100 mm Sucrose. +Berberine +Quinine

Transcription

1 8 mm Sucrose Probability (%) Wild-type Gr32a / 2 +Caffeine +Berberine +Quinine +Denatonium Supplementary Figure 1: Detection of sucrose and bitter compounds is not affected in Gr32a / flies. Proboscis Extension Response (PER) of Gr32a / to mm sucrose (first graph) and sucrose containing various bitter compounds (graph 2 to 5) is the same as in wild type flies. Probability of Extension represents the number of times flies from a given strain extended their proboscis when presented with a tastant, divided by the total number of times that the tastant was presented. Each graph represents data from at least four experiments. Error bars represent SEM (3-11 flies per experiment, 2-5 flies total for each strain and tastant tested). No differences (p<.5) were detected between the strains using the Student s t-test. Caffeine and quinine was present at mm, berberine at mm and Denatonium at 1 mm.

2 a Courtship to male b Female receptivity Courtship index Gr32a 5.9 ** * * * +/+ +/ / / / / #1 #2 #1/#2 Non-maters (%) 15 5 Gr32a +/+ / Copulation latency (min) 15 5 Gr32a +/+ / c 6 Courtship to female Courtship index * * * W: DTI Gr32a[1]: W Gr32a[1]: DTI Gr32a[2]: DTI Gr66a[1]: DTI Gr66a[2]: DTI Gr22e[1]: DTI Gr22e[2]: DTI d 6 Courtship to female 5 Courtship to male 5 Courtship index * * Courtship index Gr32a / / / / / / GAL4 + + GAL4VP UAS-Gr32a Gr32a / / / / / / GAL4 + + GAL4VP UAS-Gr32a + + +

3 Supplementary Figure 2: Courtship analyses of various Gr32a mutant genotypes. (a) Courtship index of males towards decapitated males. Experiments were carried out as described in Figure 2a. Gr32a +/ mutant males and Gr32a / males with either one of the two Gr32a_5.9 transgenes show intermediate male-directed courtship phenotype. (b) Gr32a / females mate with wild type males as efficiently as wild type females in one on one mating assays. A single male was provided with a single virgin female in a courtship chamber (n=2). No statistically significant difference in fraction of non-maters (left panel) and latency time (time to copulation; right panel) was observed between wild type and Gr32a / (p>.5; chi-square test for non-maters, ANOVA for copulation latency). (c) Courtship index of males lacking various Gr expressing neurons towards decapitated females. Experiments were carried out as described in Figure 3a. (n>3 for each genotype tested). All males expressing DTI show the same level of courtship as control males (containing only the UAS:DTI transgene). The Gr32a driver line exhibits slightly higher courtship than the other strains. (d) Courtship index of males towards decapitated males or females. Experiments were carried out as described in Figure 2a. Male-male courtship is significantly decreased only if the tested male has both Gr32a- Gal4/Gr32a-Gal4VP16 and UAS-Gr32a cdna. Error bars represent SEM. Statistical significance was measured by ANOVA (* p<.5, ** p<.1).

4 a b c Male GAL4VP16 Whole (1-29 µm) Male Female GAL4 Surface (1-13 µm) Inside (14-29 µm) Supplementary Figure 3: Projection pattern of Gr32a-expressing neurons beyond the SOG. (a) Individual flies (2 males and 1 female) show slightly different axonal projection patterns and position of the termini, visualized in whole mount antibody staining of brains from Gr32a-GAL4VP16; UAS-mCD8::GFP flies using anti-gfp antibody (see also Supplementary Table 4). (b) Live GFP images of male tarsi expressing a UAS-mCD8::GFP reporter under the control of the Gr32a-Gal4VP16 or Gr32a-Gal4 driver, respectively. Because Gal4VP16 is much more potent and leads to higher expression of the reporter, the signal was adjusted to obtain similar visual images in the two panels. Note that both drivers are expressed in the same set of neurons. (c) Visualization of projections of Gr32a expressing neurons and FRUM expressing nuclei. In contrast to the axons of Gr32a neurons, almost all cell body of fruitless neurons are on or close to the surface of the brain, and no termini of Gr32a neuron contact FRUM-positive cell bodies.

5 Tapping Wing vibration Attempting Wild 7.9 ± ± ±.9 Gr32a / 6.4 ± ± 2.4 * 6.6 ± 1.7 * Gr32a / : 5.9 #1/#2 8.9 ± ± ±.4 Supplementary Table 1: Gr32a / males exhibit late courtship steps towards males (wing vibration and attempting) much more frequently than wild type and Gr32a rescued males. Individual courtship steps towards decapitated males were quantified. In contrast to Gr32a / males, wild type and Gr32a / males carrying rescue transgenes rarely extend courtship beyond tapping. Numbers represent how many times each courtship step was carried out during an experiment of five minutes. n>2. * p<.5 (ANOVA).

6 Gr32a:Gal4/UAS mcd8-gfp DTI +DTI labial palp 6.8 ±.9.6 ±.6 ** foreleg 3.9 ±.2.2 ±.1 ** Gr66a:Gal4/UAS mcd8-gfp DTI +DTI labial palp 16. ± ±.6 ** foreleg.9 ±.2. ±. * Supplementary Table 2: Diphtheria toxin efficiently ablates GRNs. Cell numbers were counted based on GFP expression in flies of the indicated genotypes in the absence or presence UAS-DTI. n=5-. * p<.5, ** p<.1 (ANOVA).

7 treatment Total (n) beyond SOG (n) intact foreleg ablation 4* mid+hindleg ablation 9 9 Supplementary Table 3: The axonal projection pattern of Gr32a-expressing neuron with and without foreleg. Ablations were performed between one to days after eclosion and flies were kept for another 8 to days in vials to allow effective deafferentiation to occur. Only the fourth and fifth tarsal segments were cut off (harboring all Gr32a-expressing cell bodies) to achieve the highest possible survival rate. Compared to intact flies, and flies lacking mid and hindlegs, foreleg-ablated flies showed a significant reduction of projections into the ventrolateral protocerebrum (* p<.5; chisquare test).

8 Gr32a neuron projection status Between VP and SOG Border of VP and AL Inside of VP Total Male Female Supplementary Table 4: Projection patterns of Gr32a-expressing neurons beyond the SOG. The end positions of Gr32a axon beyond the SOG were analyzed in detail. In general, the axons are shorter in male than in female and termini were more difficult to visualize in males. This is likely due to somewhat more robust CNS staining in females than males due to better antibody penetration in the former. Intensity of GFP expression in the cellbodies on the foreleg did not show any apparent difference between the sexes.

9 Supplementary Video: Movie of a mating competition experiment. A wild type and a Gr32a / male are let to compete for a virgin female. The mutant, but not the wild type, male performs occasionally courtship towards the male competitor. The female is easily discernable by the white eye. Both males have red eyes, but the mutant male was marked by a blue dot on thorax, which is difficult to see in the movie, but easily recognizable when directly viewed under a stereomicroscope. Methods: Movie was recorded by COLOUR C/CS MOUNT CAMERA.

10 Methods Gr32a targeting: Upstream (5 arm; ) and downstream (3 arm; ) regions of Gr32a were amplified by Elongase (Invitrogen) from Oregon R DNA. These fragments were cloned into BsiWI SpeI sites and NotI NheI sites, respectively, of CMC5 vector (a gift from C. M. Chen and G. Struhl). This vector contains two polylinkers flanking the mini white gene with a I SceI site, flanked by FRT sites and containing conventional P element repeats. This construct was designed to replace the 2/3 of Gr32a coding region with the eye color marker, white. Virgin female flies carrying targeting construct insertions on 3rd chromosome were crossed to w 1118 ;7FLP; 7I SceI, and 3 day old progeny were heat shocked at 38 C for 6 min. Approximately 8 white eyed adult virgin females were crossed to w 1118 ;7FLP males and progeny were heat shocked as described above. Of the 28 red eyed flies that were obtained, 6 were simple gene replacements. Genomic DNAs from wild type Ore R and Gr32a mutant flies were subjected to PCR amplification primers annealing to sequences within the Gr32a coding region ( ) and the 5 upstream arm ( ). We also confirmed the gene replacement with genomic Southern hybridization. Genomic DNA was digested with BglII or XmaI, fragments were separated on 1 % agarose gels by electrophoresis and transferred to nictorcellulose membranes (Amersham, Inc.), which were hybridized with probes derived from regions upstream of the 5 arm or downstream of the 3 arm (corresponding to nucleotides and , respectively). Transgenesis: The Gr32a_5.9 rescuing constructs contain genomic fragments from 3776 nt upstream of the Gr32a translation initiation codon to 585 nt downstream of the Gr32a translation termination codon. They were obtained by PCR, sequenced and cloned into the pcaspr 4 transformation vector to generate two independently derived transgenic lines (#1 and #2). For the generation of a Gr32a GAL4VP16 driver, a segment containing the 378 nt upstream of the Gr32a translation initiation codon was inserted into a transformation vector containing the GAL4VP16 coding sequence (a gift from Michael Green). Courtship assays: All mutant males, mutant males rescued with transgenes and DTI expressing males used in mating assays carried a w + containing X chromosome to

11 eliminate any effects of different eye pigmentation on mating performance (except for Gr32a[2] Gal4; UAS dti males). Wild type males (control) derived from a cross of Ore R females to w 1118 males. w 1118 males and females were used as mating targets. They were collected as virgins shortly after eclosion, kept separately for 2 5 (female) or 3 7 days (male), unless indicated otherwise. Male subjects were kept in isolation until used for mating behavior. All flies were kept at 25 C on 12:12 light:dark cycle. Copulation latency and non maters experiments were performed in a small Plexiglas mating chamber (4 3 mm). Copulation latency was measured from the time a single male was aspirated into the mating chamber until he successfully mounted the female. A male was considered a non mater if he did failed to copulate within 3 min. The courtship index (CI) is given as percentage of time a male performs vigorous courtship behaviors (wing vibration, licking, and attempting) during 3 seconds. Competition experiments were performed by placing two males in a chamber with a single female and scoring the male which successfully mated. One of the males was marked with a dot of blue ink on the thorax shortly after eclosion at the time of collection in order to distinguish between genotypes. The blue dot did not affect success rate (data not shown). Courtship experiments with fertilized females were carried out as follows: Females were mated and kept in food vials for 1 day. They were then decapitated and used in as described above. Females exposed to males were kept with fru 3 mutant males under heavily crowded conditions to facilitate hydrocarbon exchange between the male and females. females were placed with fru 3 mutant males in 21 mm diameter and a 3 mm high food vial for 1 day. fru 3 mutant males court females at very low intensity and cannot copulate. Immunostaining: UAS mcd8::gfp was driven by Gr32a Gal4VP16 to visualize axonal projections. Animals were aged for at least days before dissection. Antibody staining was conducted as described by Laissue et al. 15, except that 5% heat inactivated goat serum was added to the blocking solution (protocol provided by Leslie Vosshall). Primary antibodies were rabbit anti GFP (Molecular Probes; A 6455) and mouse anti nc82 as a neuropil marker. Secondly antibodies were goat anti rabbit

12 ALEXA 488 and goat anti mouse Cy3. Confocal Microscopy was performed on a Zeiss LSM. For leg ablations, both fore legs or all four mid/hindlegs were cut off above the third tarsal segment with a razor blade, removing all Gr32a expressing cell bodies, which are located on the fourth and fifth tarsal segments. The flies were let to recover for 8 to days, before their brain was dissected and used for immunostaining. Statistical analyses: Statistical analysis of behavioral assays was done using ANOVA. To determine statistically significant differences between genotypes for non maters and for statistically significant differences in established projection beyond the SOG in normal and leg ablated animals, we employed the chi square test. 15. Laissue, P.P., et al. Three-dimensional reconstruction of the antennal lobe in Drosophila melanogaster. J Comp Neurol 45, (1999).