DEV031377 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1-S8 -

• Supplemental Figure S1 -

  Fig. S1. Organization of the nerve fiber layer of OSNs. Adult antennal lobes stained with antibody against Futsch (22C10). Confocal sections at the nerve fiber layer are stacked to show controls (A) and Or83b-nulls (B), Or83b-Gal4>IMPTNT-V (C) and Or83b-Gal4>TNT-G (D). The imperfect (IMPTNT-V, C) and active (TNT-G, D) toxins were expressed conditionally using tub-Gal80^ts and rearing flies throughout development at 18°C and raising them to 29°C for 6 days PE. In controls (A,C), large axonal bundles can be seen (arrowhead) traversing the surface of the antennal lobe from the antennal nerve (asterisk) to the antennal commissure (AC). These are disorganized in experimental antennal lobes (B,D). Genotypes: (A) Or83b-Gal4, UAS mCD8::GFP; (B) Or83b-Gal4, UAS mCD8::GFP; Or83b⁻/Or83b⁻; (C) w; tubGal80^ts, tubGal80^ts/UAS IMPTNT-V; Or83b-Gal4, UAS 2XeGFP/+; (D) w; tubGal80^ts, tubGal80^ts/UAS-TNT-G; Or83b-Gal4, UAS-2XeGFP/+. Scale bar: 15 µm.

• Supplemental Figure S2 -

  Fig. S2. Neurodegeneration affects the axons of OSNs but not the soma in the antenna. (A-F) A 15 µm region of interest (ROI) (red dotted squares in A-E) was chosen to quantify the pixels occupied by the Or22a axons just proximal to entry into the DM2 glomerulus (encircled in B). Mean±s.e.m. is represented in F. A schematic of the right III antennal segment (Ar, Arista). (G-J) The region of the third segment of adult antenna in the diagram is shown. Cell bodies of the OSNs are visualised by GFP expression at 6 days PE. (K) Cell numbers (n=5 animals each) are plotted. Genotypes: (A-E) Or22a-Gal4, UAS-mCD8::GFP/UAS-Or83b; tub-Gal80^ts, Or83b⁻/tub-Gal80^ts, Or83b⁻; (G) w; Or22a-Gal4, UAS-mCD8::GFP/+; +/+; (H) w; Or22a-Gal4, UAS-mCD8::GFP/+; Or83b-/Or83b-; (I) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-IMPTNT-V; tubGal80^ts, tubGal80^ts/+; (J) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-TNT-G; tubGal80^ts, tubGal80^ts/+. Scale bar: 20 µm in J for G-J.

• Supplemental Figure S3 -

  Fig. S3. Rescue of the phenotype of Or83b-nulls by targeted expression of Or83b only until eclosion. (A-B′) Or22a neurons in Or83b-null animals examined 4 (A,B) or (A′,B′) 6 days PE. In A, Or83b was ectopically expressed during pupal stage but switched 'OFF' after eclosion. (C) The pixel intensity of the terminal arbors was estimated. Mean±s.e.m., n=5. The timeline beneath indicates the rearing conditions of animals. Genotypes: (A,A′) Or22a-Gal4, UAS-mCD8::GFP/UAS-Or83b; tub-Gal80^ts, Or83b⁻/tub-Gal80^ts, Or83b⁻; (B,B′) Or22a-Gal4, UAS-mCD8::GFP/+; tub-Gal80^ts, Or83b⁻/tub-Gal80^ts, Or83b⁻.

• Supplemental Figure S4 -

  Fig. S4. Effect of TNT-G expression on GFP intensity and glomerular volume. (A-D) Antennal lobes stained with anti-Bruchpilot (Brp) antibody. Neurons marked by Or47b-Gal4; UAS-mCD8::GFP are visualized by anti-GFP (green in A,C). (E,F) The intensity of GFP expression was estimated in regions of interests (ROIs) (green circles in F) within the VA1v glomerulus where IMPTNT-V (A) or TNT-G (C) was expressed (ROIs, as shown by blue arrowheads in A,C). Bar chart (E) shows the mean and s.e.m. of fluorescence intensities (n=5). (G,H) Relative volume of VA1v and VA6 glomeruli calculated as described in Materials and methods from the Bruchpilot antibody (nc82)-stained preparations (B,D; see H). Bars indicate the mean and s.e.m. of relative glomerular volume (VA1v/VA6; n=5). **P<0.005. Genotypes: (A,B) w; Or47b-Gal4(15.7), UAS mCD8::GFP/UAS IMPTNT-V; tubGal80^ts, tubGal80^ts/+; (C,D) w; Or47b-Gal4(15.7), UAS mCD8::GFP/UAS TNT-G; tubGal80^ts, tubGal80^ts/+.

• Supplemental Figure S5 -

  Fig. S5. High-magnification images of Or47b OSN MARCM clones. (A-C) Reconstructions (Amira 5.0) of 4-day-old OSNs induced by MARCM and visualized by mCD8::GFP expression. (B,C) OSNs expressing TNT-G. Preparations were examined at 4 days PE. (A′-C′) Magnifications of the terminals in the boxed regions from A-C. The fragmentation and beading is apparent in TNT-G-expressing axons (B′,C′). Genotypes: (A,A′) FRT19A/FRT19A, tubGal80^ts, hsFlp; Or47b-Gal4(15.7), UAS-mCD8::GFP/+; (B-C′) FRT19A/FRT19A, tubGal80^ts, hsFlp; Or47b-Gal4(15.7), UAS-mCD8::GFP/UAS-TNT-G. Scale bar: 20 µm in C for A-C.

• Supplemental Figure S6 -

  Fig. S6. Co-expression of p35 or Wlds does not rescue neurodegeneration induced by silencing neurons. (A-F) Or83b-Gal4 drives expression of GFP and TNT-G (A-C) or Kir₂.₁ (D-F). Appropriate confocal sections are shown to demonstrate degeneration in VC2, DC3 and VA6 (labeled in A). Baculovirus p35 (B,E) or Wlds (C,F) was co-expressed with TNT-G (B,C) or Kir₂.₁ (E,F). (G,H) Or83b⁻ animals with Or22a OSNs labeled with GFP. The control image from Fig. 5H is reproduced in G for ease of comparison. (H) Ectopic expression of UBP2 rescues the degeneration observed in controls at 6 days PE. (I) Mean±s.e.m. (n=5) of pixel intensity over the DM2 glomerulus demonstrates a rescue of degeneration (**P<0.005; see Table S4). (J-M) Animals expressing IMPTNT-V (J,K) or TNT-G (L,M) were stained with anti-Draper (red). Fluorescence intensity within the VAIv glomeruli was estimated in the ROIs (white rectangle in K,M). (N) Mean±s.e.m. (n=7) is plotted (**P<0.005; see Table S8). Timeline indicates the rearing conditions of animals in all experiments except G-I which were reared throughout at 25°C. Genotypes: (A) w; tubGal80^ts, tubGal80^ts/UAS TNT-G; Or83b-Gal4, UAS 2XeGFP/+; (B) w; tubGal80^ts, tubGal80^ts/UAS-TNT-G; Or83b-Gal4, UAS-2XeGFP/UAS-p35; (C) w; tubGal80^ts, tubGal80^ts/UAS-TNT-G; Or83b-Gal4, UAS-2XeGFP/UAS-Wlds; (D) w; tubGal80^ts, tubGal80^ts/+; Or83b-Gal4, UAS-2XeGFP/UAS-Kir2.1; (E) w; tubGal80^ts, tubGal80^ts/UAS-p35; Or83b-Gal4, UAS-2XeGFP/UAS-Kir2.1; (F) UAS-Wlds; tubGal80^ts, tubGal80^ts/+; Or83b-Gal4, UAS-2XeGFP/UAS-Kir2.1; (G) w; Or22a-Gal4, UAS-mCD8::GFP/+; Or83b⁻/Or83b⁻; (H) w; Or22a-Gal4, UAS-mCD8::GFP/UAS-UBP2; Or83b⁻/Or83b⁻; (J,K) w; Or47b-Gal4(15.7), UAS-mCD8::GFP/UAS IMPTNT-V; tubGal80^ts, tubGal80^ts/+; (L,M) w; Or47b-Gal4(15.7), UAS-mCD8::GFP/UAS TNT-G; tubGal80^ts, tubGal80^ts/+. Scale bars: 10 µm in C for A-F and in M for J-M.

• Supplemental Figure S7 -

  Fig. S7. Wingless immunoreactivity in the adult antennal lobe. (A-B′) Five-day-old adult animals stained with anti-Wg (red, A′,B′). A few cell bodies peripheral to the antennal lobe are shown (arrows in A′,B′). Appropriate confocal sections are shown to demonstrate expression within glomeruli (one glomerulus has been marked with dotted lines in B and B′). Genotypes: w;; Or83b-Gal4, UAS-2XeGFP.