, SD = 4.907. BONT-C: n=7, mean = 28.75, SD = 4.809. WT Canton-S: n=9, mean = 26.91, SD = 3.473.
-
Figure 10—source data 1
Mating duration data of control and experimental genotypes.
- https://cdn.elifesciences.org/articles/108225/elife-108225-fig10-data1-v1.xlsx
Figure 10—figure supplement 1
Prot-GFP fluorescent marker in male and female reproductive systems in control and TRH-AD/VT019028-GAL4-DBD neuron-silenced flies.
(A) Control. (B) TRH-AD/VT019028-GAL4-DBD neurons silenced with BONT-C. Matings were interrupted 10 min after initiation. No Prot-GFP sperm is observed in the ejaculatory duct of the experimental male. (C and D) Wild-type Canton-S female reproductive systems from completed matings to a (C) control male; and (D) TRH-AD/VT019028-GAL4-DBD male whose neurons were silenced with BONT-C. Abundant Prot-GFP sperm are observed in the mating to the control male (arrow), while no Prot-GFP sperm is observed in the female reproductive system after mating to the experimental male (arrow). Prot-GFP-Protamine GFP. SV-SV, ED-ED. Scale bars: A and B-75 μm; C and D-200 μm.
Figure 10—figure supplement 2
Comparison of male Drosophila fecundity between genotypes.
Bar plots show the mean ± SD for each group with individual data points overlaid on each bar. Figure 10—figure supplement 1A males were aged 5 days before copulation. Figure 10—figure supplement 1B males were aged 30 days before copulation. (A) Columns from left to right: TRH ∩ VT019028 → vGlut-: n=34, mean = 173.2, SD = 24.22. AbdB ∩ TRH, Tdc2 → vGlut-: n=20, mean = 175.3, SD = 22.92. TRH ∩ VT019028: n=32, mean = 149.0, SD = 26.10. TRH: n=28, mean = 172.8, SD = 21.11. VT019028: n=25, mean = 148.6, SD = 20.81. (B) TRH ∩ VT019028 → vGlut-: n=14, mean = 133.9, SD = 37.64. TRH ∩ VT019028: n=19, mean = 123.7, SD = 25.02. TRH: n=13, mean = 106.3, SD = 41.93. VT019028: n=9, mean = 131.0, SD = 38.88.
-
Figure 10—figure supplement 2—source data 1
Five day old male fertility data of control and experimental genotypes.
- https://cdn.elifesciences.org/articles/108225/elife-108225-fig10-figsupp2-data1-v1.xlsx
-
Figure 10—figure supplement 2—source data 2
30 day old male fertility data of control and experimental genotypes.
- https://cdn.elifesciences.org/articles/108225/elife-108225-fig10-figsupp2-data2-v1.xlsx
Figure 10—figure supplement 3
vGlut expression in accessory glands (AGs) of control and vGlut conditional mutant.
(A–C) control male. (A) vGlut; (B) 6XmCherry; (C) overlay. (D–F) experimental male conditionally silenced for vGlut in TRH-AD/VT019028-GAL4 neurons. Scale bar: 50 μm.
Videos
Video 1
Male reproductive system of a ProtB-GFP control.
Sperm with green fluorescent nuclei are visible in the ejaculatory duct of a male subjected to a mid-mating interruption.
Video 2
Male reproductive system of a ProtB-GFP experimental male in which a subset of the TRH-AD/VT019028-GAL4-DBD subset of serotonergic neurons innervating the male reproductive system have been silenced.
Sperm with green fluorescent nuclei are restricted to the seminal vesicle and never enter the ejaculatory duct from a male subjected to a mid-mating interruption.
Video 3
GCAMP8m Ca++ imaging of an ejaculatory duct experiencing spontaneous peristaltic waves of muscle contractions.
Video 4
GCAMP8m Ca++ imaging of the seminal vesicle (SV)/ejaculatory duct (ED) junction showing the initiation site of the spontaneous peristaltic waves.
Video 5
GCAMP8m Ca++ imaging of the seminal vesicle (SV)/ejaculatory duct (ED) junction showing the subtle variation in the initiation site of spontaneous peristaltic waves as compared to Video 4.
Video 6
GCAMP8m Ca++ imaging of the seminal vesicle (SV) showing spontaneous uncoordinated muscle activity.
Video 7
GCAMP8m Ca++ imaging of an accessory gland (AG) showing spontaneous uncoordinated muscle activity.
Video 8
GCAMP8m Ca++ imaging of a testes showing spontaneous uncoordinated muscle activity.
Video 9
GCAMP8m Ca++ imaging of the accessory gland (AG) showing strong coordinated muscle activity.
Tables
Table 1
Male and female fertility phenotypes.
| Genotype | Male fertile | Female fertile |
|---|---|---|
| yw; TRH-GAL4DBD/AbdB-AD, UAS-BONT-C | No | No |
| yw; Tdc2-GAL4DBD/AbdB-AD, UAS-BONT-C | Yes | No |
| yw; VT019028-GAL4-DBD/AbdB-AD, UAS-BONT-C | No | Yes |
| yw; TRH-AD; UAS-BONT-C/VT019028-GAL4-DBD | No | Yes |
| yw; AbdB-AD/UAS-BONT-C | Yes | Yes |
| yw; UAS-BONT-C | Yes | Yes |
| Canton-S wild-type | Yes | Yes |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | Tdc2-GAL4 | Cole et al., 2005 | RRID:BDSC_9313 | |
| Genetic reagent (Drosophila melanogaster) | GluRIIA-GAL4 | Deng et al., 2019 | RRID:BDSC_84637 | |
| Genetic reagent (Drosophila melanogaster) | GluRIID-GAL4 | Deng et al., 2019 | RRID:BDSC_84638 | |
| Genetic reagent (Drosophila melanogaster) | TRH-GAL4 | Deng et al., 2019 | RRID:BDSC_84694 | |
| Genetic reagent (Drosophila melanogaster) | vGlut-GAL4 | Diao et al., 2015 | RRID:BDSC_60312 | |
| Genetic reagent (Drosophila melanogaster) | vGlut-GAL4-DBD | Diao et al., 2015 | ||
| Genetic reagent (Drosophila melanogaster) | Tdc2-AD | Dionne et al., 2018 | : RRID:BDSC_601902 | |
| Genetic reagent (Drosophila melanogaster) | TRH-GAL4-DBD | Aso et al., 2014 | RRID:BDSC_70371 | |
| Genetic reagent (Drosophila melanogaster) | TRH-AD | Aso et al., 2014 | RRID:BDSC_70975 | |
| Genetic reagent (Drosophila melanogaster) | GluRIIB-GAL4 | Diao et al., 2015 | RRID:BDSC_60333 | |
| Genetic reagent (Drosophila melanogaster) | GluRIIE-GAL4 | Diao et al., 2015 | RRID:BDSC_60332 | |
| Genetic reagent (Drosophila melanogaster) | Oct-TyrR-GAL4 | Lee et al., 2018 | RRID:BDSC_77735 | |
| Genetic reagent (Drosophila melanogaster) | GluRIIC-GAL4 | Lee et al., 2018 | RRID:BDSC_83268 | |
| Genetic reagent (Drosophila melanogaster) | GluRIIA-GFP | Beckers et al., 2024 | RRID:BDSC_99517 | |
| Genetic reagent (Drosophila melanogaster) | 5HT1A-GAL4 | Deng et al., 2019 | RRID:BDSC_84588 | |
| Genetic reagent (Drosophila melanogaster) | 5HT1B-GAL4 | Deng et al., 2019 | RRID:BDSC_84589 | |
| Genetic reagent (Drosophila melanogaster) | 5-HT2A-GAL4 | Deng et al., 2019 | RRID:BDSC_84590 | |
| Genetic reagent (Drosophila melanogaster) | 5-HT2B-GAL4 | Deng et al., 2019 | RRID:BDSC_84591 | |
| Genetic reagent (Drosophila melanogaster) | 5-HT7-GAL4 | Deng et al., 2019 | RRID:BDSC_84592 | |
| Genetic reagent (Drosophila melanogaster) | TβH-GFP | Beckers et al., 2024 | RRID:BDSC_80113 | |
| Genetic reagent (Drosophila melanogaster) | fru-GAL4 | Donor: Barry Dickson, Howard Hughes Medical Institute, Janelia Research Campus | RRID:BDSC_66696 | |
| Genetic reagent (Drosophila melanogaster) | UAS-CD8-mCherry | Donor: Frank Schnorrer, Max Planck Institute of Biochemistry | RRID:BDSC_27392 | |
| Genetic reagent (Drosophila melanogaster) | ProtB-GFP | Manier et al., 2010 | RRID:BDSC_58406 | |
| Genetic reagent (Drosophila melanogaster) | AbdB-AD | Diao et al., 2024 | ||
| Genetic reagent (Drosophila melanogaster) | vGlut-AD | Gao et al., 2008 | ||
| Genetic reagent (Drosophila melanogaster) | IA2-GFP | Yu et al., 2025 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-BONT-C | Han et al., 2022 | ||
| Genetic reagent (Drosophila melanogaster) | dsx-GAL4-DBD | Shirangi et al., 2016 | ||
| Genetic reagent (Drosophila melanogaster) | vGlut-40XV5 | Stowers, 2025 | germline excision of STOP cassette. | |
| Genetic reagent (Drosophila melanogaster) | vGlut-40XMYC | Stowers, 2025 | germline excision of STOP cassette. | |
| Genetic reagent (Drosophila melanogaster) | B2RT-7XMYC-vAChT | Tison et al., 2020 | germline excision of STOP cassette. | |
| Genetic reagent (Drosophila melanogaster) | RSRT-9XV5-vGAT | Certel et al., 2022 | germline excision of STOP cassette. | |
| Genetic reagent (Drosophila melanogaster) | OAMB-GAL4 | McKinney et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | OAα2 R-GAL4 | McKinney et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | OAβ1 R-GAL4 | McKinney et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | OAβ2 R-GAL4 | McKinney et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | OAβ3 R-GAL4 | McKinney et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | FRT-F3-OAMB-10XV5 | Bakshinska et al., 2025 | ||
| Genetic reagent (Drosophila melanogaster) | B3RT-Tdc2-LexA | Sherer et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | B3RT-vGlut-LexA | Sherer et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | RSRT-STOP-RSRT-6XV5-vMAT | Sherer et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | UAS-B3 | Sherer et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | 20XUAS-R | Sherer et al., 2020 | ||
| Genetic reagent (Drosophila melanogaster) | 20XUAS-B2 | Williams et al., 2019 | ||
| Genetic reagent (Drosophila melanogaster) | 13XLexAop-6XmCherry | Shearin et al., 2014 | ||
| Genetic reagent (Drosophila melanogaster) | Tdc2-GAL4-DBD | this study | Octopamine split-GAL4-DBD, JK65C landing site | |
| Genetic reagent (Drosophila melanogaster) | B3RT-Tdc2-LexA germline excision | this study | Tdc2-LexA driver | |
| Genetic reagent (Drosophila melanogaster) | B3RT-vGlut-LexA germline excision | this study | vGlut-LexA driver | |
| Genetic reagent (Drosophila melanogaster) | RSRT-6XV5-vMAT germline excision | this study | constitutively expressed 6XV5-vMAT | |
| Genetic reagent (Drosophila melanogaster) | FRT-F3-OAα2R-20XV5 | this study | conditionally expressible OAα2R-20XV5 | |
| Genetic reagent (Drosophila melanogaster) | FRT-F3-OAα2R-20XV5 germline inversion | this study | constituvely expressed OAα2R-20XV5 | |
| Genetic reagent (Drosophila melanogaster) | B2RT-STOP-B2RT-OAβ2R-40XV5 | this study | conditionally expressible OAα2R-40XV5 | |
| Genetic reagent (Drosophila melanogaster) | B2RT-OAβ2R-40XV5 germline excision | this study | constitutively expressed OAβ2R-40XV5 | |
| Genetic reagent (Drosophila melanogaster) | 5-HT7-20XV5 | this study | constitutively expressed 5-HT7-20XV5 | |
| Genetic reagent (Drosophila melanogaster) | MHC-GCAMP8m | this study | Muscle-specific expression of GCAMP8m, VK00027 landing site | |
| Antibody | SYN 3 C11 supernatant | (DSHB Cat# 3 C11, RRID:AB_528479) | Mouse monoclonal | Dilution 1:20 |
| Antibody | nc82 (Brp) supernatant | (DSHB Cat# nc82, RRID:AB_231486) | Mouse monoclonal | Dilution 1:40 |
| Antibody | Dlg 4 F3 supernatant | (DSHB Cat# 4 F3 anti-discs large, RRID:AB_528203) | Mouse monoclonal | Dilution 1:40 |
| Antibody | Mouse anti-V5 | Novus NBP2-52703 | Mouse monoclonal | Dilution 1:200 |
| Antibody | Rabbit anti-V5 | Novus NBP2-52653 | Rabbit monoclonal | Dilution 1:200 |
| Antibody | Rat anti-V5 | Novus NBP2-81037 | Rat monoclonal | Dilution 1:200 |
| Antibody | Human anti-V5 | Novus NBP2-81035 | Human monoclonal | Dilution 1:200 |
| Antibody | Rat anti-MYC 9E10 | Novus NBP2-81020 | Rat monoclonal | Dilution 1:200 |
| Antibody | Rabbit anti-MYC 9E10 | Novus NBP2-52636 | Rabbit monoclonal | Dilution 1:200 |
| Antibody | Mouse anti-MYC 4 A6 | Millipore Sigma 05–724 | Mouse monoclonal | Dilution 1:200 |
| Antibody | Mouse anti-vGlut | Banerjee et al., 2021 | Mouse monoclonal | Dilution 1:10 |
| Antibody | Mouse anti-GFP 3E6 | Thermo-Fisher A11120 | Mouse monoclonal | Dilution 1:200 |
| Antibody | Rabbit anti-GFP | Thermo-Fisher G10362 | Rabbit monoclonal | Dilution 1:200 |
| Antibody | Rat anti-mCherry 16D7 | ThermoFisher M11217 | Rat monoclonal | Dilution 1:200 |
| Antibody | Rabbit anti-Tdc2 | pab0822-P, Covalab | Rabbit polyclonal | Dilution 1:500 |
| Antibody | Rat anti-5-HT YC5 | Abcam ab6336 | Rat monoclonal | Dilution 1:300 |
| Antibody | Goat anti-mouse Alexa 405 | Thermo-Fisher A31553 | Goat polyclonal | Dilution 1:400 |
| Antibody | Goat anti-mouse Alexa 488 | Jackson Immunoresearch 115-545-166 | Goat polyclonal | Dilution 1:400 |
| Antibody | Donkey anti-rat Alexa 488 | Jackson Immunoresearch 712-546-153 | Donkey polyclonal | Dilution 1:400 |
| Antibody | Goat anti-rabbit Alexa 488 | Thermo-Fisher A32731 | Goat polyclonal | Dilution 1:400 |
| Antibody | Goat anti-mouse Alexa 568 | Thermo Fisher A11031 | Goat polyclonal | Dilution 1:400 |
| Antibody | Goat anti-rabbit Alexa 568 | Thermo Fisher A11036 | Goat polyclonal | Dilution 1:400 |
| Antibody | Donkey anti-rat Alexa 568 | Thermo-Fisher A78946 | Donkey polyclonal | Dilution 1:400 |
| Antibody | Goat anti-mouse Alexa 647 | Thermo-Fisher A32728 | Goat polyclonal | Dilution 1:400 |
| Antibody | Goat anti-rabbit Alexa 647 | Thermo-Fisher A32733 | Goat polyclonal | Dilution 1:400 |
| Antibody | Goat anti-rat Alexa 647 | Thermo-Fisher A48265 | Goat polyclonal | Dilution 1:400 |
| Antibody | Goat anti-mouse ATTO 647 N | Rockland 610-156-121 | Goat polyclonal | Dilution 1:100 |
| Antibody | Goat anti-rabbit ATTO 647 N | Rockland 611-156-122 | Goat polyclonal | Dilution 1:100 |
| Antibody | Goat anti-rat ATTO 647 N | Rockland 612-156-120 | Goat polyclonal | Dilution 1:100 |
| Antibody | Donkey anti-human Alexa 790 | Jackson Immunoresearch 709-655-149 | Donkey polyclonal | Dilution 1:400 |
| other | Stain, Phalloidin iFluor 405 | Abcam AB176752 | Dilution 1:800 | |
| Chemical compound, drug | Triton X-100 | Sigma-Aldrich T8787 | X-100 | |
| Chemical compound, drug | Bovine serum albumin | Lampire-cat#–7500804 | ||
| Chemical compound, drug | Acryloyl-X SE | Thermo Fisher A20770 | ||
| Chemical compound, drug | Ammonium persulfate | Sigma-Aldrich 215589 | ||
| Chemical compound, drug | Acrylamide 40% solution | Sigma-Aldrich 01697 | ||
| Chemical compound, drug | N,N'-Methylenebisacrylamide (bis) 2% solution | Sigma-Aldrich M1533 | ||
| Chemical compound, drug | DAPI | Thermo Fisher D1306 | 2μg/ml | |
| Chemical compound, drug | Proteinase K | New England Biolabs P81075 | Dilution 1:20 | |
| Software, algorithm | Image J | Schindelin et al., 2012 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108225/elife-108225-mdarchecklist1-v1.docx
-
Supplementary file 1
Donor Plasmid Sequences.
- https://cdn.elifesciences.org/articles/108225/elife-108225-supp1-v1.zip
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Two classes of amine/glutamate multi-transmitter neurons innervate Drosophila internal male reproductive organs
eLife 14:RP108225.
https://doi.org/10.7554/eLife.108225.3
