Supplemental Information. Proprioceptive Opsin Functions. in Drosophila Larval Locomotion
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1 Neuron, Volume 98 Supplemental Information Proprioceptive Opsin Functions in Drosophila Larval Locomotion Damiano Zanini, Diego Giraldo, Ben Warren, Radoslaw Katana, Marta Andrés, Suneel Reddy, Stephanie Pauls, Nicola Schwedhelm-Domeyer, Bart R.H. Geurten, and Martin C. Göpfert
2 Supplemental Information Proprioceptive Opsin Functions in Drosophila Larval Locomotion Damiano Zanini, Diego Giraldo, Ben Warren, Radoslaw Katana, Marta Andrés, Suneel Reddy, Stephanie Pauls, Nicola Schwedhelm-Domeyer, Bart R. H. Geurten, and Martin C. Göpfert Figure S1 (Related to Figure 1) Figure S1. Locomotion and contraction defects in opsin mutants. (A) Locomotion. (B) Body contraction. Same data as in Figure 1B,C. Instead of comparing to wild-type, however, comparisons are shown here against w 1118 larvae, 2
3 which crawl slightly slower than the wild-type. Opsin effects can nonetheless be discerned. N = 30 w 1118 larvae, otherwise as specified in the legend to Fig. 1B,C. Note that genetic rescues partly show significant differences from controls because they are partly outperforming them. Colour codes, abbreviations, and statistics as in Fig. 1B,C. 3
4 Figure S2 (Related to Figure 2) Figure S2. Opsin expression and localization in proprioceptors. (A, B) Additional examples to those shown in Fig. 2. (A) ninae (left) and Rh6 (right) expression in lch5. (B) Anti-NINAE (left) and Anti-Rh6 (right) staining of the inner dendritic segments of lch5 receptors, whereby the receptor neurons are counterstained with anti-hrp. For NINAE, staining is shown for wild-type (WT) larvae, whereas a staining from a Rh6 1, P{Rh6 + } rescue larva is shown for Rh6. Lch5 receptors 2 and 4 show only weak anti- Rh6 staining that can be seen at higher magnification as shown in Fig. 2C. For additional details, see legend to Fig. 2A,B. (C) Rh7 opsin gene expression in chordotonal proprioceptors. Left: Sketch of lch5, depicting chordotonal receptor dendrites (D), somata (S), and axons (A). Right: GFP signals in lch5 receptors revealed driving a UAS-6xGFP reporter with PBac{IT.Gal4}Rh G4, a Gal4 insertion in the coding region of the Rh7 gene. Receptors of lch5 are counterstained with anti-hrp and GFP signals are enhanced with anti-gfp. 4
5 Figure S3 (Related to Figure 4) Figure S3. Ciliary ion channel localization in wild-type proprioceptors. Lch5 stained with anti-iav, anti-nompc, and anti-hrp antibodies. Iav localizes to the proximal ciliary region, between the two HRP bands (arrowheads, left panel). NOMPC localizes to the cilium tips, distally to the second HRP band, yet it protrudes in the proximal ciliary region in lch5 receptors 2 and 4 (arrows, middle panel), which also show weak anti-rh6 signals (Fig. 2C). The extended ciliary NOMPC localization in lch5 receptors 2 and 4 was consistently seen in controls and wild-type, documenting a partial overlap between Iav and NOMPC in some receptor cilia of lch5. 5
6 Figure S4 (Related to Figures 1 and 3) Figure S4. Locomotion and proprioceptor latency. (A) Locomotion effects caused by ablating chordotonal (Ch) receptors through crossing UAS-hid, rpr to the chordotonal receptor driver Dhc93AB-Gal4. (B) Locomotion defects caused by RNAi 6
7 knockdown of ninae in chordotonal receptors by crossing UAS-ninaE-RNAi to the chordotonal receptor driver dnai2-gal4. (C) Locomotion phenotypes of ninae 17, Rh6 1 double mutants compared to the single mutants. (D) Locomotion in santa-maria 1 mutants that fail to synthesize the retinal chromophore compared to wild-type (WT) and w 1118 controls. (E) Locomotion effects caused by ablating TRPA1-expressing neurons by crossing UAS-hid, rpr to trpa1-gal4. (F) Locomotion in ninae 17 and Rh6 1 mutants compared to wild-type and w 1118 controls at an elevated temperature of 29 C where opsins do not contribute to thermosensing. (G) Targeted ablation of the chordotonal receptors by driving UAS-hid, rpr with Dhc93AB-Gal4. Actin-based rods supporting lch5 receptor dendrites are stained with phalloidin (magenta) and neurons with anti-hrp (yellow). Whereas the actin-based rods persist, lch5 receptors are lost. (H) lch5 response latency. Example traces showing lch5 compound action currents (black) in response to a 3 m deflection (red) applied to the stimulus-conveying cap cells. Sample size in panels (A-H): N = 20 to 43 larvae per strain. Significant differences (Fisher's exact permutation tests with Bonferroni correction): * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant. 7
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