Supplemental Information Ascending SAG Neurons Control

Neuron, Volume 83
Supplemental Information
Ascending SAG Neurons Control
Sexual Receptivity of Drosophila Females
Kai Feng, Mark T. Palfreyman, Martin Häsemeyer, Aaron Talsma, and Barry J. Dickson
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Supplemental Figure S1 (Related to Figure 1). Only VT3280 Targets the SPSNs.
(A) Double labeling of VT3280-GAL4 (red) and FruP1.LexA (green) in the uterus. The
structure of uterus is visualized by phalloidin staining (blue).
(A' and A") Higher magnification views of the region indicated by dashed (A') and
solid (A") boxes in (A) for the red channel.
(B) Abdominal ganglion of VT3280-GAL4 UAS-mCD8-GFP female stained with antiGFP (green) and nc82 (magenta).
(C) Receptivity and remating within 1 hour. *** P < 0.0001, Fisher’s exact test.
(D) Eggs laid per female within 48 hours, shown as mean ± SEM, *** P < 0.0001,
Student’s t-test.
(E) Reproductive tracts of females carrying the indicated GAL4 and UAS-mCD8GFP, stained wtih anti-GFP (green) and phalloidin (blue). None of the five lines
appear to label SPSNs, although some epithelial-like cells are labeled by VT50405GAL4 and VT45154-GAL4 and innervations in the oviduct from the abdominal nerve
are labeled by VT7068-GAL4.
(F) Receptivity and remating within 1 hour for GAL4 UAS-SPR-IR females. P > 0.05
for every line compared to the no-GAL4 control, Fisher’s exact test.
Scale bars: (A), (A'), and (E), 100 µm; others, 10 µm.
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Supplemental Figure S2 (Related to Figure 3). Intersectional Strategies to Target
SAG Neurons
(A) Receptivity of virgin females within 30 minutes, each carrying the indicated splitGAL4 combination and one copy of a UAS-EGFP-Kir2.1 transgene. One copy of
UAS-Kir2.1 is sufficient to completely abolish the receptivity with SAG-1 but has
only a mild effect with SAG-2.
(B) Receptivity of virgin females within 1 hour, each carrying the indicated splitGAL4 combination and one copy of a UAS-shibirets transgene. *** P < 0.0001; n.s.
P > 0.05, Fisher’s exact test.
(C) Intensity of fluorescence in individual SAG cells labeled by the SAG-1 and SAG2. Tissues were stained with antibody against tdTomato and imaged from the ventral
side under identical conditions (resulting in lower light penetration to dorsally located
cells). ** P < 0.01; * P < 0.05, Student’s t-test.
(D) Receptivity and remating within 30 minutes for females carrying the indicated
GAL4 or split-GAL4 driver and UAS-NaChBac. *** P < 0.001; ** P < 0.01; * P <
0.05; n.s. P > 0.05, for remating rates compared with the no-GAL4 control, Fisher’s
exact test with Bonferroni correction.
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Supplemental Figure S3 (Related to Figure 4). Characterization of SAG and
LexA Reagents for SPSN and SAG.
(A-C and A’-C’) Ventral (A-C) and lateral (A’-C’) views of abdominal ganglia of
SAG-2 UAS-mcD8-tdTomato females stained with anti-DsRed (red) and nc82 (blue),
showing variable location of SAG soma (arrowheads). With SAG-1 we observed one
sample with 2 dorsal SAGs, 12 with one dorsal and one ventral SAG, and 13 with two
ventral SAGs, as well as one sample with only a single dorsally-located SAG. With
SAG-2, the corresponding numbers were 3, 8, 9, and 1, respectively.
(D and E) Abdominal ganglia of female (D) and male (E) SAG-2-GAL4 UAS-mCD8tdTomato females stained with anti-DsRed (red) and anti-Dsx (green). Insets show
higher magnification views of two SAGs from a female sample.
(F and G) Abdominal ganglia of fruP1.lexA lexAop-CD2-GFP SAG-2-GAL4 UASmCD8-tdTomato female, stained with anti-GFP (green) and anti-DsRed (red).
(H) Receptivity of virgin females within 1 hour. *** P < 0.0001, Fisher’s exact test.
(I) Eggs laid per female within 48 hours, shown as mean ± SEM. *** P < 0.0001,
Student’s t-test.
Scale bars: 10 µm.
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Supplemental Figure S4 (Related to Figure 6). SAG Responses Triggered by
Stimulation of Attenuated Light Dose on the Soma of the SPSNs.
(A) Representative traces from virgins, females mated to wild type males (SP+) and
females mated to sex peptide null males (SP0) from the same animals in Figure 6A.
The full 1-second response (from black line onwards) is shown.
(B) Triggered EPSPs during 1s stimulation at varying light power.
(C) Triggered APs during the first 100ms of 1s stimulation at varying light power.
(D) Delay to first response at 0.79mW/mm2. Each point represents one of the two
repeats of an individual animal.
(E) Receptivity scored as copulation with wild-type males within 1 hr. *** P <
0.0001; ** P < 0.01, compared with virgin, Fisher’s exact test.
(F) EPSPs triggered for the light doses for virgin (grouped data) and selected SP0–
mated rejectors (individual animals).
Horizontal bars in (D) indicate the mean. Data in (B), (C), and (F) are plotted as mean
± SEM. n=8 virgin, n=10 SP+, n=10 SP0 and n=4 SP0 rejectors.
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Supplemental Table S1. Stochastic Labeling and Silencing.
Each column represents an individual fly, each row a distinct cell cluster. Data are the
number of cells labeled in each cluster, expressed as a fraction of the maximum
number of cells in the cluster. Orange color indicates neuronal clusters with at least
80% of the maximum number of cells labeled, which was taken as a threshold for
assessing correlation with receptivity.
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Supplemental Movie S1 (Related to Figure 2). SAG Morphology in the VNC.
3D reconstruction of confocal stacks showing the morphology of a single SAG clone
(as in Figure 2E) in the VNC.
Supplemental Movie S2 (Related to Figure 2). SAG Morphology in the Brain.
3D reconstruction of confocal stacks showing the morphology of a single SAG clone
(as in Figure 2D) in the brain, from the same animal as Movie S1.
Supplemental Movie S3 (Related to Figure 4). Close Apposition of SPSN and
SAG Neurons.
3D reconstruction of confocal stacks showing overlap of arborizations between SAG
and SPSN in the abdominal ganglion. Anti-GFP staining of myr-GFP driven by
VT3280-lexA3 is in green; Anti-DsRed staining of mCD8-tdTomato driven by SAG-2
is in red.
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