DEV020644 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Schematic illustration of the Drosophila visual system. Three layers are depicted (central panel). In this work, we examine the morphology of R cells at two levels, in the retina (left-most panel) and the lamina (right-most panel). The Drosophila compound eye is composed of reiterated facets, designated ommatidia, each of which contains eight R cells (designated R1-R8). We examine the targeting of six of these, R1-R6, whose axons project into the lamina. In the retina, R cell rhabdomes form chevron-shaped clusters in each ommatidium, while in the lamina, R cell axons form circular clusters of synaptic terminals centered around their post-synaptic targets, the lamina neurons.

• Supplemental Figure S2 -

  Fig. S2. SdhA mutant R cells in whole-eye clones develop normally, and then degenerate. (A-L) Cross sections of the lamina neuropil in wild-type (A,C,E,G,I,K) and SdhA¹¹¹⁰ eye-specific mosaic (B,D,F,H,J,L) animals, at (A-D) 72 hours after puparium formation, (E-H) 0 days after eclosion, and (I-L) 5 days after eclosion. Photoreceptor terminals are stained with the R cell-specific antibody mAb24B10 (green), and the synaptic vesicle marker CSP (magenta; A,B,E,F,I,J), or for the active-zone assembly molecule bruchpilot (red; C,D,G,H,K,L). Insets show single synaptic cartridges. SdhA mutant R cell terminal displayed normal labeling at 72 hours after puparium formation, but progressively lost CSP staining and displayed disordered bruchpilot localization in adults. Scale bar: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Early development in SdhA mutants is normal. (A,B) Third instar larval eye discs. R cells, magenta; Bar homeodomain protein, a marker of R1 and R6, green. (C-F) Third instar larval optic lobes. (C,D) R cell axons (red), and a pan-neuronal marker, anti-elaV (green). Carets indicate the lamina plexus; vertical bar, lamina cortex. (E,F) R cells (green), labelled with anti-horse radish peroxidase and a marker for glia, anti-repo (magenta). Ep, epithelial glia; Ma, marginal glia; Me, medulla glia. (G,H) DiI injections into single ommatidia during mid-pupal development. (I) Quantification of dye injection data. (A,C,E,G) Wild type; (B,D,F,H) sdhA mutant.

• Supplemental Figure S4 -

  Fig. S4. Standard curve showing the absorbance of a sample as a function of the concentration of retina. This curve is dependent on the amount of photopigment in each sample (gray square represent retinas isolated from white mutant flies lacking pigmentation), but there was no difference between wild-type (diamonds) and mutant (triangles) retinas. These curves were used to determine the mass of retina dissected in each ATP assay.

• Supplemental Figure S5 -

  Fig. S5. Chronic antioxidant treatment suppresses mitochondrial loss and synaptic terminal degeneration in SdhA mutant R cells. (A-D) Cross sections of the lamina neuropil from (A) 5-day-old wild-type flies grown on food treated with 200 µg/ml α-tocopherol, (B) 5-day-old SdhA¹¹¹⁰ mutant flies treated with vehicle alone, or 5-day-old (C) SdhA¹¹¹⁰ and (D) SdhA¹⁴⁰⁴ mutants treated with 200 µg/ml α-tocopherol. Laminas are stained for the active zone marker Bruchpilot (green) and mitochondrial complex V (magenta). Insets show single synaptic cartridges. Treatment with α-tocopherol suppressed defects in mitochondrial localization, and the regularity of the presynaptic zone (C,D).

• Supplemental Figure S6 -

  Fig. S6. Inhibition of pro-apoptotic pathways does not suppress synaptic degeneration in SdhA mutants. (A-F) Cross sections of lamina neuropil in (A,D) wild type overexpressing the anti-apoptotic protein Buffy, under the control of the R1-R6 specific driver Rh1GAL4, (B,E) SdhA¹¹¹⁰ mutant eye-specific mosaic animals or (C,F) SdhA¹¹¹⁰ mutant eye-specific mosaic animals overexpressing Buffy. Images were taken on the day of eclosion (A-C), and 5 days after eclosion (D-F). R cells are labeled green; vesicles, magenta. (G,H) Caspase3 staining in (G) control and (H) SdhA¹¹¹⁰ mutant eye-specific mosaic retinas. (I-K) TUNEL labeling in (I) control, (J) control treated with DNAse and (K) SdhA¹¹¹⁰ mutant eye-specific mosaic retinas. (L,M) DAPI labeling of nuclei in (L) control and (M) SdhA¹¹¹⁰ mutant eye-specific mosaic retinas. (N) Quantification of nuclear number in control and SdhA¹¹¹⁰ mutant eye-specific mosaic retinas (n, number of ommatidial sections scored). Only a subset of the total nuclei in an ommatidium can be seen in a single ommatidial section. Scale bar: 20 µm.