Drosophila Sidekick is required in developing photoreceptors to enable visual motion detection

Abstract

The assembly of functional neuronal circuits requires growth cones to extend in defined directions and recognize the correct synaptic partners. Homophilic adhesion between vertebrate Sidekick proteins promotes synapse formation between retinal neurons involved in visual motion detection. We show here that Drosophila Sidekick accumulates in specific synaptic layers of the developing motion detection circuit and is necessary for normal optomotor behavior. Sidekick is required in photoreceptors, but not in their target lamina neurons, to promote the alignment of lamina neurons into columns and subsequent sorting of photoreceptor axons into synaptic modules based on their precise spatial orientation. Sidekick is also localized to the dendrites of the direction-selective T4 and T5 cells, and is expressed in some of their presynaptic partners. In contrast to its vertebrate homologs, Sidekick is not essential for T4 and T5 to direct their dendrites to the appropriate layers or to receive synaptic contacts. These results illustrate a conserved requirement for Sidekick proteins in establishing visual motion detection circuits that is achieved through distinct cellular mechanisms in Drosophila and vertebrates.

Keywords: Lamina; Optomotor behavior; Photoreceptor; Sidekick; Visual motion detection.

Fig. 1.

Sdk is a homophilic adhesion molecule expressed in the visual system. (A) The sdk gene (top) with coding exons in black and non-coding exons in gray, showing the positions of the MB05054 Minos insertion, the EP369 P element insertion and derived Δ15 deletion, and the NP3507 GAL4 insertion. The Sdk protein (bottom) has six Ig domains, 13 FNIII domains, a transmembrane domain (TM) and a PDZ-binding motif (PDZ-BM). (B-D) Aggregates formed by S2 cells transfected with Actin5c (Act)-GAL4 and UAS-GFP (green) (B); Act-GAL4, UAS-HA-Sdk and UAS-GFP (C); and a mixture of cells transfected with Act-GAL4, UAS-HA-Sdk and UAS-GFP, and with Act-GAL4 and UAS-mCherry (red) (D). DAPI is in magenta (B,C) and anti-HA in blue (D). Inset provides an enlargement of the boxed region. Sdk-expressing cells form aggregates that lack cells not transfected with Sdk. (E,F) In situ hybridization with a sdk probe on a third-instar eye-antennal imaginal disc (E) and brain (F). sdk is expressed throughout the eye disc and enriched in the optic lobes of the brain. Posterior is towards the right in E-H′. (G) Anti-Sdk (G′, green in G) and anti-Elav (magenta) labeling of an eye-antennal disc, showing enrichment of Sdk protein posterior to the morphogenetic furrow (arrowheads). (H) Anti-Sdk (H′, green in H) and anti-Elav (blue) labeling of an eye disc containing sdk^MB05054 mutant clones marked by the absence of GFP (red). Sdk labeling is absent from the clones. An arrow indicates the morphogenetic furrow. yz sections show Sdk accumulation at the apical surface of the disc (arrowheads). (I) Western blot of extracts from w¹¹¹⁸ (control), sdk^MB05054 and sdk^Δ15 embryos, probed with anti-Sdk and anti-Tubulin (Tub). Sdk protein is not detected in either mutant. Scale bars: 100 µm in D; 30 µm in G and inset in D; 10 µm in H,H′.

Fig. 2.

Lamina neuron placement requires sdk in photoreceptors. (A) Diagram of a coronal section through the third-instar larval brain, showing neuroblasts (nb) and lamina precursor cells (LPCs), which become postmitotic (pLPCs) behind the lamina furrow (LF; arrowhead) and differentiate into lamina neurons (LN) aligned into columns along the axons of R1-R6. Glia are shown in blue. (B-E″) Confocal images of the same view of larval brains for: (B-B″) control (sdk^MB05054/+); (C-C″) sdk^MB05054; (D-D″) sdk knockdown in the eye with ey^3.5-FLP, Act>CD2>GAL4 [Sdk still accumulates in LNs (asterisk)]; and (E-E″) sdk knockdown in the lamina with NP6099-GAL4. A-E″ are labeled with anti-Sdk (B″,C″,D″,E″, green in B′,C′,D′,E′), anti-HRP to mark photoreceptor axons (red in B,B′,C,C′,D,D′,E,E′), anti-β-galactosidase (β-gal) reflecting dac-lacZ (green in B,C) or anti-Dac (green in D,E) to mark lamina neurons, anti-Repo to mark glia (blue in B,C), or anti-β-gal (blue in D,D′) or anti-GFP (blue in E,E′) to mark the domain of RNAi expression. Lamina neurons are misplaced in sdk mutants and when sdk is knocked down in the eye (empty arrowheads, C,D), but not when it is knocked down in lamina neurons or in glia with repo-GAL4. Filled arrowheads mark the lamina furrow. (F) The number of LNs per µm in or beyond the lamina plexus (LP; arrows in A-E′) in the indicated genotypes. Data are mean±s.e.m. n=15 (sdk^Δ7, a precise excision of sdk^MB05054 used as a control, and sdk^MB05054), n=10 (ey^3.5-FLP, Act>CD2>GAL4; sdk RNAi; UAS-dcr2), n=18 (NP6099>sdk RNAi; UAS-dcr2) and n=14 (repo>sdk RNAi; UAS-dcr2). ***P<0.0001 by one-way ANOVA with Tukey's post-hoc test; ns, not significant. Scale bars: 20 µm.

Fig. 3.

sdk is required in photoreceptors for sorting to the correct cartridges. (A-F) Adult laminas labeled with anti-Cysteine string protein (Csp) to mark the terminals of R1-R6 (A-C) or transmission electron micrographs of adult lamina cartridges with R1-R6 terminals pseudo-colored in yellow (D-F). (A,D) sdk^Δ7 control; (B,E) sdk^MB05054; (C,F) sdk RNAi in the eye. Many cartridges contain more or fewer photoreceptor terminals than the usual wild-type number (6), indicating mis-sorting. (G) The distribution of the numbers of R1-R6 terminals per cartridge in sdk^Δ7, sdk^MB05054 and in flies with sdk RNAi expressed in the eye (ey^3.5-FLP, Act>CD2>GAL4) or lamina (27G05-FLP, Act>CD2>GAL4); distributions for sdk mutant and RNAi in the eye differ significantly from controls (P<0.0005, t-test). n=171 cartridges from three retinas (sdk^Δ7, sdk^MB05054); n=90 cartridges from three retinas (sdk RNAi). (H) Adult lamina with sdk^MB05054 clones generated with ey-FLP and marked with anti-β-gal (green), labeled with anti-Csp (H′, magenta). Cartridge organization is abnormal in regions where photoreceptors are mutant (arrowheads). Scale bars: 5 µm in A-C,H,H′; 2 µm in D-F.

Fig. 4.

Sdk is present at contacts between photoreceptor axons and promotes growth cone polarization. (A-D) Laminas of sdk^Δ7 control (A-B′), sdk^MB05054 mutant (C,C′) and E(spl)mδ-GAL4; UAS-sdk RNAi (D,D′) pupae labeled with anti-Sdk (green) and anti-Chaoptin (Chp) to mark photoreceptor axons (blue in A, magenta in A′) and myr-tdTomato expressed in lamina neurons with GH146-GAL4 (red in A) or in R4 with E(spl)mδ-GAL4 (B′,C′,D′, magenta in B,C,D). (A) 30 h after puparium formation (APF). (B-D′) 38-40 h APF. Sdk is localized to five puncta (arrowheads) in a horseshoe shape, two of which are closely apposed to the heel of the R4 growth cone and depleted by sdk RNAi expression in R4 (open arrowheads in D,D′; enlarged in insets). The front of the R4 growth cone is indicated by an arrow in the inset in B′. The orientation of R4 growth cones is variable in sdk mutants but is not affected by knocking down sdk in R4. (E) Quantification of the fluorescence intensity of Sdk labeling at the R4:R5 contact relative to R2:R3 in control and E(spl)mδ-GAL4; UAS-sdk RNAi 30 h pupal laminas. n=67 ommatidia from three laminas, control; n=62 ommatidia from three laminas, sdk RNAi; ***P<0.0001, t-test. (F) Model depicting Sdk stabilizing the contacts between photoreceptor heels to form a scaffold from which the fronts can extend in fixed directions. (G,H) Optomotor responses were tested in a single-fly assay, in which panoramic visual stimuli elicited rotational responses from tethered flies. Graphs show responses to rotating square-wave contrast gratings, with wavelengths of 30°, 60° and 90°. (G) sdk^MB05054 compared with sdk^Δ7; (H) sdk RNAi expressed in the eye with ey3.5FLP, Act>CD2>GAL4 compared with controls (ey3.5FLP, Act>CD2>GAL4 crossed to attP; UAS-dcr2 or Act>CD2>GAL4 crossed to UAS-sdkRNAi; UAS-dcr2). Loss of sdk in the whole animal or just in the eye produced significantly reduced turning responses compared with controls at a wavelength of 30°, but not at 60° or 90°. Points represent mean responses over flies±s.e.m. n=10 (sdk^Δ7); n=13 (sdk^MB05054); n=21 (ey3.5FLP, Act>CD2>GAL4 X UAS-sdkRNAi; UAS-dcr2); n=22 (ey3.5FLP, Act>CD2>GAL4 X attP; UAS-dcr2); n=28 (Act>CD2>GAL4 X UAS-sdkRNAi; UAS-dcr2). *P<0.01, **P<0.001 by a rank sum test, Bonferroni-corrected for nine comparisons between the experimental and control genotypes. Scale bars: 5 µm in A-D′,F; 2 µm in insets in A,A′,B,B′,D,D′.

Fig. 5.

Sdk is localized to the dendrites of T4 and T5, but is not required for them to receive synaptic input. (A-D) Brains labeled with anti-Chp to mark photoreceptor axons (magenta) and anti-Sdk (green). (A) 24 h APF, (B) 42 h APF, (C) 55 h APF and (D) 72 h APF. Lower panels show enlargements of the distal medulla, with dotted lines marking the terminals of R7 and R8. During pupal stages, Sdk is localized to the lamina (La, arrow), to synaptic layers M3a and M10 in the medulla (M), and to Lo1 in the lobula (Lo), but is absent from the R7 and R8 growth cones. Arrowhead in A indicates glial cell bodies. (E) Diagram showing the projection patterns of identified Sdk-expressing neurons in the motion-detection pathways. (F) Adult brain showing subsets of neurons that express NP3507-GAL4, labeled with Citrine using the Flybow system. T4, T5 and C2 cell bodies (arrows) and neurites (arrowheads) are indicated. (G,G′) 55 h APF brain in which sdk RNAi is expressed in T4 and T5 with GMR42F06-GAL4, labeled with anti-Chp (magenta) and anti-Sdk (G′, green). Sdk is lost from the cell bodies in the lobula plate cortex (asterisk, compare with asterisk in C) and from the M10 and Lo1 layers, which contain the dendrites of T4 and T5, respectively. (H-I,K-L′) Adult brains, with the Lo1 layer (arrow in H) enlarged in H′,I,K-L′. (H-I) Endogenous Brp is tagged with V5 in Tm9 to label concentrations of presynaptic active zones (H′,I, green in H). Myr-tdTomato (magenta) labels the Tm9 neurons. (H,H′) sdk^Δ7; (I) sdk^MB05054. Brp-V5 puncta/µm² in the Lo1 layer, counted in J (mean±s.e.m.), show no significant difference (P>0.5, t-test) between sdk^Δ7 controls and mutants. n=94 sections from eight optic lobes (sdk^Δ7), 110 sections from 10 optic lobes (sdk^MB05054). (K-L′) GFP reconstituted from presynaptic Syb-GFP1-10 expressed in Tm9 and postsynaptic CD4-GFP11 expressed in T5 (K′,L′, green in K,L). Tm9 terminals are labeled with myr-tdTomato (magenta). (K,K′) sdk^Δ7; (L,L′) sdk^MB05054. The GRASP signal is taken to indicate that functional synapses are formed between Tm9 and T5 in sdk mutants. Scale bars: 20 µm.