Figure 5. DILP2 derived from L5 lamina neurons positively regulates Dm8 dendritic field elaboration.

(A) A schematic illustration showing the developmental processes of R7 and R8 photoreceptors, L5 lamina neurons and Dm8 neurons. A Dm8 neuron (purple) expands its dendritic arbors during development to reach its final size. R7 (orange), R8 (cyan) and L5 (green) growth cones undergo dynamic changes at their target locations. R8 growth cones first project to a temporary layer at 24 hr APF and then extend to a deeper layer (M3 in the adult). At 24 hr APF (after puparium formation), R7 growth cones first target the deep medulla layer where Dm8 dendrites reside, and then reshaped and stabilized at the M6 layer in the adult. L5 growth cones extend close to Dm8 dendrites by 24 APF and eventually to the M5 layer at the adult stage. (B–B"') Overexpression of a GFP-tagged DILP2 (green) in LNs led to its accumulation in the deep layer of lamina neurons. Photoreceptors were labeled by anti-24B10 (red), and medulla (Me) and the lobula (Lo) neuropil were labeled by anti-Highwire (Hiw) (blue). Scale bar, 20 μm. (C–C') The GFP-tagged DILP2 (green) is present in the growth cone of lamina neurons (red). The bottom (proximodistal) view is shown. Scale bar, 10 μm. (D–E') In situ hybridization of dilp2 in the developing pupal eye disc. In situ hybridization detected a high level of dilp2 mRNA (green) in the developing L5 lamina neurons (red) which were labeled by anti-Bsh immunostaining (red). Anti-Bsh also labeled the nuclei of L4 lamina neurons (the upper row) in addition to L5’s (the lower row). Side view (D, D') and bottom view (E, E') are shown. The dilp2 mRNA was located in the cytosol adjacent to the nucleus of L5, as shown in E and E'. Scale bar, 10 µm (in D). (F–F') The L5 cell bodies were labeled using 6–60 Gal4 driving the mCD8GFP marker (green). Photoreceptors were labeled by anti-24B10 (blue) as landmarks. (F') shows a single z-section in (F). Scale bar, 10 µm. (G–G') 6–60 Gal4 labeled L5 processes (green) in the medulla (Me), anti-24B10 staining R7/R8 (red) and anti-Hiw (blue) for neuropil landmarks. Scale bar, 10 µm (in G’). 6–60 Gal also labeled few trans-medulla neurons with processes in the medulla and lobula. (H–O) RNAi-mediated dilp2 knockdown in pan-lamina neurons or L5s caused Dm8 dendritic field reduction, as examined in adults. Single flipped-out Dm8 clones (green) with dilp2-RNAi alone (H) or driven two pan-LN driver, 9B08-Gal4 (I) and 27G05-Gal4 (J), or L5-specific 6–60 Gal4 (K), or photoreceptor-specific GMR-Gal4 (L). Misalignment of photoreceptor terminals was consistently observed with pan-LN knock-down of dilp2 (yellow arrow). (M) GMR-Gal4-driven dilp6-RNAi had minimal effects on Dm8 dendrites. (N–O) Overexpression of dilp2 in L5s or photoreceptors did not significantly alter Dm8 dendritic field sizes. Scale bar, 10 μm (in H). (P) Box plot showing the dendritic field sizes of Dm8 neurons in wild-type and dilp manipulations. Dilp2 knock-down in pan-lamina neurons or L5 neurons specifically reduced the Dm8 dendritic fields. *p<0.05; ^#p=0.002; ns, not significant, unpaired Student's t-test. N; the number of cells scored for each genotype.

Figure 5—figure supplement 1. RNAi-mediated knockdown of dilp2 in lamina neurons caused tiling phenotype of photoreceptor R7.

(A–D) The axon terminals of R7 photoreceptors displayed tiling defects after knockdown of dilp2 by the pan-LN 9B08-Gal4, but not the L5-specific 6–60 Gal4 driver. Photoreceptors were visualized by anti-24B10 (red), and R7 axon terminals were labeled by a transgene Rh3/4-Syb::GFP^sp1-10 with polyclonal anti-GFP staining (green). Neuropils were labeled by anti-Hiw (blue). The bottom views of R7 terminals are shown for wild type (A"'), knockdown of dilp2 by 6–60 Gal4 (B"'), and knockdown of dilp2 by 9B08-Gal4 (C"'). (D) Quantification of terminal fusion of R7 photoreceptors in adult wild-type and flies with dilp2 knockdown by 6–60 Gal4 and 9B08-Gal4. (E–E'') Knockdown of dilp2 by 9B08-Gal4 did not cause cell death of lamina neurons. The nuclei of lamina neurons are labeled by anti-Elav (blue), and co-stained with anti-Bsh (green) for L4 and L5. The photoreceptors are labeled by anti-24B10 (red). (F–F') The insulin-binding protein Imp-L2 is highly expressed in the surface glia of the adult optic lobe. Imp-L2 expression was visualized by the Imp-L2-Gal4 driving a mCD8-GFP (green), and co-stained with anti-Hiw (blue) to label the neuropil. La, lamina; Me, medulla; Lo, lobula; Lop, lobula plate.